Synthesis and Initial Biological Evaluation of Boron-Containing Prostate-Specific Membrane Antigen Ligands for Treatment of Prostate Cancer Using Boron Neutron Capture Therapy

Article abstract of DOI:10.1021/acs.molpharmaceut.9b00464

Boron neutron capture therapy (BNCT) is a therapeutic modality which has been used for the treatment of cancers, including brain and head and neck tumors. For effective treatment via BNCT, efficient and selective delivery of a high boron dose to cancer cells is needed. Prostate-specific membrane antigen (PSMA) is a target for prostate cancer imaging and drug delivery. In this study, we conjugated boronic acid or carborane functional groups to a well-established PSMA inhibitor scaffold to deliver boron to prostate cancer cells and prostate tumor xenograft models. Eight boron-containing PSMA inhibitors were synthesized. All of these compounds showed a strong binding affinity to PSMA in a competition radioligand binding assay (IC₅₀ from 555.7 to 20.3 nM). Three selected compounds 1a, 1d, and 1f were administered to mice, and their in vivo blocking of ⁶⁸Ga-PSMA-11 uptake was demonstrated through a positron emission tomography (PET) imaging and biodistribution experiment. Biodistribution analysis demonstrated boron uptake of 4-7 μg/g in 22Rv1 prostate xenograft tumors and similar tumor/muscle ratios compared to the ratio for the most commonly used BNCT compound, 4-borono-l-phenylalanine (BPA). Taken together, these data suggest a potential role for PSMA targeted BNCT agents in prostate cancer therapy following suitable optimization.

Full text of DOI:10.1021/acs.molpharmaceut.9b00464

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Article
Synthesis and initial biological evaluation of boron-containing
prostate specific membrane antigen ligands for treatment
of prostate cancer using boron neutron capture therapy
Sinan Wang, Charles Blaha, Raquel Santos, Tony Huynh, Thomas R. Hayes, Denis R. Beckford
Vera, Joseph E Blecha, Andrew S Hong, Miko Fogarty, Thomas A Hope, David R Raleigh, David
M. Wilson, Michael J. Evans, Henry F. VanBrocklin, Tomoko Ozawa, and Robert R. Flavell
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.9b00464 • Publication Date (Web): 05 Aug 2019
Downloaded from pubs.acs.org on August 5, 2019
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Synthesis and initial biological evaluation of boron-containing prostate
specific membrane antigen ligands for treatment of prostate cancer using
boron neutron capture therapy
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Sinan Wang†, Charles Blaha‡, Raquel Santos§, Tony Huynh†, Thomas R. Hayes†, Denis R.
Beckford-Vera†, Joseph E. Blecha†, Andrew S. Hong†, Miko Fogarty§, Thomas A. Hope†, David
R. Raleigh§,‖, David M. Wilson†, Michael J. Evans†, Henry F. VanBrocklin†, Tomoko Ozawa§,
Robert R. Flavell*,†
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†
Department of Radiology and Biomedical Imaging, University of California, San Francisco, California, USA.
‡
Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, California, USA.
§
Department of Neurological Surgery, University of California, San Francisco, California, USA.
‖
Departments of Radiation Oncology, University of California, San Francisco, California, USA.
ABSTRACT:
Boron neutron capture therapy (BNCT) is a therapeutic modality which has been used for the
treatment of cancers, including brain and head and neck tumors. For effective treatment via
BNCT, efficient and selective delivery of a high boron dose to cancer cells is needed. Prostate
specific membrane antigen (PSMA) is a target for prostate cancer imaging and drug delivery. In
this study, we conjugated boronic acid or carborane functional groups to a well-established
PSMA inhibitor scaffold to deliver boron to prostate cancer cells and prostate tumor xenograft
models. Eight boron-containing PSMA inhibitors were synthesized. All of these compounds
showed strong binding affinity to PSMA in a competition radioligand binding assay (IC50 from
555.7 nM-20.3 nM). Three selected compounds 1a, 1d and 1f were administered to mice, and
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their in vivo blocking of Ga-PSMA-11 uptake was demonstrated through a positron emission
tomography (PET) imaging and biodistribution experiment. Biodistribution analysis
demonstrated boron uptake of 4-7 µg/gram in 22Rv1 prostate xenograft tumors and similar
tumor/muscle ratios compared to the ratio for the most commonly used BNCT compound, 4-
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borono-L-phenylalanine (BPA). Taken together, this data suggest a potential role for PSMA
targeted BNCT agents in prostate cancer therapy following suitable optimization.
