PET imaging and pharmacological therapy targeting carbonic anhydrase-IX high-expressing tumors using US2 platform based on bivalent ureidosulfonamide

Carbonic anhydrase-IX (CA-IX) is attracting much attention as a target molecule for cancer treatment since high expression of CA-IX can lead to a poor prognosis of patients. We previously reported low-molecular-weight 111In/90Y complexes with a bivalent ureidosulfonamide scaffold ([111In/90Y]In/Y-US2) as cancer radiotheranostic agents for single photon emission computed tomography and radionuclide-based therapy targeting CA-IX. Here, we applied the US2 platform to positron emission tomography (PET) imaging and pharmacological therapy targeting CA-IX high-expressing tumors by introducing 68Ga and natIn, respectively. In an in vitro cell binding assay, [67Ga]Ga-US2, an alternative complex of [68Ga]Ga-US2 with a longer half-life, markedly bound to CA-IX high-expressing (HT-29) cells compared with low-expressing (MDA-MB-231) cells. In a biodistribution study with HT-29 and MDA-MB-231 tumor-bearing mice, [67Ga]Ga-US2 showed accumulation in the HT-29 tumor (3.81% injected dose/g at 60 min postinjection) and clearance from the blood pool with time. PET with [68Ga]Ga-US2 clearly visualized the HT-29 tumor in model mice at 60 min postinjection. In addition, the administration of [natIn]In-US2 to HT-29 tumor-bearing mice led to tumor growth delay and prolonged mouse survival, while no critical toxicity was observed. These results indicate that [68Ga]Ga-US2 and [natIn]In-US2 may be useful imaging and therapeutic agents targeting CA-IX, respectively, and that US2 may serve as an effective cancer theranostic platform utilizing CA-IX.


Introduction
Hypoxia in many kinds of solid tumors is caused by an imbalance between oxygen supply and consumption, and is closely associated with tumor propagation, malignant progression, and resistance to chemotherapy and radiotherapy [1][2][3][4][5]. Carbonic anhydrase (CA) isozymes, a class of zinc metalloenzymes, are found in most living organisms [5,6]. CA-IX is the protein most markedly upregulated by hypoxia through the hypoxia inducible factor-1 (HIF-1) cascade, and is strongly associated with cancer progression [7][8][9][10][11][12]. CA-IX catalyzes the reversible hydration of carbon dioxide to a bicarbonate ion and a proton [7][8][9][10][11][12], which may promote cancer cell survival [13]. CA-IX is functionally involved in diverse aspects of cancer development, such as primary tumor growth, metastatic dissemination of cancer cells, and homing and growth of metastatic lesions [14]. Concerted regulation of the intra-and extra-cellular pH is conducted by CA-IX and various other proteins, such as aquaporins and anion exchangers [9-11, 15, 16]. The wild-type von Hippel Lindau (VHL) tumor suppressor protein binds to HIF-1 hydroxylated by oxygen, leading to the degradation of HIF-1 after ubiquitination. In other words, a deficit in the VHL gene, which is often observed in patients with clear cell renal cell carcinoma, contributes to HIF-1 stabilization, leading to high-level CA-IX expression [7][8][9][10][11]. CA-IX expression is limited in normal tissues except in the gastrointestinal tract [17]. In contrast, the expression of CA-IX is markedly increased in many types of tumors in response to hypoxia [5]. Moreover, CA-IX is pathologically expressed in cancer cells and located at the cell surface. Therefore, CA-IX has emerged as an attractive target for both the therapy and diagnosis of cancer.
Recently, we reported an original theranostic platform based on ureidosulfonamide targeting CA-IX for cancer diagnosis and therapy (US2) (Fig 1) [46]. US2 consists of 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (DO2A) as the metal-chelating moiety and two ureidosulfonamide scaffolds, and is utilized to develop cancer diagnostic and therapeutic agents by conjugating with functional metals. US2 conjugated with 111 In (γ-emitter) and 90 Y (β − -emitter) ([ 111 In]In-US2 and [ 90 Y]Y-US2, respectively) were prepared for tumor single photon emission computed tomography (SPECT) imaging and radionuclide-based therapy targeting CA-IX, respectively. [ 111 In]In-US2 showed high selectivity for CA-IX high-expressing (HT-29) cells in vitro, and demonstrated higher HT-29 tumor uptake at 1−4 h postinjection than other previously reported radioligands of CA-IX [26,27,[47][48][49][50][51][52]. Moreover, in vivo SPECT imaging with [ 111 In]In-US2 clearly visualized HT-29 tumors in model mice. In addition, [ 90 Y]Y-US2 administration into HT-29 tumor-bearing mice significantly delayed tumor growth as compared with non-treated mice. Based on these encouraging results, in the present study, we utilized this US2 platform to develop novel positron emission tomography (PET) and pharmacologically therapeutic agents targeting CA-IX by conjugating with 68 Ga (β + -emitter) and nat In, respectively. 68 Ga, the most widely utilized radiometal for PET, is available from a germanium-68/gallium-68 generator and easily conjugated with various metal chelators, such as DO2A. We synthesized [ 68 Ga]Ga-US2 and evaluated its utility as a CA-IX imaging agent for PET. We performed fundamental evaluations with [ 67 Ga]Ga-US2 due to the longer half-life of 67 Ga (78 h) than that of 68 Ga (68 min). In addition, tumor growth inhibition by the administration of ureidosulfonamide-based drugs, such as SLC-0111, has been reported by several groups [30, [37][38][39]. Therefore, we synthesized [ nat In]In-US2, in which nat In was introduced in order for it to exhibit a similar biodistribution to [ 111 In]In-US2, and evaluated its utility for CA-IX high-expressing tumor therapy.

