Targeted radionuclide therapy has shown impressive results for the palliative treatment of several types of cancer diseases. The folate receptor has been identified as specifically associated with a variety of frequent tumor types. Therefore, it is an attractive target for the development of new radionuclide therapies using folate-based radioconjugates. Previously, we found that pemetrexed (PMX) has a favorable effect in reducing undesired renal uptake of radiofolates. Moreover, PMX also acts as a chemotherapeutic and radiosensitizing agent on tumors. Thus, the aim of our study was to investigate the combined application of PMX and the therapeutic radiofolate 177Lu-EC0800. Determination of the combination index (CI) revealed a synergistic inhibitory effect of 177Lu-EC0800 and PMX on the viability of folate receptor–positive cervical (KB) and ovarian (IGROV-1) cancer cells in vitro (CI < 0.8). In an in vivo study, tumor-bearing mice were treated with 177Lu-EC0800 (20 MBq) and a subtherapeutic (0.4 mg) or therapeutic amount (1.6 mg) of PMX. Application of 177Lu-EC0800 with PMXther resulted in a two- to four-fold enhanced tumor growth delay and a prolonged survival of KB and IGROV-1 tumor-bearing mice, as compared to the combination with PMXsubther or untreated control mice. PMXsubther protected the kidneys from undesired side effects of 177Lu-EC0800 (20 MBq) by reducing the absorbed radiation dose. Intact kidney function was shown by determination of plasma parameters and quantitative single-photon emission computed tomography using 99mTc-DMSA. Our results confirmed the anticipated dual role of PMX. Its unique features resulted in an improved antitumor effect of folate-based radionuclide therapy and prevented undesired radio-nephrotoxicity. Mol Cancer Ther; 12(11); 2436–45. ©2013 AACR.

Targeted radionuclide therapy has shown impressive results for the palliative treatment of several cancer diseases. It is based on the use of particle-emitting radioisotopes (e.g., 177Lu, 90Y, 131I) in conjunction with tumor-targeted biomolecules (e.g., peptides, antibodies; ref. 1). A prominent example of a successfully used radiopharmaceutical in clinical routine are somatostatin-based radiopeptides (e.g., 177Lu-DOTATATE, 90Y-DOTATOC) for the treatment of neuroendocrine tumors (2). Moreover, radiolabeled antibodies such as 90Y-Ibritumomab (Zevalin) and 131I-tositumomab (Bexxar) are approved for the treatment of non-Hodgkins lymphoma (3).

The development of new targeting strategies for the treatment of further tumor types is of high interest and would have a critical impact on the future management of these cancer diseases. In this respect, the folate receptor is an attractive target as it has been identified as specifically associated with a variety of cancer types, such as ovarian, endometrial, lung, brain, breast, and colorectal cancer (4, 5). The vitamin folic acid has been used as a targeting ligand because it binds to the folate receptor with high affinity followed by endocytotic internalization of the therapeutic payload into cancer cells (6). While folic acid conjugates of highly toxic chemotherapeutics have been successfully used in clinical trials (7, 8), application of therapeutic folic acid radioconjugates is currently being developed in preclinical studies.

Substantial expression of the folate receptor in the proximal tubule cells of the kidneys results in commonly high and specific renal uptake of folate-based radioconjugates (9, 10). As a consequence there is an inherent risk of damage to the kidneys by particle radiation. However, we have shown in several preclinical studies that administration of the antifolate pemetrexed (PMX) resulted in a tremendous reduction of the radiofolate's retention in the kidneys whereas accumulation in tumor xenografts remained unaffected (11–13). The exact mechanism of this interaction is still not completely clear. However, in numerous preclinical studies, we observed an interrelation between the kidney reducing effect and the time point of preinjected PMX (14) as well as the molar amount of PMX and the folate radioconjugate, respectively (15). However, the reduced kidney uptake of radiofolates was not a result of PMX's antifolate activity as the effect was maintained even if PMX was applied in combination with the antidote thymidine (16). These facts suggest that PMX's kidney reducing effect is based on a competition among PMX and the folate radioconjugate for folate receptor binding sites in the kidneys.

PMX is a multitargeted antifolate, which is clinically approved for the treatment of pleural mesothelioma and non–small cell lung cancer in combination with cisplatin (17–19). Moreover, PMX is currently being tested in a number of clinical trials for the treatment of several other cancer types (reviewed in ref. 20), among those is also ovarian cancer (21). PMX has been used in combinations with gemcitabine, tyrosine kinase inhibitors, antibodies, or even external radiation (22–24). A combination of PMX with external radiotherapy was based on the observation that PMX acts as a radiosensitizing agent in variable types of cancer cells in vitro and in vivo (25–28).

