Research ArticleExperimental Studies

Development and Pre-clinical Evaluation of New 68Ga-NOTA-folate Conjugates for PET Imaging of Folate Receptor-positive Tumors

IBRAHIM ALJAMMAZ, BASIM AL-OTAIBI, NOURAH AL-HOKBANY, SUAD AMER and SUBHANI OKARVI
Anticancer Research November 2014, 34 (11) 6547-6556;

Abstract

In an attempt to develop new folate radiotracers with favorable biochemical properties for detecting folate receptor-positive cancers, we synthesized 68Ga-NOTA- and 68Ga-NOTAM-folate conjugates using a straightforward and a one-step simple reaction. Radiochemical yields were greater than 95% (decay-corrected) with total synthesis time of less than 20 min. Radiochemical purities were always greater than 98% without high-performance liquid chromatography (HPLC) purification. These synthetic approaches hold considerable promise as a rapid and simple method for 68Ga-folate conjugate preparation with high radiochemical yield in a short synthesis time. In vitro tests on the KB cell line showed that significant amounts of the radioconjugates were associated with cell fractions. Biodistribution studies in nude mice bearing human KB xenografts, demonstrated a significant tumor uptake and favorable biodistribution profile for 68Ga-NOTA-folate over the 68Ga-NOTAM-folate conjugate. The uptake in the tumors was blocked by excess injection of folic acid, suggesting a receptor-mediated process. These results demonstrate that the 68Ga-NOTA-folate conjugate may be useful as a molecular probe for detection and staging of folate receptor-positive cancers, such as ovarian cancer and their metastasis, as well as monitoring tumor response to treatment.

Advancement of scintigraphic imaging is highly determined by the development of more specific radiotracers. Receptor-targeted radiotracers have shown great potential for imaging tumors and other recurring diseases in nuclear medicine (1). The Membrane-associated-folic acid receptor is a glycosylphosphatidylinositol protein overexpressed in approximately 100% of ovarian adenocarcinomas and various epithelial cancers including cervical, colorectal and renal cancers (2, 3). Meanwhile, this receptor is highly restricted in most normal tissues, which make these tumors excellent candidates for molecular targeting through the folate receptor system. Folate enters into cells by high affinity folate receptors in addition to the classical reduced folate carrier-mediated transport system (4). Folic acid (FA) has been mainly conjugated with chelates covalently via its gamma carboxylate of the glutamic acid fragment (5-8). These conjugates have shown considerable success for delivering various single-photon emission computed tomography (SPECT) radionuclides to folate-receptor-positive KB cell tumor xenografts in athymic mouse models. Among these conjugates, 67Ga-deferoxamine-folate (67Ga-DF-folate) (7, 9, 10) and 111In-diethylenetriamine pentaacetic acid (111In-DTPA-folate), which appeared to be feasible for targeting tumor folate receptors in vivo (11, 12). However, 111In-DTPA-folate provided tumor-selective radionuclide delivery in vivo that has led to the initial clinical study for imaging of ovarian malignancy and differentiation between malignant and benign ovarian masses (13). Due to its favorable physical properties and wide availability of 99mTc, numerous 99mTc-labeled-folate conjugates have also been synthesized and biologically evaluated (14-17). For instance, the folate conjugates of 99mTc-6-hydrazino-nicotinamido-hydrazido (99mTc-HYNIC), 99mTc-ethylenedicysteine (99mTc-EC), 99mTc-EC20 and 99mTc-DTPA have shown the capability of imaging folate receptor-positive tumors. Recently, our group described the synthesis and evaluation of 99mTc-mercaptoacetyltriglycine-folate (99mTc-MAG3-folate) and 99mTc-mercaptoacetyldiglycine-folate (99mTc-MAG2-folate) conjugates (17). These conjugates, in particular, 99mTc-MAG3-folate retained sufficient binding affinity to breast tumor MDA-MB 231 cells in vitro and in vivo. With the increased demand of positron-emission tomography (PET), there has been a great interest in the development of positron-emitting radiopharmaceuticals for the early detection and characterization of cancer, molecular assessment of treatment effects and more fundamental understanding of the disease process (18). Therefore, several 18F-labeled folate derivatives were synthesized in high radiochemical yield and preclinically characterized (19, 20). These derivatives exhibited very high binding affinities to KB cells and demonstrated significant tumor uptake and favorable pharmacokinetics as molecular probes for detecting folate receptor-positive cancers. Lately, 68Ga-labeled DF and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-folate conjugates were synthesized in high radiochemical purity (21, 22). Imaging of folate-receptor-positive tumor bearing mice with 68Ga-DF and DOTA-folate were feasible and the characteristic of 68Ga-DOTA-folate was similar to 111In-DTPA-folate, which has been used in clinical trials.

Due to the exceptionally high stability of GaIII-NOTA complexes with log K=31 in comparison with their corresponding DOTA complexes (log K=21) (23, 24) and in an attempt to develop new folate conjugates capable of being labeled with metallic radionuclides that possess favorable properties for detecting PET imaging of folate receptor-positive cancers, we here report the synthesis and preclinical evaluation of new 68Ga-NOTA-folate conjugates using 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and 1,4,7-triazacyclononane-1,4,7-triacitamide (NOTAM) as chelating groups.