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KEYWORDS: Boron neutron capture therapy (BNCT), prostate cancer, prostate specific
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membrane antigen (PSMA) inhibitor, carborane, boron uptake
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■ INTRODUCTION
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Boron neutron capture therapy is a cancer treatment method based on the selective uptake of
boron-containing drugs into cancer cells over normal cells, followed by selective neutron beam
exposure to the cancerous regions.1,2,3 This irradiation initiates the emission of cytotoxic alpha
particles and lithium ions. The released high linear energy transfer (LET) α-particles can travel a
distance of only around 9 µm in tissue, which is approximately the diameter of a cell. Therefore,
their short-range cytotoxicity can significantly improve tumor control with concomitant sparing
of the surrounding normal tissues.4,5 This therapeutic modality has been successfully used for
treatment of patients with primary brain and head and neck tumors.6,7,8 Clinically used BNCT
reagents include 4-borono-L-phenylalanine (BPA) and sodium borocaptate (BSH).9,10,11
However, due to various limitations including relatively low target to background ratio binding
of boron-labeled therapeutic agents, BNCT has yet to emerge as a standard of care treatment
modality for most cancer types.
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Prostate cancer (PCa) is one of the most prevalent noncutaneous cancer in men.12,13 Prostate
specific membrane antigen, also known as glutamate carboxypeptidase II (GCPII) or N-acetyl-L-
aspartyl-L-glutamate peptidase, is a cell surface enzyme which is highly expressed on prostate
cancer cells.14 Recently, urea based inhibitor agents targeting the enzymatic domain of PSMA
have been developed for both imaging and therapy of prostate cancer.15,16,17,18 Therapy with
radionuclides has been demonstrated using 177Lu and 225Ac, which exhibit successful tumor
control but also have side effects including xerostomia and bone marrow suppression.19,20 PSMA
targeted therapeutics and imaging agents have been extended beyond radionuclides, including
nanoparticles and antibody drug conjugates.21,22,23
The high target to background ratio of PSMA targeted imaging and therapeutic agents,
coupled with the high efficacy of BNCT, suggest the potential for boron labeled PSMA targeting
agents as effective BNCT agents. In principle, this approach could combine the benefit of high
target to background ratio of PSMA directed therapeutics with the spatial localization offered by
a neutron beam. Therefore, this approach could be used for treatment of oligometastatic prostate
cancer, potentially the minimizing off target side effects of radionuclides including xerostomia
and bone marrow suppression. Boron labeled PSMA targeting agents have been reported, their
binding to the enzyme characterized, and initial biodistribution studied using radioiodinated
derivatives.24,25 However, these studies did not determine the degree of boron uptake in tumors,
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which is necessary to predict therapeutic efficacy. We hypothesize that by using boron-
containing target-specific molecules, the localized and enhanced expression of PSMA on PCa
cells in comparison to normal tissue could enable preferential and adequate delivery of boron to
PCa for BNCT. We designed a series of boron labeled agents targeting PSMA including three
components – a urea-based PSMA binding group, a linker moiety, and a substituent containing
one, ten, or twenty boron atoms (Figure 1). In this study, we report the synthesis of a series of
boron-labeled agents targeting PSMA, and their initial in vitro and in vivo evaluation in
biodistribution assays.
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Figure 1. (A) The design of boron labeled PSMA targeted agents includes a urea based
inhibitory element, connected to a boron containing substituent via a linker moiety. (B)
Structures of boron labeled PSMA agents evaluated in this study.
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■ EXPERIMENTAL SECTION
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2.1. Materials.
o-Carborane was purchased from Boron Specialties (PA, U.S.A.). Decaborane and all other
chemicals were purchased from Sigma-Aldrich (MO, U.S.A.). RPMI-1640 medium, Fetal bovine
serum (FBS) and penicillin-streptomycin solutions were purchased from Life Technologies (NY,
U.S.A.).
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2.2 Chemistry.
Details for the synthesis and characterizations of all compounds are provided in the
supplementary information.
2.3 Cell Culture
The PSMA-expressing human prostate cancer cell line 22Rv1 was obtained from ATCC.
22Rv1 cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum
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(FBS), 100 units of penicillin and 100 µg/mL streptomycin in a humidified incubator at 37 C
and 5% CO2. Cells were removed from flasks for passage or for transfer to 48-well assay plates
by incubating them with 0.25% trypsin.
2.4 IC50 Measurement
IC50 values of the boron-containing compounds 1a-h were determined by screening in a
competition radioligand binding assay against 68Ga-PSMA-11. 68Ga-PSMA-11 was synthesized
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as previously reported using at Ge/68Ga generator and a manual synthesis model, using 5 µg
precursor yielding approximately 10-35 mCi per batch.26 2-(phosphonomethyl) pentanedioic acid
(PMPA) is a known PSMA inhibitor and has been chosen as a positive control.27 22Rv1 cells
were plated in a 48-well plates (250 µL/well) 48 h before testing (triplet) in RPMI1640 medium
supplemented with 10% fetal bovine serum. The cell number was about 250,000 per well when
the assay was performed. The growth medium was removed and washed with PBS for three
times. Various concentrations (0.01-100000 nM) of the tested compounds in serum free
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RPMI1640, together with 10 µCi (5 ng) of Ga-PSMA-11, were added to cells. The cells were
incubated in this buffer for 1 h at 37o C. Then the radioactive medium was removed by pipette,
and cells were washed by PBS twice, and 250 µL of 5N NaOH was added to lyse the cells. The
lysate was transferred to small vials, and the bound radioactivity was counted using a Hidex
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gamma counter. IC50 values were determined by nonlinear regression using GraphPad Prism
software (GraphPad Software).