Chemistry
The precursor for radiolabeling (US2) and corresponding nat In complex ([ nat In]In-US2) were synthesized according to our previous report [46].    13

Measurement of partition coefficient
The experimental determination of partition coefficients was performed in 1-octanol and phosphate-buffered saline (PBS) (pH 7.4). 1-Octanol (3 mL) and PBS (3 mL) were pipetted into a 15-mL test tube containing [ 67 Ga]Ga-US2 (50 kBq, 41 GBq/μmol). The test tube was vortexed for 2 min and centrifuged (4,000 ×g, 5 min). Aliquots (0.5 mL) from the 1-octanol and PBS phases were transferred into two test tubes for counting. The remaining PBS phase (1 mL), newly prepared 1-octanol (3 mL), and PBS (2 mL) were pipetted into a new test tube. The vortexing, centrifuging, and counting were repeated until consistent partition coefficient values were obtained (usually the sixth partition). The amount of radioactivity in each tube was measured with a γ counter (Wallac 1470 Wizard; PerkinElmer, Massachusetts, U.S.A.). The partition coefficient was calculated using the equation: log P ow = log[count 1-octanol /count PBS ].

Animals
All animal experiments were performed in accordance with our institutional guidelines and were approved by the Kyoto University Animal Care Committee. Male BALB/c-nu/nu nude mice and male ddY mice were purchased from Japan SLC (Shizuoka, Japan). Animals were administered materials without anesthesia. Animal sacrifice and blood collection were performed by decapitation. Animals were housed in a sterile environment with a 12-h light-dark cycle, fed standard chow, and had free access to water. All efforts were made to minimize suffering.

Analysis of stability in mouse plasma
The blood (5 mL) was collected from ddY mice (male, 5 weeks old) and centrifuged (1,200 ×g, 10 min) in venous blood collection tubes (Becton, Dickinson and Company, New Jersey, U.S. A.). The plasma (200 μL) was separated and [ 67 Ga]Ga-US2 (185 kBq, 149 GBq/μmol) was added to it. The solution was incubated at 37˚C for 1 and 4 h. After the addition of MeCN (200 μL), it was centrifuged (10,000 ×g, 5 min). The supernatant was filtered with a Cosmonice Filter (S) (0.45 μm, 4 mm) (Nacalai Tesque), and the filtrate was analyzed by RP-HPLC. The analytical method of RP-HPLC was the same as written in the radiolabeling section.

In vitro cell binding assay
HT-29, MDA-MB-231, RCC4-VHL, and RCC4-VA cells were incubated in 12-well plates (2 × 10 5 cells/well) at 37˚C in an atmosphere containing 5% CO 2 for 24 h. After incubation, cells were then incubated at 37˚C in an atmosphere containing 5% CO 2 and 21% O 2 (normoxic conditions) or 1% O 2 (hypoxic conditions) for another 24 h. After removing the medium, [ 67 Ga]Ga-US2 (18 kBq, 50−82 GBq/μmol) in the medium (1 mL) was added to each well inside the hypoxic chamber (miniMACS Anaerobic Workstation; Don Whitley Scientific, West Yorkshire, U.K.), and the plates were incubated under normoxic or hypoxic conditions for 2 h. Nonspecific binding was evaluated by the addition of acetazolamide (50 μM). After incubation, each well was washed with 1 mL of PBS (pH 7.4) (Nacalai Tesque), and the cells were lysed with 1 M NaOH (0.5 mL × 2). Radioactivity bound to cells was measured using a γ counter (PerkinElmer). The protein concentration was determined using BCA Protein Assay Kit (Thermo Fisher Scientific).