Materials and Methods

The chemicals used in the study were all analytical reagent grade purchased from Aldrich (St. Louis, MO, USA) and were used without further purification unless stated. Acetonitrile (ACN) was kept over molecular sieves. Commercial generator based on a SnO2 phase adsorbing was obtained from iThemba Lab (Cape Town, South Africa). Sep-Pak cartridges were purchased from Waters-Millipore (Milford, MA, USA). TLC-SG sheets were purchased from Gelman Sciences Inc (Ann Arbor, MI, USA). High-Performance Liquid Chromatography (HPLC) analysis was carried out on Luna, Phenomenex C-18 reversed-phase column (analytical, 250 mm × 4.6 mm) (JASCO, Tokyo, Japan). The solvent system used was isocratic (eluant: ACN/H2O, 95/5 with 0.1% TFA at flow rate of 1.0 ml/min). A Jasco chromatographic system equipped with a variable wavelength ultraviolet monitor and in tandem with a Canberra flow through radioactivity detector was used (Meriden, CT, USA). Ultraviolet absorption was monitored at 254 nm. Chromatograms were acquired and analyzed using the BORWIN software (JASCO, Tokyo, Japan). Melting points were determined on a Thomas-Hoover Unimelt capillary melting point apparatus (Labequip, Ontario, Canada). Mass spectroscopy was run on Quattra electrospry mass spectrometer (ES-MS, Perkin Elmer, Waltham, MA, USA).

1,4,7-Triazacyclononane-N,N’,N”-triacetic acid (NOTA, 2)

Compound 2 in Figure 1 was synthesized utilizing the method reported previously (25) with minor modification and briefly described below. The hydrochloric acid salt of 1,4,7-Triazacyclononane (TACN.HCl, 500 mg, 2.1 mmol) was dissolved in ACN (10 ml) followed by the addition of triethylamine (TEA, 1.16 ml, 8.4 mmol). To the stirred mixture, bromoacetic acid (0.88 g, 6.3 mmol) in ACN (4 ml) was added drop-wise over the course of 5 min. The reaction mixture was then stirred and refluxed for 3 h. The brown solution was concentrated by rotary evaporation to leave a brown solid product which was washed with ACN (10 ml) to leave a creamy precipitate. This was then filtered, washed with ACN (10 ml) and dried in vacuo to yield 466 mg of NOTA as an oily material.

N-Succinimidyl-1,4,7-triazacyclononane-N,N’,N”-triacetic acid (NOTA-NHS, 3)

NOTA (220 mg, 0.72 mmol) was dissolved in ACN (10 ml) followed by the addition of N-hydroxysuccinimide (NHS, 78 mg, 0.72 mmol) and dicyclohexylcarbodiimide (DCC, 154 mg, 0.72 mmol). The reaction mixture was stirred at ambient temperature for 3 h. The by-product dicyclohexylurea was then removed by filtration and the filtrate was dried by rotary evaporation to furnish 246 mg of NOTA-NHS as an oily product.

1,4,7-Triazacyclononane-N,N’,N”-triacetic acid-folate (NOTA-folate, 5)

The γ-hydrazide-folate conjugate 4 (folate-NH2-NH2, 273 mg, 0.6 mmol) was dissolved in dimethylsulfoxide (DMSO, 3 ml) followed by the addition of TEA (83 μl, 0.6 mmol) and NOTA-NHS (240 mg, 0.6 mmol). The reaction mixture was stirred while shielded from light for 3 h at 60°C. The product was precipitated by addition of ACN (2 ml), centrifuged and then washed several times with diethyl ether (ether) before drying under vacuum into a 350 mg of NOTA-folate as a yellow powder.

1,4,7-Triazacyclononane-N,N’-diacetamide (NOTAM, 6)

Compound 6 in Figure 2 was synthesized utilizing the method described above for compound 2. TACN.HCl salt (500 mg, 2.1 mmol) was dissolved in ACN (10 ml) followed by the addition of TEA (0.87 ml, 6.3 mmol). To the stirred mixture, bromoacetamide (0.57 g, 4.2 mmol) in ACN (4 ml) was added drop-wise over the course of 5 min. The reaction mixture was then stirred and refluxed for 3 h. The brown solution was concentrated by rotary evaporation to leave a brown solid product which was washed with ACN (10 ml) to leave a creamy precipitate. This was then filtered, washed with ACN (10 ml) and dried in vacuo to yield 348 mg of NOTAM as an oily material.

N-Succinimidyl-1,4,7-triazacyclononane-N,N’-diacetamide (NOTAM-NHS, 8)

NOTAM 6 (348 mg, 1.42 mmol) was dissolved in ACN (10 ml) followed by the addition of TEA (0.4 ml, 1.42 mmol). To the stirred mixture, bromoacetic acid (0.2 g, 1.42 mmol) in ACN (3 ml) was added drop-wise over the course of 5 min. The reaction mixture was then stirred and refluxed for 3 h. The brown solution was concentrated by rotary evaporation to leave a brown solid product which was washed with ACN (8 ml) to leave a creamy precipitate. The creamy precipitate was then filtered, washed with ACN (10 ml) and dried in vacuo to yield 432 mg of NOTAM 7 as an oily material. The oily material (120 mg, 0.39 mmol) was dissolved in ACN (6 ml) followed by the addition of NHS (43 mg, 0.39 mmol) and DCC (85 mg, 0.39 mmol). The reaction mixture was stirred at ambient temperature for 3 h. The by-product dicyclohexylurea was then removed by filtration and the filtrate was dried by rotary evaporation to furnish 155 mg of NOTAM-NHS as an oily product.