2.5 Log P Measurement
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Log P measurements were made using the “shake-flask” method25,28. The ICP matrix was
prepared using 2% H2SO4, 2% HNO3 and 0.3% triton in Millipore water. 50 µL of 1 mM
compound in PBS (pH = 7.4) was added to each of three vials containing 50 µL of 1-octanol.
The vials were shaken on a vortex mixer for 10 min, followed by centrifugation for a further 10
min. Aliquot (30 µL) of each PBS and octanol layer were transferred to 15 mL Falcon tubes and
diluted to 10 mL with ICP matrix buffer or 0.3 mL of EtOH and 9.7 mL of ICP matrix buffer
respectively. The boron content was measured by Inductively Coupled Plasma Optical-Emission
Spectrometry (ICP-OES, Thermo Scientific iCAP 7000 series). Log P values were calculated as
Log{[boron content per mL(1-octanol)]/[ boron content per mL(buffer)]}.
2.6 Internalization Studies
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When cells reached 70% confluency in a T75 flask, medium was removed, and the cells
were washed twice with PBS. 20 mL 100 µM compound in serum free RPMI 1640 was added
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and the cells were incubated at 37 C for 1 hour. The medium was removed and the cells were
washed with PBS twice. The cells were detached with 0.25% trypsin solution. The cells were
washed three times with PBS and then treated with a solution of 50 mM glycine and 100 mM
NaCl at pH 3 for 2 min at 37 °C and 5% CO2. Cells were then centrifuged (3 min at 12,000 rpm),
and the supernatant was collected. This treatment was repeated two additional times, and the
combined supernatants were diluted to 10 mL with ICP matrix. The pellet was digested by 400
µL 1:1 H2SO4/HNO3 mixture overnight, then diluted to 10 mL by ICP matrix. The boron content
was measured by Inductively Coupled Plasma Optical-Emission Spectrometry (ICP-OES,
Thermo Scientific iCAP 7000).
2.7 Inoculation of Mice with Xenograft
All animal studies were conducted according to an Institutional Animal Care & Use
Program (IACUC) approved protocol. Five to six weeks old, male athymic mice (nu/nu,
homozygous; Jackson Laboratories), housed in aseptic conditions, received subcutaneous tumor
cell inoculation, as approved by the University of California, San Francisco Institutional Animal
Care and Use Committee. In brief, 5 million 22Rv1 cells in a 200 µL 1:1 mixture of complete
medium and matrigel (Fisher Scientific, IL) was injected in the left thigh of the animals. All
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mice were subjected to undergo PET imaging as well as biodistribution analysis when the tumor
reached a size of 300 - 500 mm3.
2.8 Serum protein binding assay
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25 µl of 1 mM compound in PBS was added to 0.5 ml mouse serum. The mixture was
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vortexed for 5 mins and incubated at 37 C for 30 mins. Then 0.5 ml acetonitrile was added to
precipitate the protein. The mixture was then centrifuged (3 min at 12,000g), and the
supernatants and pellet were collected respectively. ICP matrix was prepared by 2% H2SO4, 2%
HNO3, 0.3% triton in millipore water. The supernatants were diluted to 10 ml by ICP matrix. The
pellet was digested by 400 µl 1:1 H2SO4/HNO3 mixture overnight, then diluted to 10 ml by ICP
matrix. The boron content was measured by ICP-OES.
2.9 Toxicity Test
Cell toxicity assay was performed using a CellTiter-Glo kit to measure the viability of cells
after incubating with various concentrations of compounds. 22Rv1 cells were plated in a 96-well
plates (100 µL/well) 48 h before testing (triplet) in RPMI1640 medium supplemented with 10%
fetal bovine serum. The growth medium was removed and washed with PBS for three times.
Various concentrations (0.01-100000 nM) of the tested compounds in PBS and blank were added
to cells. The cells were incubated in this buffer for 4 h at 37o C. Then 100 µL of CellTiter-Glo
buffer was added to each well. The mixture was incubated at room temperature for 15 minutes
and the luminescence was measured using a luminometer.
For animal toxicity experiments, non-tumor bearing male athymic mice were administered
graded intraperitoneal doses of the selected boronated compounds to determine the maximum
tolerated dose (5 mg/mouse, 7.5 mg/mouse, 15 mg/mouse or 30mg/mouse), and were monitored
for 24 hours. Each testing group contained two mice. If animals showed physical symptoms as
outlined by our IACUC guidelines such as loss of activity, animals were euthanized.