We hypothesized that PMX would have a dual role if it was combined with therapeutic folate radioconjugates. First, it was expected to prevent radionephropathy by reducing the absorbed radiation dose of folate radioconjugates in the kidneys. Second, PMX was believed to enhance the therapeutic efficacy of folate-based radionuclide tumor therapy by its action as a chemotherapeutic and radiosensitizing agent.

The goal of this study was to show the anticipated dual role of PMX. For this purpose, we used an established DOTA-folate conjugate (EC0800; ref. 29), which was radiolabeled with 177Lu (T1/2 = 6.7 days, Eav(β): 134 keV, E(γ): 113 keV, 208 keV). 177Lu-EC0800 was applied in combination with PMX using 2 folate receptor positive human cancer cells (KB and IGROV-1) in vitro and as human tumor xenografts in athymic nude mice. Moreover, the protective effect of PMX on the kidneys to reduce the absorbed radiation dose was investigated in non–tumor-bearing mice.

Preparation of 177Lu-EC0800

The DOTA-folate conjugate (EC0800; ref. 29) was kindly provided by Endocyte Inc. (Supplementary Fig. S1). The radiosynthesis of 177Lu-EC0800 and the evaluation of its stability were carried out as previously reported (Supplementary Methods; ref. 30). It was shown that 177Lu-EC0800 was stable (>95%) over at least 24 hours in PBS and human blood plasma.

Cell culture

KB cells (human cervical carcinoma cells, ACC-136), and PC-3 (human prostate carcinoma cells, ACC-465) were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ). IGROV-1 cells (human ovarian carcinoma cells) were a kind gift from Dr. Gerrit Jansen (Department of Rheumatology, VU University Medical Center, Amsterdam, the Netherlands). All 3 cell lines were authenticated by DMSZ based on DNA profiling. KB and IGROV-1 cells are known to express the folate receptor (31, 32), whereas folate receptor negative PC-3 cells (33) were used as a negative control. PC-3 cells were cultured in RPMI 1640 medium (Amimed) whereas KB and IGROV-1 cells were cultured in folate-free RPMI medium referred to as FFRPMI (without folic acid, vitamin B12, and phenol red; Cell Culture Technologies GmbH). Cell culture media were supplemented with 10% fetal calf serum, l-glutamine, and antibiotics (penicillin/streptomycin/fungizone). Routine culture treatment was conducted twice a week. Uptake and internalization studies of 177Lu-EC0800 as well as clonogenic and MTT assays were conducted as previously reported in the literature in detail in the Supplementary Methods (29, 34).

MTT assay

Cell viability was assessed using an MTT assay (35). In brief, KB, IGROV-1, and PC-3 cells were treated with 200 μL FFRPMI medium (without supplements) containing 177Lu-EC0800 (0.001–5.0 MBq/mL) and/or PMX (0.01–100 μmol/L; pemetrexed, Alimta, LY231514; Eli Lilly, Supplementary Fig. S1). The control cells were incubated with FFRPMI medium only. After 4 hours incubation at 37°C, the cells were washed once with 200 μL PBS followed by addition of FFRPMI or RPMI medium (with supplements). Cells were then allowed to grow for 4 days at 37°C without changing medium. Analysis of the viability was conducted as previously reported using an MTT reagent and a microplate reader (Victor X3; Perkin Elmer; ref. 36). To quantify cell viability, the absorbance of the test samples was expressed as percentage of the absorbance of the control cell samples (= 100%). Dose–response curves were analyzed using the software GraphPad Prism (version 4.0). Inhibition of cell viability was expressed as the half-maximal inhibitory concentration of PMX (IC50 in μmol/L) and as the half-maximal inhibitory activity concentration of 177Lu-EC0800 (IAC50 in MBq/mL) by measuring dose–response curves of PMX and 177Lu-EC0800. The dose–response curves were used to determine the combination index (CI) according to Chou and colleagues (Supplementary Methods; ref. 37).

Animal studies

The in vivo experiments were approved by the local veterinarian department and conducted in accordance with the Swiss law of animal protection. Female athymic nude mice (4–5 week-old CD-1 Foxn-1/nu; Charles River Laboratories) were fed with a folate-deficient rodent diet (Harlan Laboratories) starting 5 days before tumor cell inoculation (38). For therapy experiments, endpoint criteria were defined as (i) a tumor volume >1000 mm3, (ii) body weight loss of >15%, (iii) active ulceration of the tumor xenograft, or (iv) abnormal behavior of the mice and signs of unease.