Tumor model
Under anesthesia with isoflurane (2% in an air mixture), BALB/c-nu/nu nude mice (male, 5 weeks old) were subcutaneously inoculated with MDA-MB-231 cells (1 × 10 7 cells/mouse), in 150 μL of DMEM and Geltrex (Thermo Fisher Scientific) at a 1:1 ratio, in the left flank. Fifteen days later, HT-29 cells (1 × 10 7 cells/mouse) were also subcutaneously injected into the right flank of MDA-MB-231 tumor-bearing mice. For the PET/CT and pharmacological therapy studies, BALB/c-nu/nu nude mice were subcutaneously inoculated with only HT-29 cells (5 × 10 6 cells/mouse) in the right flank. All efforts were made to minimize suffering.
The % injected dose/g of samples was calculated by comparing the sample counts with the count of the initial dose.

PET/CT
A solution of [ 68 Ga]Ga-US2 (1.1−3.5 MBq, 46−216 GBq/μmol) in saline (100 μL) was directly injected into the tail vein of HT-29 tumor-bearing mice. PET and CT images were collected using the G8 PET/CT system (PerkinElmer) (PET conditions: 10 min × 1 frame; CT conditions: accurate full angle mode in 60 kV/615 μA) at 60 and 120 min postinjection. Cross-calibration between the dose calibrator and the imaging systems was performed according to the manufacturer's specifications. The images were acquired as static whole-body scans using G8 acquisition software and reconstructed with maximum-likelihood expectation maximization (MLEM) according to a previous report [53]. Acquired PET and CT data were analyzed using PMOD software (Version 3.3; PMOD Technologies, Zürich, Switzerland).

Pharmacological therapy
[ nat In]In-US2 was solubilized in 37.5% PEG400/12.5% EtOH/50% saline prior to administration. A dose of 50 mg/kg [ nat In]In-US2 or vehicle was administered via intraperitoneal injection using a volume of 100 μL/mouse three times a week for 2 weeks (n = 12). The tumor volume and body weight were measured three times a week for 4 weeks after the injection of [ nat In]In-US2. The tumor volume was calculated using the formula: V = [length × (width) 2 ]/2. The initial tumor volumes (on day 0) for the [ nat In]In-US2 and vehicle groups were 72.6 ± 7.2 and 74.4 ± 8.0 mm 3 (mean ± standard error), respectively. Mice were euthanized when the tumor volume reached 1,000 mm 3 or the body weight had decreased by over 20% from the original weight.

Statistical analysis
All data were analyzed with GraphPad Prism or Microsoft Excel. Differences at the 95% confidence level (P < 0.05) were considered significant.

Chemistry and radiolabeling
[ nat Ga]Ga-US2 was prepared by reacting the precursor with [ nat Ga]Ga(NO 3 ) 3 �nH 2 O in MES buffer (0.1 M, pH 5.5) at a 38% yield. The yield suggested that [ nat Ga]Ga-US2 was dissolved in the solvent to some extent. The 1 H NMR, 13 C NMR, and HRMS were consistent with the assigned structures. synthesized at a 51% radiochemical yield (decay-corrected). The 68 Ga-labeling reaction was performed by incubating the precursor with [ 68 Ga]GaCl 3 in 1.2 M acetate buffer (pH 4.0) at 90˚C (Fig 2). [ 68 Ga]Ga-US2 was synthesized at a 30% radiochemical yield (decay-corrected) with over 95% radiochemical purity. The radiochemical identity of the 67/68 Ga complex was verified by comparative RP-HPLC using the corresponding nat Ga complex as a reference. The retention times between the 67/68 Ga-labeled compound (22.8/22.7 min) and corresponding nat Ga complex (22.7 min) suggest that the desired 67/68 Ga-labeled ureidosulfonamide derivatives were successfully synthesized.

Hydrophilicity and in vitro stability
We examined the hydrophilicity of radiogallium-labeled US2, and the log P ow value for [ 67 Ga] Ga-US2 was −1.79 ± 0.02. In addition, the in vitro stability of [ 67 Ga]Ga-US2 was evaluated by incubating it in the mouse plasma at 37˚C for pre-determined times. Almost all [ 67 Ga]Ga-US2 existed as an intact form (> 95%) until 4 h in vitro (Fig 3).