2.10 In Vivo PET Imaging Studies.
Approximately three weeks after implantation, animals with tumors reaching 300-500 mm3
were anesthetized by isoflurane inhalation and were administered 200 μL of PBS, compound 1a
(30 mg/mouse in 200 μL of PBS), 1d (5 mg/mouse in a mixture of 40 μL of DMSO and 160 μL
of PBS) or compound 1f (7.5 mg/mouse in a mixture of 40 μL of DMSO and 160 μL of PBS)
through a single intraperitoneal injection. BPA (5 mg/mouse) was administered to mice through
oral gavage due to its low solubility in buffer. Approximately 15 minutes after the administration
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of these compounds, 100-150 μCi (3.70-5.55 MBq, 50 – 75 ng) of 68Ga-PSMA-11 was
administered through tail vein injection. The animals were imaged with 10 min acquisition by a
microPET/CT imaging system (Inveon, Siemens, Germany) at 1 h post injection of the first
administration. PET imaging data were acquired in list mode and reconstructed with the iterative
OSEM 2-D reconstruction algorithm provided by the manufacturer. Imaging data were viewed
and processed using open source Amide software.
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2.11 Biodistribution Studies.
The 22Rv1 tumor bearing mice were sacrificed at the 1 hour or 4 hour time points post
injection of 68Ga-PSMA-11. Blood was collected by cardiac puncture. Major organs (brain, bone,
heart, kidney, liver, lung, muscle, pancreas, salivary, skin, spleen and subcutaneous tumor) were
harvested, weighed, and counted in an automated gamma counter (Hidex). The percent injected
dose per gram of tissue (% ID/g) was calculated by comparison with standards of known
radioactivity.
2.12 Organ Digestion and ICP Mass
Tissue samples were digested for 2 days at room temperature in 1 mL of a 1:1 mixture of
concentrated sulfuric and nitric acids. After digestion, 1.5 mL of a 5 % Triton X-100 solution in
water was added to each sample. The samples were then sonicated for 90 min. 1 mL of the
sample solution was transferred to a 15 mL centrifuge tube and diluted to 10 mL with ICP matrix.
The samples were processed for boron analysis by ICP-OES.
■ RESULTS
3.1 Synthesis of Compound 1a-1f.
The syntheses of compounds 1a-f are outlined in scheme 1. Various boron-containing
activated esters were coupled to a common synthetic intermediate 12 to yield the final products.
Starting materials 2a and 2b are commercially available. Starting materials 2c24, 329, 530, 931,
1232 and reagent B10H12(MeCN)231 were obtained following known literature procedures. Briefly,
compound 3 was reacted with B10H12(MeCN)2 followed by deprotection to generate intermediate
2d. Sonogashira coupling of compound 5 with trimethylsilyl acetylene followed by a
deprotection formed intermediate 7. Reacting 7 with B10H12(MeCN)2 followed by deprotection
gave carborane containing intermediate 2e. Mixing compound 9 with 4-nitrophenyl
chloroformate produced carbonate 10. Then 10 was reacted with L-lysine tert-butyl ester
hydrochloride followed by deprotection to give di-carborane containing intermediate 2f. Each of
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the obtained boron containing intermediates 2a-f was mixed with N,N
′ -
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Dicyclohexylcarbodiimide (DCC) and N-Hydroxysuccinimide (NHS) in THF and stirred
overnight, the precipitation was removed by filtration. Then, compound 12 was added to the
solution, the mixture was stirred for 24 hours. After purification by chromatography, each of
compound 13a-f was reacted with TFA in DCM to remove the protecting groups, yielding the
final products 1a-f.
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O
O
H
O
OH
OH
HO
OH
O
B
B
OH
O
2a
2b
2c
O
O
O
H
O
H
OH
b
a
O
4
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3
O
c
O
d
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O
O
O
Br
Si
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O
OH
H
H
f
e
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O
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2e
H
H
O
N
O
N
NO2
H
H
O
O
H
O
H
h
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O
g
i
OH
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O
OH
O
O
NH
O
NH
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H
O
H
2f
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O
O
NH2
R
NH
R
NH
O
O
O
HO
O
O
O
O
O
j
O
k
+
O
R
OH
O
O
HO
OH
O
N
H
N
N
N
N
H
N
H
H
H
H
O
O
O
O
O
O
2a-f
12
13a-f
1a-f
Scheme 1. Reagents and conditions for the synthesis of compounds 1a-f:
o
Key synthetic intermediates are outlined. (a) B10H12(MeCN)2, AgNO3, PhMe, 100 C. (b) Pd/C,
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H2, MeOH, rt. (c) trimethylsilyl acetylene, PdCl2(PPh3)2, Et3N, 70 C. (d) Bu4NF, THF, rt. (e)
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B10H12(MeCN)2, AgNO3, PhMe, 100 oC. (f) Pd/C, H2, MeOH, rt. (g) 4-nitrophenyl
chloroformate, Et3N, PhMe, 70 oC. (h) L-lysine tert-butyl ester, Et3N, THF, rt. (i) TFA, DCM, rt.
(j) DCC, NHS, THF, rt. (k) TFA, DCM.
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3.2 Synthesis of Compounds 1g and 1h.
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The synthesis of compound 1g and 1h is outlined in scheme 2. The starting material 15 was
obtained following a known literature procedure.33 Intermediate 10 was reacted with compound
12 or 15 followed by trifluoroacetic acid (TFA) deprotection to generate final compounds 1g or
1h respectively.