Biodistribution study and dosimetric calculations

Biodistribution studies over 72 hours were conducted as previously reported (Supplementary Methods and Table S1; ref. 29). These datasets were used to estimate the equivalent absorbed radiation dose to the tumor xenografts and kidneys upon injection of 177Lu-EC0800 (Supplementary Methods). Based on the biodistribution results the accumulation of radioactivity in KB and IGROV-1 tumors was taken as equal. For estimation of the kidney dose, it was assumed that in the case of preinjected PMX kidney uptake was reduced to 25% of control values.

Biodistribution studies conducted with 3H-PMX are reported in the Supplementary Methods.

Investigation of potential radiotoxicity

Groups of 6 mice were injected with only PBS (group A), with 177Lu-EC0800 (20 MBq, 1 nmol; group B) or with 177Lu-EC0800 (20 MBq, 1 nmol) and PMXsubther (0.4 mg; group C). From day 21 after start of the therapy, the animals were fed with a standard rodent diet. For studying plasma parameters, blood was taken from the sub-lingual vein collected in heparinized vials at day 50, 130, and 180 or before euthanasia. Blood plasma parameters such as creatinine, blood urea nitrogen, alkaline phosphatase, and total bilirubin were determined using a Fuji Dri-Chem 40000i analyzer (Polymed Medical Center AG). Quantitative single-photon emission computed tomography (SPECT) to determine renal uptake of 99mTc-DMSA in mice was conducted in week 3, 15, and 23 of the experiment.

SPECT experiments were conducted with a 4-head multiplexing multi-pinhole camera (NanoSPECT/CT; Bioscan Inc.) using collimators of 4 × 9 holes of a diameter of 1.4 mm. Dimercaptosuccinic acid (DMSA)-kits (TechneScan) were purchased from Mallinckrodt Inc. The 99mTc-radiolabeling was conducted according to the instructions of the manufacturer using 99mTc-pertechnetate, which was freshly eluted from a 99mTc-generator (Mallinckrodt Inc.). SPECT images of the kidneys (30-mm scan length) were acquired 2 hours after injection of 99mTc-DMSA (∼30 MBq/mouse). After acquisition, SPECT data were reconstructed iteratively with HiSPECT software (version 1.4.3049; Scivis GmbH) using a γ-energy of 140 keV for 99mTc. Quantification of renal uptake of 99mTc-DMSA was carried out as previously reported using InVivoScope postprocessing software (version 2.0; Bioscan Inc.; ref. 39).

Therapy studies in tumor-bearing mice

Twenty mice were inoculated with KB tumor cells (4.5 × 106 in 100 μL PBS, model I) and another 20 mice were inoculated with IGROV-1 tumor cells (6.0 × 106 in 100 μL PBS, model II). At the start of the therapy 4 days later, the average tumor size reached a volume of ∼65 mm3 (KB xenografts) and ∼40 mm3 (IGROV-1 xenografts). For each study, 4 groups of 5 mice were injected with only PBS (group A), with 2 × 0.8 mg PMX (i.e., PMXther, group B), with 1 × 20 MBq 177Lu-EC0800 combined with 1 × 0.4 mg PMX (i.e., PMXsubther, group C), and with 1 × 20 MBq 177Lu-EC0800 combined with 2 × 0.8 mg PMX (i.e., PMXther, group D).

Body weights and tumor volumes were measured 3 times a week. The tumor volume (V) was determined by measuring 2 perpendicular diameters with a digital caliper and calculated by using the formula V = [0.5 × (L × W2)], where L is the length of the tumor (large diameter) and W is the width (small diameter; ref. 40). The values were indicated as the average of the individual relative tumor volumes (RTV = Vx/V0; Vx = volume at day x, V0 = volume at day 0) and of individual relative body weight (RBW = Wx/W0; Wx = weight at day x; W0 = weight at day 0). The therapeutic efficacy was expressed as the percentage of tumor growth inhibition (%TGI = 100 − (T/C × 100), where T is the mean RTV of the treated mice and C is the mean RTV of untreated control mice at day 17 when the first mouse in the control group was euthanized. Tumor growth delay (TGD) was calculated for the time that passed until the RTVs reached 4-fold the value of day 0. The tumor growth delay index (TGDI) was calculated as the ratio of TGDs among treated animals and control animals (41).