In vitro cell binding
A cell binding assay was performed to evaluate the in vitro CA-IX affinity of [ 67 Ga]Ga-US2 under both normoxic and hypoxic conditions (Fig 4). According to our previous study, HT-29 and RCC4-VA cell lines were used as CA-IX high-expressing cells, while MDA-MB-231 and RCC4-VHL cell lines were used as CA-IX low-expressing ones, under both normoxic and hypoxic conditions [46].  [46]. In addition, the addition of acetazolamide, a classical CA inhibitor, significantly blocked binding to the CA-IX high-expressing cells (9.67 and 10.8% initial dose/mg protein), suggesting the in vitro CA-specificity ( Fig 4A). In response to hypoxia, the binding of [ 67 Ga]Ga-US2 to HT-29 cells was markedly enhanced (27.5 to 60.4% initial dose/mg protein), which was consistent with CA-IX expression (CA-IX/GAPDH = 1.75 to 3.32) [46], suggesting that [ 67 Ga]Ga-US2 could detect hypoxic regions of tumors. The selective binding to CA-IX high-expressing cells was also observed under hypoxic conditions, and its binding was blocked by acetazolamide ( Fig 4B).

In vivo tumor uptake
A biodistribution study was carried out to assess the in vivo uptake into the CA-IX-expressing tumor in the cancer model mice (Fig 5 and S1 Table). According to our previous report, HT-29 and MDA-MB-231 tumors were used as CA-IX high-and low-expressing ones, respectively [46]. [ 67 Ga]Ga-US2 showed significantly higher accumulation in the HT-29 tumor (2.98 −4.63% injected dose/g) than that in the MDA-MB-231 tumor (1.64−2.58% injected dose/g) at all evaluated timepoints, indicating the selective accumulation in CA-IX high-expressing tumors. Among normal organs, marked kidney uptake was observed (13.3−19.9% injected dose/g). Radioactivity in the HT-29 tumor at 60 min postinjection (3.81% injected dose/g) was significantly decreased by the coinjection of acetazolamide (1.80% injected dose/g), indicating the in vivo specificity of [ 67 Ga]Ga-US2 for CA ( Fig 5B). Moreover, both the HT-29 tumor/ blood and HT-29 tumor/muscle ratios remained above 1.5 after 30 min postinjection, suggesting favorable properties for in vivo imaging of solid tumors (S1 Table).

PET/CT
PET/CT study with [ 68 Ga]Ga-US2 targeting CA-IX high-expressing tumors was performed (Fig 6). [ 68 Ga]Ga-US2 clearly visualized the HT-29 tumors at 60 and 120 min postinjection probably due to its favorable tumor/blood and tumor/muscle ratios demonstrated in the biodistribution study (S1 Table), while no marked difference in radioactivity biodistribution between 60 and 120 min postinjection was observed. However, a high level of radioactivity accumulation in the kidneys and bladder was observed, suggesting renal excretion of [ 68 Ga] Ga-US2. Radioactivity biodistribution on PET corresponded with the results of the biodistribution study with [ 67 Ga]Ga-US2 (Fig 5).

Pharmacological therapy
Pharmacological therapy with [ nat In]In-US2 targeting CA-IX high-expressing tumors was carried out (Fig 7). The injected dose of [ nat In]In-US2 was 50 mg/kg mouse and administered via intraperitoneal injection. The therapeutic effect was evaluated by monitoring the tumor volume in mice using a caliper three times a week. As shown in Fig 7B,   In-US2 (Fig 7D). Moreover, on day 28, mice were sacrificed, and their spleen, kidneys, and liver were removed and weighed (S1 Fig). No marked difference in organ weight between mice treated with [ nat In]In-US2 and vehicle was observed, suggesting no critical toxic effect of the nat In complex on these organs.