H
O
H
O
O
NH
NH2
O
NH
NO2
O
O
O
H
O
O
O
HO
O
O
O
b
a
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+
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O
O
O
HO
OH
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N
N
N
N
N
H
H
H
H
H
H
O
O
O
O
O
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1g
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O
H2N
H
NH
O
O
N
NO2
NH
O
H
O
O
O
O
O
O
O
O
H
a
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+
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N
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H
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H
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H
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O
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O
H
N
O
NH
O
HO
O
O
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b
HO
OH
N
N
H
H
1h
O
O
Scheme 2. Reagents and conditions for the synthesis of compounds 1g, 1h.
(a) Et3N, THF, rt. (b) TFA, DCM, rt.
3.3 In Vitro Binding Studies
The relative binding affinities of compounds 1a-h were determined using a 68Ga-PSMA-11
competitive radioligand binding assay (Figure 2). The compounds that have been prepared are
shown in Table 1. PMPA was selected for comparison and as a positive control.27 In general,
most of the compounds had a very high binding affinity, improved compared to PMPA, ranging
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from approximately 20 – 550 nM. The relatively high IC50 for PMPA observed compared against
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a prior study may be due in part to the high affinity of the Ga-PSMA-11 ligand used and/or
differences in cell type.25 Based on the IC50 values of compounds 1d/1e and 1g/1h, compounds
with shorter linkers had higher binding affinities than compounds with longer linkers. The
dicarborane compound 1f had an increased IC50, possibly due to its branched structure and bulky
size.
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Based on these encouraging binding assays, three compounds were advanced for in vivo
evaluation. Compound 1a was selected for further study due to its good binding affinity,
excellent water solubility and as a representative for the boronic acid PSMA inhibitor series, as a
complement to the more hydrophobic carborane series. Carborane containing compound 1d was
selected since it showed the highest binding affinity. Compound 1f was selected due to its
highest boron content among the compounds (20 boron atoms per molecule).
Figure 2. IC50 of compounds 1a-1h determined by a competitive binding assay against 68Ga-
PSMA-11 on PSMA expressing 22Rv1 cells.
Table 1: IC50 of compounds 1a-1h for PSMAα,β,γ
.
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O
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Entry
R
IC50 (nM)
Entry
R
IC50 (nM)
H
O
N
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H
O
1a
130.3±28.9
1f
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363.8±8.7
1g
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O
H
H
O
O
N
1h
128.0±8.4
O
H
O
H
1d
1e
20.3±2.4
control
2-PMPA
523.5±84.0
536.8±58.7
O
H
α Cell line in all cases is 22Rv1. β The competitive binding compound is 68Ga-PSMA-11. γ Cells
were plated 48 h before testing (in triplicate). Various concentration (0.01-100000 nM) of
compounds with 10 µCi of 68Ga-PSMA-11 were added to cells and incubated for 1h. Cells were
washed and lysed. A gamma counter was used to measure the bound fraction to the lysed cells.
3.4 LogP Hydrophobicity Assay, Internalization Assay and Serum Protein Binding Assay
The logP values of compound 1a, 1d and 1f were measured to assess their hydrophilicity
using the shake flask method and analysis of the resulting fractions via ICP-OES. Compared
against the carborane containing compounds 1d and 1f, the boronic acid compound 1a showed
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increased hydrophilicity. A cellular internalization assay was performed in 22Rv1 cells. In the
internalization assay, we found that a large portion of compound 1a was bound at the membrane
instead of being internalized into the cells. For compound 1d and 1f, most of the compounds
were internalized into the cells (Table 2). The distribution of compounds 1a, 1d and 1f in rat
serum protein was measured. We found that only a small percentage of the tested compounds are
bound to serum protein (supplemental table S2).
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Table 2. Property of selected compounds
Internalization (x109 boron/cell)α
Compound
LogP
pellet
membrane
0.093 ± 0.053
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0.14 ± 0.063
α
100 µM selected compounds were incubated with cells for 1 h.
3.5 Cell Toxicity Assay and Acute Toxicity Assays
Compounds 1a, 1d and 1f were selected for cell toxicity assay using the CellTiter-Glo assay.
After incubating 22Rv1 with these three tested compounds for 4 hours, no obvious toxicity was
observed for all of the three compounds even up to 100 µM, suggesting a low cell toxicity of
these compounds (supplemental table S3 and figure S1).
A preliminary acute toxicity assay was performed prior to biodistribution and imaging
studies. Compound 1a was dissolved in 200 μL of PBS. Compound 1d or 1f was dissolved in a
mixture of 40 μL of DMSO and 160 μL of PBS. Non-tumor bearing male athymic mice were
administered graded doses of the selected boronated compounds via intraperitoneal injection (5
mg/mouse, 7.5 mg/mouse, 15 mg/mouse or 30mg/mouse), and were monitored closely.