Statistical analysis

Statistics was conducted by using a t-test (Microsoft Excel software). All analyses were 2-tailed and considered as type 3 (2 sample unequal variance). A P-value of <0.05 was considered as statistically significant.

Cancer cell lines

The human cervical KB cancer cell line is a subclone of HeLa cells (42), known to express the folate receptor at very high levels. IGROV-1 cells are human ovarian cancer cells that express the folate receptor at a somewhat lower level than KB cells (12, 31, 36). The human prostate cancer cell line PC-3 does not express the folate receptor and was used as a negative control. These facts were confirmed in uptake/internalization studies of 177Lu-EC0800 (Supplementary Fig. S2).

Cell survival upon exposure to 177Lu-EC0800

Clonogenic assays revealed plating efficiencies of 11%, 14%, 18% and 16%, for KB, IGROV-1, and PC-3 cells. At a radioactivity concentration of 1.0 MBq/mL 177Lu-EC0800 (16 nmol/L) with a 4 hours exposure, the survival fraction of KB and IGROV-1 cells was 0.12 and 0.43, respectively. The survival fraction of folate receptor negative PC-3 cells was still 0.98, even after the treatment with a 5-fold higher radioactivity concentration. Reduction of cell survival was completely suppressed by preincubating folate receptor positive tumor cells with excess folic acid to block folate receptors (Fig. 1A; Supplementary Fig. S3).

Figure 1.

A, survival of folate receptor–positive KB and IGROV-1 cells and folate receptor–negative PC-3 cells upon exposure to177Lu-EC0800 (1 MBq/mL; 16 nmol/L) in the presence and absence of excess folic acid (FA). B and C, dose–response curves of KB and IGROV-1 tumor cells incubated with variable activity concentrations of 177Lu-EC0800 (indicated as 177Lu) and PMX.

Figure 1.

A, survival of folate receptor–positive KB and IGROV-1 cells and folate receptor–negative PC-3 cells upon exposure to177Lu-EC0800 (1 MBq/mL; 16 nmol/L) in the presence and absence of excess folic acid (FA). B and C, dose–response curves of KB and IGROV-1 tumor cells incubated with variable activity concentrations of 177Lu-EC0800 (indicated as 177Lu) and PMX.

Close modal

Cell viability upon exposure of 177Lu-EC0800 combined with PMX

MTT assays were conducted to determine IAC50 and IC50 values of 177Lu-EC0800 and PMX. The inhibition of cell viability was found to be dependent on the concentration of PMX in all cell lines. The IC50 value of PMX amounted to 1.22 ± 0.13 μmol/L and 0.93 ± 0.17 μmol/L for KB and IGROV-1 cells, respectively. The IAC50-values of 177Lu-EC0800 revealed activity concentrations of 0.054 ± 0.004 MBq/mL for KB and 0.83 ± 0.08 MBq/mL for IGROV-1 tumor cells (Fig. 1B and C).

Combination index

The interactions between 177Lu-EC0800 and PMX were calculated according to the results obtained with KB and IGROV-1 cells, which were exposed to 177Lu-EC0800 and PMX as single agents or simultaneously. The concentrations of the test agents, alone and in combination, required to reduce cell viability to 55% and 70% of controls were determined. All calculations revealed values of the CI below 0.8, indicating a synergistic effect between 177Lu-EC0800 and PMX (Supplementary Table S2).

Biodistribution studies

Biodistribution studies in KB and IGROV-1 tumor-bearing mice showed a relatively high uptake of 177Lu-EC0800 in tumor xenografts [KB: 5.94 ± 1.20% ID/g and IGROV-1: 6.58 ± 1.50% ID/g; 4 hours past injection (p.i)], and a ∼10-fold higher accumulation in the folate receptor positive kidneys. PMX treatment reduced renal uptake of 177Lu-EC0800 up to 7-fold, while simultaneously allowing for greater tumor uptake (Table 1). Dosimetric estimation revealed a dose of 0.38 Gy/MBq to the tumor xenografts and 4.84 Gy/MBq to the kidneys if 177Lu-EC0800 was applied as a single agent. Under the assumption of a 4-fold reduced renal uptake of 177Lu-EC0800 in combination with PMX, the kidney dose was reduced to 1.21 Gy/MBq whereas the tumor dose remained unaffected (Supplementary Methods and Fig. S4).

Table 1.