Figure 1.
Figure 1.

Synthesis of NOTA-folate conjugate precursor 5.

1,4,7-Triazacyclononane-N,N’,N”-triacetamide-folate (NOTAM-folate, 9)

The folate-NH2-NH2 conjugate 4 (181 mg, 0.39 mmol) was dissolved in dimethylsulfoxide (DMSO, 3 ml) followed by the addition of TEA (54 μl, 0.39 mmol) and NOTAM-NHS (155 mg, 0.39 mmol). The reaction mixture was stirred while shielded from light for 3 h at 60°C. The product was precipitated by addition of ACN (2 ml), centrifuged and then washed several times with diethyl ether (ether) before drying under vacuum into a 70 mg of NOTA-folate as a yellow powder.

Reference Ga-conjugates (Ga-NOTA- and Ga-NOTAM-folates)

Reference compounds Ga-NOTA- and Ga-NOTAM-folate conjugates were prepared by reacting compounds 5 and 9 (5 mg each, 6.75 μmol) with gallium chloride (GaCl3, 7.0 μmol) in acetic acid (0.1% in EtOH, pH~4.5, 500 μl) at 80°C for 30 min. The reference compound conjugates were precipitated by addition of ACN (2 ml), centrifuged and then washed several times with ACN before drying under vacuum into yellow powders.

Radiochemistry. 68Ge/68Ga generator was eluted with suprapure HCl (0.6 M, 6 mL) in 0.5 ml fractions. The two fractions with the highest 68GaCl3 37-300 MBq) were generally used for labelling purposes (26, 27). The synthetic approaches for the preparation of 68Ga-NOTA- and 68Ga-NOTAM-folate conjugates were simple and straightforward. Fractionated solutions of 68GaCl3 solutions were reacted in sealed vials with NOTA- and NOTAM-folate conjugates (50 μg each) in acetic acid (0.1% in EtOH, pH~4.5, 200 μl) at 80°C for 10 min. The reaction mixtures were dried by rotary evaporation to leave yellowish residues which were then re-solubilized in NaCl (0.9% saline, 1 ml each) before passing through 0.22 μm pore membrane filter for further studies. Similarly, 67Ga-NOTA- and 67Ga-NOTAM-folate conjugates were prepared by incubating 67GaCl3 (185-370 MBq) with NOTA- and NOTAM-folate conjugates (50 μg each) in acetic acid (0.1% in EtOH, pH~4.5, 200 μl) at 80°C and different time range (5-60 min). 67/68Ga-NOTA- and 67/68Ga-NOTAM-folate conjugates were obtained in quantitative radiochemical yields and purities as assessed by HPLC and confirmed by TLC.

Figure 2.
Figure 2.

Synthesis of NOTAM-folate conjugate precursor 9.

Partition coefficient. One hundred μl of 68Ga-NOTA- and 68Ga-NOTAM-folate conjugates were added into test tubes containing 1 ml of each n-octanol and buffered H2O (pH=7.3). The tubes were shaken for 1 min. After partial separation of the phases by gravity, 0.7 ml of each phase was transferred to separate tubes and centrifuged at 5,000 rpm for 5 min. Duplicate 0.2 ml aliquots of each phase was taken for radioactivity measurement and the partition coefficient was determined by the function: Partition coefficient=Log10 (counts in n-octanol layer/counts in aqueous layer). The calculation of the electronic charge density distribution was performed using the Extend Hückel MM2 program (Chem-Biodraw Ultra II Office 2008, Cambridge Soft, USA).

Stability in plasma. For stability in plasma, 68Ga-NOTA- and 68Ga-NOTAM-folate conjugates (100 μl, 20 μCi each) were incubated with human plasma (500 μl) in duplicate at 37°C for 4 h. This was followed by precipitation using a mixture of ACN/EtOH (400 μl, 1/1 v/v) and centrifugation at 5,000 rpm for 5 min. The supernatant layer was then analyzed by HPLC under the conditions described above.