Discussion
We previously reported the utility of bivalent ureidosulfonamide (US2) for CA-IX-targeting radiotheranostics. In the present study, we applied this US2 platform to PET imaging and pharmacological therapy targeting CA-IX high-expressing tumors.
[ 67 Ga]Ga-US2 showed fast clearance from the blood and muscle, suggesting favorable pharmacokinetics for in vivo imaging of solid tumors. The rapid pharmacokinetics of radiogalliumlabeled US2 due to its low molecular weight are significant for 68 Ga-PET, while imaging with antibody-based probes showing slow clearance from the blood pool requires longer-half-life radionuclides, such as 124 I, 131 I, 111 In, and 89 Zr [18][19][20][21][22]. However, [ 67 Ga]Ga-US2 markedly accumulated in the kidney and lung as well as [ 111 In]In-US2, which might lead to unclear imaging of such types of cancer. Their high accumulation might be due to the expression of other CA isozymes on the cell surface in these organs [57], since radioactivity accumulation in them was decreased by acetazolamide (S1 Table). Blocking of the transmembrane isozymes in normal organs by nonradioactive CA ligands at appropriate doses may improve the contrast of the tumor image, since the number of CA molecules in normal organs might be lower than in tumors [24]. PET imaging of mice provided a clear image of the HT-29 tumor; however, a high level of radioactivity in the kidneys was observed, as demonstrated in the biodistribution study (Fig 6). Moreover, SPECT/CT studies with [ 67 Ga]Ga-US2 using HT-29 and MDA-MB-231 tumor-bearing mice were also carried out to evaluate CA-IX selectivity of radiogalliumlabeled US2 (S2 Fig). The SPECT image at 60 min postinjection showed greater radioactivity accumulation in the HT-29 tumor than the MDA-MB-231 tumor, indicating CA-IX-selective tumor visualization of radiogallium-labeled US2. SPECT with [ 67 Ga]Ga-US2 showed a similar pharmacokinetics profile to that of PET with [ 68 Ga]Ga-US2. SPECT with [ 67 Ga]Ga-US2 suggests the efficacy of the other modality using US2, reinforcing the utility of the US2 platform. These results with [ 67/68 Ga]Ga-US2 indicate that [ 68 Ga]Ga-US2 may be a useful CA-IX-targeting PET probe and that the US2 platform can provide a CA-IX-PET imaging strategy by conjugating with 68 Ga.
Recently, 68 Ga has been attracting much attention as a useful radionuclide for PET imaging because it can be easily obtained from a commercial generator. However, its short half-life (68 min) limits the selection of targeting ligands. High-molecular-weight compounds such as antibodies, generally showing long retention in the blood pool, are inappropriate for 68 Ga-PET, since it is difficult to reach a high signal-to-background ratio at an early time post-administration. Radiogallium-labeled US2 showed rapid pharmacokinetics; however, its tumor accumulation was less than clinically utilized tumor-imaging probes, such as [ 68 Ga]Ga-PSMA-11 targeting prostate-specific membrane antigen [58]. We recently identified a small-molecule CA-IX ligand, imidazothiadiazole sulfonamide (IS), with favorable properties for CA-IX imaging [59]; thus, a small-molecule 68 Ga-PET probe based on IS may provide a clearer image of CA-IX-expressing tumors than [ 68 Ga]Ga-US2.
For the therapeutic application of US2, we synthesized [ nat In]In-US2 and administered it to HT-29 tumor-bearing mice. Nonradioactive indium was conjugated with US2, because [ 111 In] In-US2 showed greater tumor accumulation than [ 67 Ga]Ga-US2 in the biodistribution study. In vivo CA-IX inhibition by sulfamate, sulfonamide, and coumarin inhibitors has been shown to reverse the effect of tumor acidification, leading to a delay in tumor growth, while the effects depended on the treatment schedule, administered dose, and tumor cell line [23,30,38,40,60,61]. The unsubstituted sulfonamides and their bioisosteres, including ureidosulfonamide, bind to the Zn 2+ ion of the enzyme by substituting the non-protein zinc ligand, such as H 2 O, to generate a tetrahedral adduct, leading to the inhibition of enzyme activity [5]. [ nat In]In-US2 therapy showed tumor growth delay and survival prolongation in HT-29 tumor-bearing mice, indicating therapeutic effects on the HT-29 tumor (Fig 7C and 7D). Meanwhile, no marked change in the whole body or organ weight by [ nat In]In-US2 was observed, suggesting no critical toxicity of [ nat In]In-US2 (Figs 7E and S1). Therefore, the US2 platform can also provide a tumor therapeutic application targeting CA-IX.
Pharmacological therapy with [ nat In]In-US2 targeting CA-IX showed limited therapeutic effects: no remission or shrinkage of tumors. Combination therapy with anti-tumor drugs, such as cisplatin, doxorubicin, and fluorouracil has shown greater therapeutic effects on tumors [62][63][64]. It is generally accepted that hypoxia in tumors often causes resistance to antitumor drugs through the HIF-1 cascade; therefore, the inhibition of CA-IX, closely associated with hypoxia, can enhance the effect of anti-tumor drugs, leading to a decrease in the dose of anti-tumor drugs for therapy, which may help realize safer cancer treatments. Thus, [ nat In]In-US2 administration may enhance the effects of conventional therapies.

Conclusions
According to our previous study using 111 In