Compound 1a demonstrated no acute toxicity up to 30 mg per mouse. For compound 1d and 1f,
the maximum tolerated dose was 5 mg per mouse and 7.5 mg per mouse, respectively. When 7.5
mg 1d/mouse or 15 mg 1f/mouse was administered, one mouse in each group demonstrated
acute toxicity requiring euthanasia (supplemental table S4). No obvious hemorrhages or
damaged organs were observed on gross dissection.
3.6 Outline of In Vivo PET Imaging and Biodistribution Assays
Next, we determined the ability of the boron labeled PSMA agents to bind to PSMA in vivo
using a PET imaging assay, and determined the boron biodistribution by harvesting the organs
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and recording ICP-OES of the resulting tissues (Figure 3). The in vivo Ga-PSMA-11 blocking
assay and boron biodistribution studies were performed in athymic mice bearing 22Rv1 tumor
xenografts three weeks post inoculation. The study population consisted of nine groups (n = 4
mice for each group). Vehicle (PBS) was used as a negative control. Boronophenylalanine (BPA)
is the most widely used compound in BNCT clinical trials, and was included for comparison.34
Tested compounds were administered followed by the injection of approximately 100 µCi (3.7
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MBq) of Ga-PSMA-11. For the vehicle group, mice were imaged after 1 hour post injection
and were immediately sacrificed for organ collection after imaging. For each of BPA and
candidate compounds 1a, 1d and 1f, mice were split to either the 1 hour group or the 4 hour
group. For the 1 hour group, mice were sacrificed and their organs were collected at 1 hour post
injection. For the 4 hour group, mice were imaged at 1 hour post injection and were subsequently
sacrificed at 4 hour post injection for organ collection.
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Figure 3. Flow chart of PET imaging, Ga-PSMA-11 biodistribution and boron biodistribution
assay performed in mice bearing 22Rv1 xenograft tumors.
3.7 In Vivo PET Imaging and 68Ga-PSMA-11 Biodistribution Analysis
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Analysis of the micro PET/CT images demonstrated that for the vehicle and BPA group,
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there was high Ga-PSMA-11 uptake in the tumor, as expected based on the known expression
of PSMA in 22Rv1 xenografts.35,36 On the other hand, boron-containing PSMA inhibitors 1a, 1d,
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and 1f blocked Ga-PSMA-11 uptake in the tumors, demonstrating the strong in vivo binding
between boron-containing PSMA inhibitors and PSMA (Figure 4A). The 68Ga-PSMA-11
biodistribution showed high uptake in kidney, spleen and tumor, similar with previously
published results.36 As expected, the addition of BPA did not significantly block uptake in target
organs including kidney, spleen and tumor. Unexpectedly, there was an apparent increase in the
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blood and tumor concentration of Ga-PSMA-11 with BPA treatment. The reasons for this are
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treatment. In contrast, 1a and 1d significantly reduced the uptake of Ga-PSMA-11 in kidney,
spleen and tumor. Compound 1f reduced uptake of 68Ga-PSMA-11 in spleen and tumor, but there
was still moderate amount of uptake at kidney (Figure 4B). The tumor/blood ratio and
tumor/muscle ratio showed a decrease when inhibitors were administered (Figure 4C). For
compound 1a and 1d, the ratio markedly increased after 4 hour compared to 1 hour, while for
compound 1f, the ratio only slightly increased, which indicates a longer circulation life time of 1f.
Similar results were obtained through region of interest analysis of the micro PET/CT images
(supplemental table S5 and S6). Taken together, these data indicate that boron labeled PSMA
inhibitors 1a, 1d, and 1f are able to block 68Ga-PSMA-11 uptake in target tissues including
tumor and kidney, suggesting a high degree of receptor occupancy in vivo.
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Figure 4. (A) PET imaging results showed efficient blocking of 68Ga-PSMA-11 uptake by boro-
PSMA compounds, but not by BPA. (B) 68Ga-PSMA-11 biodistribution as determined by
collection of organs and gamma counting. (C) Tumor/muscle uptake ratio of 68Ga-PSMA-11.
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3.8 In Vivo Boron Biodistribution Analysis
The organs collected were also subjected to ICP-OES to assay total boron content (Figure
5A and Figure S2 in Supplementary). As expected, the vehicle group resulted in minimal boron
uptake in tissue when no boron compounds were administered. The known BNCT compound
BPA demonstrated accumulation at blood, kidney, pancreas and especially liver. For 1a, the
blood boron content significantly decreased at 4 hour compared to 1 hour from 7.8 µg/gram
tissue to 1.7 µg/gram, suggesting quick clearance of this compound. There was more uptake in
kidney than in liver, suggesting primarily renal elimination, as expected based on its hydrophilic
character. The tumor boron uptake was 4.1 µg/gram tissue, which is similar to boron uptake in
muscle (3.8 µg/gram). For compound 1d, the blood boron content slightly decreased at 4 hour
compared to 1 hour from 22.5 µg/gram tissue to 17.1 µg/gram tissue, indicating a longer
circulation time in vivo. More uptake was observed in liver than in kidney, suggesting primarily
hepatobiliary clearance. The boron uptake in tumor was 4.4 µg/gram tissue at 1 hour, similar to
the boron uptake in muscle (4.4 µg/gram). However, the boron uptake in tumor was 4.2 µg/gram
tissue at 4 hour, two fold higher than the boron uptake in muscle (2.2 µg/gram tissue) (Figure
5C). For compound 1f, the blood boron content significantly increased at 4 hour compared to 1
hour from 17.4 µg/gram tissue to 78.3 µg/gram tissue, suggesting a slow absorption and long
circulation time for the compound in mice. The liver had greater uptake than kidney, again
suggesting hepatobiliary clearance. The tumor boron uptake is 3.3 µg/gram tissue at 1 hour and
3.4 µg/gram tissue at 4 hour, which was lower than the boron uptake in muscle (5.1 µg/gram
tissue and 5.1 µg/gram tissue, respectively). Overall, the more hydrophobic compounds 1d and
1f had a longer circulation time in blood. Similar total tumor uptake, ranging from 3.3 – 7.1
µg/gram was seen between BPA, compounds 1d, and 1f, while compound 1a demonstrated
decreased tumor uptake at four hours.