In vitro cell binding. KB cells, a human oral carcinoma cell line that overexpresses the folate receptor, was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The KB cell line was grown in folate-deficient RPMI-1640 culture media with 10% fetal bovine serum (FBS) in tissue culture flasks (Fisher Scientific, Waltham, MA, USA). Twenty-four h prior conducting the cell-binding assay, media was replaced with RPMI-1640 without further addition of FBS. Confluent cultures were harvested by trypsinization, and 6×106 cells were suspended in 1.8 ml of sterile saline for binding assay. Approximately 300,000 cells (in 0.3 ml of sterile saline) were incubated with various amounts of 68Ga-NOTA- and 68Ga-NOTAM-folate conjugates ranging from 0.3-18 nM in duplicate for 60 min at 37°C. Incubation was terminated by dilution with cold saline (0.3 ml) and cells were pelleted by centrifugation. The cell pellets were then washed with cold saline to remove unbound radioactivity and centrifuged to collect supernatants. Radioactivity in the cell pellets (total bound) and washings (unbound) were measured in a γ-well counter (Perkin Elmer, Waltham, MA, USA). Non-specific binding, was determined in the presence of approximately 100 fold excess of unlabeled FA. Specific binding was calculated by subtracting the non-specific bound radioactivity from that of the total binding. The data were analyzed by a non-linear regression analysis program (Graph-Pad Software Inc., San Diego, CA, USA) using one-site binding equation. All binding data were corrected for non-specific binding and presented as the mean±standard deviation (S.D.). For internalization measurement, the cell-pellets were incubated again with 300 μl of acidic buffer (0.02 M sodium acetate in saline, pH 4.0) (2) at 37°C for 20 min. Then, cells were separated by centrifugation and washed with cold acidic buffer. The amount of cell surface-bound (acidic-wash) and intracellular radioactivity (acid-resistant) were determined by measuring the radioactivity of the supernatant and cell pellet, respectively, in a γ-well counter.

Figure 3.
Figure 3.

Synthesis of the Ga-NOTA- and Ga-NOTAM-folate conjugates (Ga=67/68/natGa).

In vivo biodistribution and tumor targeting. Approval for the animal protocol used in this study was obtained from the Institutional Animal Care and Use Committee. Animal biodistribution experiments were performed according to the international regulations governing the safe and humane use of laboratory animals in research (28). Human KB xenografts mouse models were used for in vivo tumor targeting experiments. For the implantation of tumor xenografts, approximately 3×106 KB cells in suspension of 100 μl sterile saline were injected subcutaneously into the right thigh of each mouse. Tumors were allowed to grow for 2-3 weeks by which tumors had reached weights of ~500 mg. Mice were injected via the lateral tail vein with 100 μl of the radiotracers formulated in saline. Each dose contained ~20 μCi (740 kBq) of the 68Ga-NOTA- and 68Ga-NOTAM-folate radiotracers. For the blocking studies, each animal was intravenously injected with excess cold FA (~100 μg) 10 min prior to the radiotracers injection. Animals were sacrificed at different time intervals and the tissues of interest were dissected, weighed and assayed for radioactivity. The percentage of the injected dose per gram (% ID/g) was then calculated by counting all tissues in a γ-well counter using a stored sample of the injection solution as a standard to estimate the total dose injected per mouse.

Statistical analysis. Data are expressed as mean±S.D., where appropriate. For data comparisons, a Student's t-test was performed of the mean values using the Graph-Pad Software (Graph-Pad Software Inc., San Diego, CA, USA). A probability value of p<0.05 was considered statistically significant.

Results and Discussion

Organic chemistry. As there exists debate on the affinity of the α regioisomer to have high affinity for the FR (11, 29), derivatization of the folate at the γ-carboxyl moiety revealed that its affinity for the folate receptors remains unaltered and comparable to folic acid (5). Therefore, γ-hydrazide-folate conjugate 4 was synthesized to serve as a linkage between folic acid and chelating moieties and characterized as reported in detail previously (29). Due to the exceptionally high stability of GaIII-NOTA complexes with log K being 31 in comparison with their corresponding DOTA complexes (log K=21) (23, 24) and in an attempt to develop new folate radiotracers with favorable biochemical properties for detecting folate receptor-positive cancers, we have synthesized NOTA- and NOTAM-folate conjugates and chemical purities of the γ-isomers were found to be greater than 98%, as assessed by HPLC and MS analysis. The synthetic approaches for the preparation of NOTA and NOTAM-folates 5 and 9 are outlined in Figures 1 and 2. Starting from compound TACN 1, NOTA 2 was obtained in 74% yield. NOTA 2 was converted to its corresponding ester (NOTA-NHS, 3) in 85% yield, which was then conjugated to folate-NH2-NH2 4 to furnish NOTA-folate conjugate precursor 5 in 56%. The calculated molecular masses for compounds 2, 3, and 5 were 303, 400 and 740, respectively, which were in agreement with the found ES-MS [M+1]+=304, 401 and 741, respectively. Similarly, starting from compound TACN 1, NOTAM 6 was obtained in 68% yield. NOTAM 6 was converted to its corresponding ester (NOTAM-NHS, 8) in 85% yield, which was then conjugated to folate-NH2-NH2 4 to furnish NOTAM-folate conjugate precursor 9 in 23%. The calculated molecular masses for compounds 6, 8, and 9 were 243, 398 and 738, respectively, which were in agreement with the found ES-MS [M+1]+=244, 399 and 739, respectively. Both conjugate precursors 5 and 9 appear to decompose beyond 185°C and the base peak m/z=373 and 371 correspond to the γ moiety of the conjugated folate precursors -(CH2-CH2-CO-NH-NH-NOTA) and -(CH2-CH2-CO-NH-NH-NOTAM), respectively.

The reference Ga-NOTA- and Ga-NOTAM-folate conjugates were obtained as yellow powder with overall chemical yields of 75% and 80%, respectively, which appear to decompose beyond 185°C. Chemical purities of Ga-NOTA- and Ga-NOTAM-folate conjugates were greater than 97% as determined by HPLC with retention times of 13.3 and 13.6 min, respectively (Figure 4). Elemental analysis calculated for Ga-NOTA- and Ga-NOTAM-folate conjugates were: C,45.98; H,4.94; N,20.77 and C,46.10; H,5.20; N,24.29, found:C,46.31; H,4.52; N, 20.23. and C,45.65; H, 4.96; N,23.89, respectively.

Radiochemistry. Folate receptor can be used a tumor-associated molecular target since it is over-expressed in ovarian and endometrial carcinomas. Thus, we designed and developed 68Ga-folate radiotracers utilizing 68Ga generator to facilitate production of some PET radiopharmaceuticals in PET centers lacking cyclotrons. The synthetic approaches for preparation of 68Ga-NOTA- and 68Ga-NOTAM-folate conjugates entailed a straightforward and a simple one-step reaction (Figure 3). The eluted 68GaCl3 was reacted with NOTA- and NOTAM-folate conjugates in acetic acid (0.1% in EtOH, pH~4.5) at 80°C for 10 min to provide, after EtOH evaporation and re-constitution in saline, 68Ga-NOTA- and 68Ga-NOTAM-folate radiotracers in quantitative radiochemical yields (decay corrected) in less than 20 min. Radiochemical purities of 68Ga-NOTA- and 68Ga-NOTAM-folate conjugates were always greater than 98% as determined by HPLC and confirmed by TLC with retention times of 13.6 and 13.9 min, respectively (Figure 4). On the other hand, NOTA- and NOTAM-folate conjugates were labeled with 67Ga (T1/2~78 h) to give the possibility of investigations at a later time point. The 67GaCl3 was reacted with NOTA- and NOTAM-folate conjugates using similar conditions and the optimum reaction time was found to be 30 min. These radiotracers were obtained in less than 40 min and the overall radiochemical yields (decay-corrected) and purities were quantitative as assessed by HPLC and TLC. In comparison to the generator produced 68GaCl3, longer reaction time was required for 67GaCl3, which might be attributed to the impurities (Zn, Cu, etc.) present in the final 67GaCl3 solution obtained using the cyclotron 68Zn(p,2n)67Ga nuclear process. The optimized synthetic approach appears to be faster and advantageous over other synthetic strategies reported previously for the preparation of folate analogs (22, 31) and holds considerable promise in the synthesis of 67/68Ga-DOTA-folate conjugate analogs, where the overall radiochemical yield and purity were 10% and 92%, respectively.

In addition, the calculated partition coefficient for 68Ga-NOTA- and 68Ga-NOTAM-folate conjugates were found to be −1.92±0.13 and −1.26±0.09, respectively, representing at least 30% lower lipophilic characteristic for the former, in comparison with the latter conjugate. Moreover, the calculated charged densities for 68Ga-NOTA- and 68Ga-NOTAM-folate conjugates were found to be around 0.70 and 0.23, respectively, which correlate well with partition coefficient measurements.

Figure 4.
Figure 4.

HPLC chromatograms of 68Ga-NOTA- (A) and 68Ga-NOTAM-folate (B) conjugates.

Stability in plasma. The proteolytic degradation of the 68Ga-NOTA- and 68Ga-NOTAM-folate conjugates was determined in human plasma in vitro. HPLC analysis of the human plasma revealed that 68Ga-NOTA- and 68Ga-NOTAM-folate conjugates remained sufficiently stable (>99%) during incubation at 37°C for at least 4 h demonstrating a high in vitro stability of these radioconjugates.

In vitro cell binding. The binding affinities (Kd) of 68Ga-NOTA- and 68Ga-NOTAM-folate conjugates for positive-folate receptor were evaluated using the KB human oral carcinoma cell line. The Kd values of these conjugates were determined by saturation assays (Figure 5). The results demonstrate that binding affinities of 68Ga-NOTA- and 68Ga-NOTAM-folate conjugates are 54.36±6.31 nM and 78.01±13.0 nM, respectively. These results appeared to be well at least ten-fold lower than binding affinities for their corresponding 68Ga-DOTA conjugates. Blocking experiments performed with excess amount of native FA showed negligible binding on cell surface, which indicate the high specificity of 68Ga-NOTA- and 68Ga-NOTAM-folate conjugates for the over-expressed folate receptor in vitro.

Figure 5.
Figure 5.

Determination of binding affinity (Kd) values of 68Ga-NOTA-(A) and 68Ga-NOTAM-folate (B) conjugates for positive-folate receptor using KB human oral carcinoma cell line.

It is important for therapeutic radiopharmaceuticals that radiolabeled molecules be internalized into the tumor cells after binding to the cell surface receptors. In vitro cellular internalization studies were performed to determine the extent of internalization of 68Ga-NOTA- and 68Ga-NOTAM-folate conjugates by human cancer KB cells using acidic buffer. A rapid and significant internalization was observed for both conjugates with 35.22±0.50% and 45.0±1.13% of the cell surface bound conjugates internalized after incubation in acidic buffer for 20 min at 37°C, respectively. These results appear to be at least two-fold higher than their corresponding 68Ga-DOTA conjugates (~16.5%). These findings of cell-binding and internalization into tumor cells demonstrate that, despite the modifications in the folate for 68Ga chelation, these conjugates maintained their integrity and high affinity and specificity towards positive folate receptor.