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Figure 5. (A) Boron biodistribution. Tissues were harvested as indicated in figure 3,
homogenized and boron content was determined by ICP-OES. (B) Boron uptake in tumor (C)
Tumor/muscle uptake ratio of boron. (D) Tumor/blood uptake ratio of boron.
■ DISCUSSION
In this report, we demonstrate the synthesis and initial in vitro and in vivo evaluation of a
series of boron-containing PSMA inhibitors based around a common urea skeleton. Although all
these eight Boro-PSMA compounds share the same urea-based PSMA binding skeleton, the
alteration of the side chain significantly influenced their binding affinity, cell internalization
ability and pharmacokinetics. Overall, all compounds demonstrated equal to or improved ability
to bind PSMA based on a competition radioligand binding assay, when compared against known
high affinity PSMA inhibitor, PMPA. The very high binding affinity of these compounds is not
surprising given the well-known versatility of the urea-based PSMA binding scaffold.37,38,39
Based on these encouraging initial results, three compounds were advanced to initial in vivo
testing using imaging and boron biodistribution assays. Compound 1a was selected as a
hydrophilic, single boron containing agent. Compound 1d was selected as a representative of the
carborane group, with the highest overall IC50 of 20.3 nM. Compound 1f was selected as it had
two carborane substituents and the largest number of boron atoms (20) as well as reasonable
binding affinity. All three compounds demonstrated high PSMA binding in vivo as demonstrated
by PET imaging and 68Ga-PSMA-11 biodistribution analysis, while BPA did not demonstrate in
vivo PSMA binding. However, the boro-PSMA compounds demonstrated differing
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pharmacokinetics and overall tumor boron uptake. The hydrophilic compound 1a demonstrated
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rapid renal clearance, and efficient binding of PSMA in vivo as judged by the blocking of Ga-
PSMA-11 uptake in tumor. However, the overall tumor boron uptake was the lowest of all
compounds tested at four hours, despite the highest administered dose, possibly due to only a
single boron atom per molecule. Overall, compound 1a is a very hydrophilic molecule due to its
three carboxylic acids and the boronic acid group. In contrast, the more hydrophobic carborane
compounds 1d and 1f demonstrated primary hepatobiliary clearance, while still demonstrating
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efficient Ga-PSMA-11 blocking in vivo. The in vivo half life of these compounds appeared to
be significantly greater than 1a as judged by the high residual boron content in the blood at four
hours after administration. Compounds 1d and 1f demonstrated similar overall boron uptake in
tumor when compared against BPA, the most commonly used agent for BNCT. However, an
unexpected result is that the increased boron content per molecule did not increase the uptake of
1f in tumor when compared against 1d, possibly because of slow absorption. For compound 1d
and 1f, the boron concentration in the blood was still high after 4 hours, indicating an increased
boron uptake at tumor after 4 hours, but also a higher risk of toxicity.
In this study, we assayed the receptor engagement through competition binding assays with
68Ga-PSMA-11 PET, and direct tumoral uptake through biodstribution assays and ICP-MS of
total tissue boron content. Another alternative way to determine biodistribution of boron
containing compounds could be considered for future studies using direct labeling of the Boro-
PSMA derivatives with chelators (DOTA, DTPA), that can coordinate metal imaging probes
such as 68Ga for PET or Gd for MRI.40,41
To date, there have been two reports of boro-PSMA inhibitors. Byun’s group reported the
synthesis of carborane containing PSMA inhibitors and a PSMA-inhibitor X-ray structural
characterization.24 However, the compounds were not further evaluated in animal models.
Valliant’s group also produced a series of carborane containing PSMA inhibitors, but the IC50
were not improved compared to PMPA.25 In contrast, the boro-PSMA inhibitors in our study
exhibited a significantly enhanced IC50 up to 26-fold reduced compared to PMPA. We also
further revealed their structure activity relationship, proved their in vivo binding of PSMA,
determined their boron biodistribution and tumor uptake in a 22Rv1 xenograft mice model.