In vivo biodistribution and tumor uptake. The biodistribution data in nude mice bearing human KB cell line xenografts for 68Ga-NOTA- and 68Ga-NOTAM-folate conjugates at 1, 2, 4 and 24 h post injection (p.i.) are shown in Tables I and II. Both 68Ga-NOTA- and 68Ga-NOTAM-folate conjugates displayed moderate clearance from the blood and most of the other organs. A low to moderate uptake of both 68Ga-NOTA- and 68Ga-NOTAM-folate conjugates was exhibited in the kidney (2.97±1.12 and 5.56±0.78% ID/g, respectively, 4 h p.i.), which are known to express folate receptor. Surprisingly, kidney uptake was found to be much lower than the 67/68Ga-DOTA-folate conjugate analogs (85-103% ID/g, 4h p.i.) reported previously (22, 31). The exact reason for the low uptake is unclear, but we assumed that this behavior may be attributed to the nature of 68Ga-NOTA and 68Ga-NOTAM as chelating groups and overall charge on 68Ga-NOTA- and 68Ga-NOTAM-folate complexes (32). More studies are required to validate this assumption. High tumor uptake was observed for both 68Ga-NOTA- and 68Ga-NOTAM-folate conjugates at all time-point intervals, however, the maximum uptake was observed at 4 h p.i. (17.75±4.27 for the former and 8.65±0.56% ID/g for the latter, respectively). The high tumor uptake for both conjugates at 24 h p.i. (6.08±0.65 and 7.33±0.31% ID/g) indicates a slow wash-out from the tumor, which is comparable to 67/68Ga-DOTA-folate conjugate analogs previously reported (22, 31). Tumor-to-blood, tumor-to-muscle and tumor-to-intestine ratios for 68Ga-NOTA- and 68Ga-NOTAM-folate conjugates at 4 h p.i. were 6.65, 31.14, 5.04 and 1.92, 5.62, 2.19, respectively, rendering the former conjugate more suitable as a tumor imaging agent than the latter. It is worth mentioning that the tumor uptake of 68Ga-NOTA-folate conjugate is superior to uptake profile reported for 67/68Ga-DOTA-folate conjugates (22, 31), while radioactivity accumulation in the liver and intestine was below 10% ID/g at 4 h p.i. In addition, receptor blocking studies in which 100 μg dose of FA was injected 10 min prior the administration of both conjugates, significantly reduced the uptake in the target organs and tumors. 68Ga-NOTA- 68Ga-NOTAM-folate conjugates uptake was reduced in tumor (17.75±4.27 vs. 2.01±0.56% and 8.65±0.56 vs. 1.12±0.25% ID/g, respectively, p<0.05) by approximately 85%, indicating receptor-specificity of these radioconjugates. Uptake by the kidneys of both radioconjugates was dramatically reduced by at least 70%.

The favorable biodistribution profile of 68Ga-NOTA-folate conjugate warrant further evaluation and may tempt one to infer that this PET radiotracer may be useful as a molecular probe for detecting and staging of folate receptor-positive cancers, such as ovarian cancer and their metastasis as well as monitoring tumor response to treatment. Low kidney uptake may also, offer this radioconjugate as potential candidate for radionuclide therapy when labeled with therapeutic radionuclides, such as 90Y or 177Lu, which is the subject of another study.

View this table:
Table I.

Biodistribution of 68Ga-NOTA-folate conjugate in tumor-bearing nude mice.

View this table:
Table II.

Biodistribution of 68Ga-NOTAM-folate conjugate in tumor-bearing nude mice.

Conclusion

The synthesis of 68Ga-NOTA- 68Ga-NOTAM-folate conjugates was performed using a simple one-step reaction. The overall radiochemical yields and purities for these radioconjugates were quantitative with total synthesis time of less than 20 min. In vitro binding studies on the KB cell line showed superior affinity of 68Ga-NOTA-folate over 68Ga-NOTAM-folate conjugates for folate receptor. In a nude mice model bearing human KB cell line xenografts, 68Ga-NOTA-folate demonstrated significant tumor uptake and favorable pharmacokinetics over the 68Ga-NOTAM-folate conjugate. These results demonstrate that the 68Ga-NOTA-folate conjugate may be useful as molecular probe for detecting folate receptor-positive cancers, such as ovarian cancer, using positron emission tomography, however, further evaluation is warranted.

Acknowledgements

The Authors wish to thank Mr. Mohamed Al-Amoudi for the MS analysis. This project was supported by the International Atomic Energy Agency and the King Faisal Specialist Hospital & Research Center (RAC # 2040027)

  • Received June 26, 2014.
  • Revision received August 3, 2014.
  • Accepted August 6, 2014.

References

  1. ↵
    1. Okarvi MS
    : Peptide-based radiopharmaceuticals: Future tools for diagnostic imaging of cancers and other diseases, Med Res Rev 24: 357-368, 2004.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Campbell IG,
    2. Jones TA,
    3. Foulkes WD,
    4. Trowsdale J
    : Folate-binding protein is a marker for ovarian cancer, Cancer Res 51: 5329-5338, 1991.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    1. Antony A
    : The Biological Chemistry of Folate Receptors, Blood 11: 2807-2820, 1992.
    OpenUrl
  4. ↵
    1. Westerhof GD,
    2. Jansen G,
    3. Emmerik N,
    4. Kathmann I,
    5. Rijksen G,
    6. Jackman AL,
    7. Schornagel J
    : Membrane transport of natural folates and antifolate compounds in murine L1210 leukemia cells: Role of carrier and receptor-mediated transport systems, Cancer Res 51: 5507-5513, 1991.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Leamon CP,
    2. Low PS
    : Membrane folate-binding proteins are responsible for folate-protein conjugate endocytosis into cultured cells, J Biochem 291: 855-860, 1993.
    OpenUrl
    1. Leamon CP,
    2. Pastan I,
    3. Low PS
    : Cytotoxicity of folate-pseudomonas exotoxin conjugates toward tumor cells, J Biol Chem 268: 24847-24854, 1993.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Wang S,
    2. Lee R J,
    3. Mathias CJ,
    4. Green MA,
    5. Low PS
    : Synthesis, Purification, and Tumor cell uptake of 67Ga-Deferoxamine, a potential radiopharmaceutical for tumor imaging, Bioconjugate Chem 7: 56-62, 1996.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Ilgan S,
    2. Yang DJ,
    3. Higuchi T,
    4. Zareneyrzi F,
    5. Bayhan H,
    6. Yu D,
    7. Kim E,
    8. Podolff DA
    : Imaging tumor folate receptors using 111In-DTPA-methotrexate, Cancer Bioth Radiopharm 13: 177-184, 1998.
    OpenUrl
  9. ↵
    1. Mathias CJ,
    2. Wang S,
    3. Lee RJ,
    4. Waters DJ,
    5. Low PS,
    6. Green MA
    : Tumor-selective radiopharmaceutical targeting via receptor-mediated endocyctosis of 67Ga-Deferoxamine-folate, J Nucl Med 37: 1003-1008, 1996.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Mathias CJ,
    2. Wang S,
    3. Waters DJ,
    4. Low PS,
    5. Green MA
    : Receptor-mediated targeting of 67Ga-Deferoxamine-folate to folate-receptor-positive human KB tumor xenografts, Nucl Med Biol 26: 23-25, 1999.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Wang S,
    2. Luo J,
    3. Lantrip DA,
    4. Waters DJ,
    5. Mathias CJ,
    6. Green MA,
    7. Fuchs PL,
    8. Low PS
    : Design and synthesis of 111In-DTPA-folate for use as a tumor-targeted radiopharmaceutical, Bioconjugate Chem 8: 673-679, 1997.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Mathias CJ,
    2. Wang S,
    3. Waters DJ,
    4. Turek JJ,
    5. Low PS,
    6. Green MA
    : Indium-111-DTPA-folate as a potential folate–receptor-targeted radiopharmaceutical, J Nucl Med 39: 1579-1585, 1998.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Siegel BA,
    2. Dehdashti F,
    3. Mutch DG,
    4. Podoloff DA,
    5. Wendt R,
    6. Sutton GP,
    7. Burt RW,
    8. Ellis PR,
    9. Mathias CJ,
    10. Green MA,
    11. Gershenson DM
    : Evaluation of 111In-DTPA-folate as a receptor-targeted diagnostic agent for ovarian cancer: Initial clinical results, J Nucl Med 44: 700-707, 2003.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Guo W,
    2. Hinkle GH,
    3. Lee RJ
    : 99mTc-HYNIC-Folate: A Novel Receptor-Based Targeted Radiopharmaceutical for Tumor Imaging, J Nucl Med 40: 1563-1569, 1999.
    OpenUrlAbstract/FREE Full Text
    1. Mathias CJ,
    2. Hubers D,
    3. Low PS,
    4. Green MA
    : Synthesis of 99mTc-DTPA-folate and its evaluation as a folate-receptor-targeted radiopharmaceutical, Bioconjugate Chem 11: 253-257, 2000.
    OpenUrlCrossRefPubMed
    1. Leamon CP,
    2. Parker MA,
    3. Vlahov IR,
    4. Xu L,
    5. Reddy JA,
    6. Vetzel M,
    7. Douglas N
    : Synthesis and biological evaluation of EC20: A new folate-derived 99mTc-based radiopharmaceutical. Bioconjugate Chem 13: 1200-1210, 2002.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Okarvi SM,
    2. AlJammaz I
    : Preparation and in vitro and in vivo evaluation of 99mTc-lalbeled folate and methotrexate conjugates as tumor imaging agents, Cancer Bioth Radiopharm 21: 49-60, 2006.