Taken together, these experiments demonstrate similar overall uptake of Boro-PSMA
compounds 1d and 1f when compared against BPA, the most commonly used BNCT agent.
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When compared against the previously reported studies evaluating BPA uptake in tumor, our
BPA results demonstrate a tumor/muscle uptake ratio of 2.0 at 4 hour time point, consistent with
other prior reports ranging between 1.5 and 4.4.42 In our study, the BPA uptake at tumor is
7.1±4.0 µg/gram at 4 hour. Overall, the BPA biodistribution results are similar to prior reports
conducted under similar conditions.43,44
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A tumor uptake of at least 20-50 µg/gram boron is typically thought to be necessary for
effective tumor therapy. The boron ratio in tumor/normal tissue needs to be greater than 3:1 for
selectivity.3 However, the boron uptake did not reach the requirements, suggesting that the
current compounds and/or dosing regimens are not sufficient for therapy. In order to deliver
sufficient boron to the tumor, both high affinity binding and high abundance targets are required.
In this study, a large amount of boron compound was administered, saturating PSMA binding
sites in vivo as verified using the 68Ga-PSMA-11 binding and blocking assay. Several approaches
could be used to improve boron delivery to tumor. Firstly, the timing and route of administration
of the compounds could be varied. The very high blood content of compounds 1d and 1f at the
latest time point tested, as well as the increased tumor to muscle ratio at four hours compared
against one hour, suggests that a longer incubation time could improve the overall boron delivery
as well as tumor to blood and tumor to muscle ratios. The second potential route for
improvement would be to include larger numbers of boron atoms per PSMA-inhibitor. While
compound 1f contains 20 atoms, larger substitutents such as dendrimers and nanoparticles have
been reported.45,46,47,48,49 In principle, these substituents could be appended to the urea-based
PSMA agent to achieve higher boron delivery. Finally, the use of animal models with increased
PSMA expression would likely yield improved tumor boron uptake. While the 22Rv1 model was
selected due to convenient, rapid and predictable tumor growth, its overall PSMA expression is
low compared against other cell lines such as LNCaP or PC3-pip.50 These improvements to the
method represent important areas for future investigation. Following optimization of the
pharmacokinetics, and dosing regimens, initial treatment studies could be performed in mice
bearing appropriate tumor xenografts.
■ CONCLUSION
In this study, a series of boronic acid- and carborane-containing PSMA inhibitors were
successfully synthesized. The in vitro binding data demonstrated high binding affinities to
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PSMA for all the boron-containing inhibitors. In particular, compound 1d showed a 26 fold
lower IC50 value that the well-known PSMA inhibitor PMPA. PET imaging data demonstrated
blocking of 68Ga-PSMA-11 uptake by boron containing PSMA compounds, demonstrating the in
vivo binding of the PSMA inhibitors to their target. The boron uptake and distribution assay
showed around 4 µg boron/gram tumor uptake and similar tumor/muscle ratios compared to the
ratio for the standard compound BPA. Although the uptake is below the required 20 µg
boron/gram tumor threshold, these data suggest that with further optimization, treatment of
oligometastatic prostate cancer with Boro-PSMA is feasible.
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■ ABBREVIATIONS
BNCT: boron neutron capture therapy; PSMA: prostate specific membrane antigen; PET:
positron emission tomography; LET: linear energy transfer; BPA: 4-borono-L-phenylalanine;
BSH: sodium borocaptate; PCa: prostate cancer; GCPII: glutamate carboxypeptidase II; FBS:
fetal bovine serum; PMPA: 2-(phosphonomethyl) pentanedioic acid; ICP-OES: inductively
coupled plasma optical-emission spectrometry. IACUC: Institutional Animal Care & Use
Program; DCC: N,N′-Dicyclohexylcarbodiimide; NHS: N-Hydroxysuccinimide; TFA:
trifluoroacetic acid.
■ ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI:
1
13
Synthesis procedure, H and C NMR spectra of compounds 1a-h, supplementary figures and
tables.
■ AUTHOR INFORMATION
Corresponding Author
*Robert Flavell, Tel: +1-415-3533638; E-mail: robert.flavell@ucsf.edu.
Notes
The authors have declared that no competing interest exists
■ ACKNOWLEDGMENTS
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This work was supported by an American Cancer Society Individual Research Grant (IRG-97-
150-13), and the New Directions in Prostate Cancer Research Award of the University of
California, San Francisco Prostate Cancer Research Program. This publication was supported by
the Helen Diller Family Comprehensive Cancer Center Support Grant of the National institutes
of Health under P30 CA 82103. Dr. Flavell was additionally supported by the David Blitzer
Young Investigator Award of the Prostate Cancer Foundation and a Physician Research Training
Grant from the Department of Defense (PC 150932). We thank Dr. Rob Franks and Dr. Brian
Dreyer of the Marine Analytical Laboratory in the University of California, Santa Cruz for
assistance with ICP measurements, Kyoughee Seo in the University of California, San Francisco
for help with mice dissection.
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■ REFERENCES
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