    OpenUrl
  18. ↵
    1. Varagnolo L,
    2. Stokkel MP,
    3. Mazzi U,
    4. Pauwels EK
    : 18F-Labeled radiopharmaceuticals for PET in oncology, excluding FDG, Nucl Med Biol 27: 103-112, 2000.
    OpenUrlCrossRefPubMed
  19. ↵
    1. AlJammaz I,
    2. Al-Otaibi B,
    3. Amer S,
    4. Okarvi S
    : Rapid synthesis and in vitro and in vivo evaluation of folic acid derivatives labeled with fluorine-18 for PET imaging of folate receptor-positive tumors, J Nucl Med Biol 38: 1019-1028, 2011.
    OpenUrl
  20. ↵
    1. AlJammaz I,
    2. Al-Otaibi B,
    3. Amer S,
    4. Al-Hokbany N,
    5. Okarvi S
    : Novel Synthesis and Preclinical Evaluation of Folic Acid Derivatives Labeled with 18F[FDG] for PET Imaging of Folate Receptor-Positive Tumors, J Nucl Med Biol 39: 864-870, 2012.
    OpenUrl
  21. ↵
    1. Mathias CJ,
    2. Lewis MR,
    3. Reichert DE,
    4. Laforest R,
    5. Sharp TL,
    6. Lewis JS,
    7. Yang Z,
    8. Waters DJ,
    9. Snyder PW,
    10. Low PS,
    11. Welch MJ,
    12. Green MA
    : Preparation of 66Ga and 68Ga-labeled Ga(III)-deferoxamine-folate as a potential folate-receptor-targeted PET radiopharmaceuticals, J Nucl Med Biol 30: 725-731, 2003.
    OpenUrl
  22. ↵
    1. Fani M,
    2. Wang X,
    3. Nicolas G,
    4. Medina C,
    5. Raynal I,
    6. Port M,
    7. Maecke H
    : Development of new folate-based PET radiotracers: preclinical evaluation of 68Ga-DOTA-folate conjugates, Eur J Nucl Med Mol Imaging 38: 108-119, 2011.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Clarke ET,
    2. Martell AE
    : Stabilities of the Fe(III), Ga(III) and In(III) chelates of N,N,N-triazacyclononanetricaetic acid, Inorganica Chimica Acta 181: 273-280, 1991.
    OpenUrl
  24. ↵
    1. Clarke ET,
    2. Martell AE
    : Stabilities of trivalent metal ion complexes of the tetraacetate derivatives of 12-, 13- and 14-membered tetraazamacrocycles, Inorganica Chimica Acta 190: 37-46, 1991.
    OpenUrl
  25. ↵
    1. Chong H,
    2. Garmestani K,
    3. Ma D,
    4. Milenic DE,
    5. Overstreet T,
    6. Brechbiel MW
    : Synthesis and biological evaluation of novel macrocyclic ligands with pendent donor groups as potential yttrium chelators for radioimmunotherapy with improved complex formation kinetics, J Med Chem 45: 3458-3464, 2002.
    OpenUrlPubMed
  26. ↵
    1. Breeman WA,
    2. de Jong M,
    3. de Blois E,
    4. Bernard BF,
    5. Konijnenberg M,
    6. Krenning EP
    : Radiolabelling DOTA-peptides with 68Ga: Eur J Nucl Med Mol Imaging 32: 478-485, 2005.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Zhernosekov KP,
    2. Filosofov DV,
    3. Baum RP,
    4. Aschoff P,
    5. Bihl H,
    6. Razbash AA,
    7. Jahn M,
    8. Jennewein M,
    9. Rosch F
    : Processing of generator-produced 68Ga for medical application, J Nucl Med 48: 1741-1748, 2007.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Guide for the Care of and Use of Laboratory Animals, Washington, DC: National Academy Press 1996.
  29. ↵
    1. Viola-Villegas N,
    2. Vortherms A,
    3. Doyle RP
    : Targeting gallium to cancer cells through the folate receptor, Drug Target Insights 3: 13-25, 2008.
    OpenUrl
    1. AlJammaz I,
    2. Al-Otaibi B,
    3. Okarvi S,
    4. Amartey J
    : Novel synthesis of [18F]-fluorobenzene and pyridinecarbohydrazide-folates as potential PET radiopharmaceuticals, J Label Compd Radiopharm 49: 125-137, 2006.
    OpenUrlCrossRef
  31. ↵
    1. Müller C,
    2. Vlahov IR,
    3. Santhapuram KR,
    4. Leamon CP,
    5. Schibli R
    : Tumor targeting using 67Ga-DOTA-Bz-folate-investigations of methods to improve the tissue distribution of radiofolates, J Nucl Med Biol 38: 715-723, 2011.
    OpenUrl
  32. ↵
    1. Miao Y,
    2. Fisher D,
    3. Quinn T
    : Reducing renal uptake of 90Y- and 177Lu-labeled alpha-melanocyte stimulating hormone peptide analogues, Nucl Med Biol 33: 723-733, 2006.
    OpenUrlCrossRefPubMed
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Development and Pre-clinical Evaluation of New 68Ga-NOTA-folate Conjugates for PET Imaging of Folate Receptor-positive Tumors
IBRAHIM ALJAMMAZ, BASIM AL-OTAIBI, NOURAH AL-HOKBANY, SUAD AMER, SUBHANI OKARVI
Anticancer Research Nov 2014, 34 (11) 6547-6556;
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