Biodistribution data 4 hours after injection of 177Lu-EC0800 (3 MBq, 1 nmol) in KB (model I) and IGROV-1 tumor-bearing nude mice (model II)

177Lu-EC0800 (model I)
ControlPMXsubtheraPMXtherb
Blood 0.08 ± 0.01 0.06 ± 0.01 0.06 ± 0.01 
Liver 6.61 ± 1.04 4.28 ± 0.69 3.46 ± 0.38 
Kidneys 51.0 ± 6.6 9.66 ± 1.14 8.12 ± 0.88 
Tumor 5.94 ± 1.20 7.83 ± 0.98 9.01 ± 1.60 
Tumor-to-blood 71.6 ± 19.2 131.1 ± 24.6 161.7 ± 17.3 
Tumor-to-liver 0.91 ± 0.22 1.88 ± 0.41 2.64 ± 0.65 
Tumor-to-kidney 0.12 ± 0.02 0.86 ± 0.14 1.11 ± 0.16 
 177Lu-EC0800 (model II) 
ControlPMXsubtheraPMXtherb
Blood 0.16 ± 0.04 0.08 ± 0.00 0.08 ± 0.02 
Liver 7.60 ± 2.04 2.75 ± 0.19 2.37 ± 0.68 
Kidneys 67.0 ± 13.4 11.4 ± 1.2 9.30 ± 1.96 
Tumor 6.58 ± 1.50 9.30 ± 1.96 9.55 ± 0.88 
Tumor-to-blood 42.7 ± 7.8 122.7 ± 28.0 127.9 ± 40.6 
Tumor-to-liver 0.88 ± 0.16 3.40 ± 0.78 4.32 ± 1.48 
Tumor-to-kidney 0.10 ± 0.02 0.81 ± 0.13 1.04 ± 0.54 
177Lu-EC0800 (model I)
ControlPMXsubtheraPMXtherb
Blood 0.08 ± 0.01 0.06 ± 0.01 0.06 ± 0.01 
Liver 6.61 ± 1.04 4.28 ± 0.69 3.46 ± 0.38 
Kidneys 51.0 ± 6.6 9.66 ± 1.14 8.12 ± 0.88 
Tumor 5.94 ± 1.20 7.83 ± 0.98 9.01 ± 1.60 
Tumor-to-blood 71.6 ± 19.2 131.1 ± 24.6 161.7 ± 17.3 
Tumor-to-liver 0.91 ± 0.22 1.88 ± 0.41 2.64 ± 0.65 
Tumor-to-kidney 0.12 ± 0.02 0.86 ± 0.14 1.11 ± 0.16 
 177Lu-EC0800 (model II) 
ControlPMXsubtheraPMXtherb
Blood 0.16 ± 0.04 0.08 ± 0.00 0.08 ± 0.02 
Liver 7.60 ± 2.04 2.75 ± 0.19 2.37 ± 0.68 
Kidneys 67.0 ± 13.4 11.4 ± 1.2 9.30 ± 1.96 
Tumor 6.58 ± 1.50 9.30 ± 1.96 9.55 ± 0.88 
Tumor-to-blood 42.7 ± 7.8 122.7 ± 28.0 127.9 ± 40.6 
Tumor-to-liver 0.88 ± 0.16 3.40 ± 0.78 4.32 ± 1.48 
Tumor-to-kidney 0.10 ± 0.02 0.81 ± 0.13 1.04 ± 0.54 

NOTE: Data are presented as average ± SD (n = 3).

aPMXsubther: 0.4 mg per mouse, injected 1 hour before 177Lu-EC0800.

bPMXther: 0.8 mg per mouse, injected 1 hour before 177Lu-EC0800.

Short after injection of 3H-PMX, the uptake in the kidneys was relatively high (∼2.7% ID/g, 1 hour p.i.) but a significant washout was observed within the following hour (∼0.8% ID/g, 2 hours p.i.). In the tumor tissue accumulation of 3H-PMX was low (∼1.1% ID/g, 1 hour p.i.) but more constant (0.6% ID/g, 2 hours p.i.) over time (Supplementary Methods and Fig. S5).

Investigation of potential radiotoxicity

In a separate study, radiotoxicity of 177Lu-EC0800 and the kidney protective effect of a subtherapeutic amount of PMX (0.4 mg) were investigated. Nontumor-bearing nude mice were monitored over 6 months. The kidney dose of 177Lu-EC0800 (20 MBq/mouse) was ∼97 Gy (group B). If PMXsubther was preinjected, the kidney dose was significantly reduced to ∼24 Gy (group C). At day 50 of the study, plasma parameters of treated mice (groups B/C) were in the same range as those of control mice (group A). However, at day 130, levels of blood urea nitrogen, alkaline phosphatase, and total bilirubin from mice treated with 177Lu-EC0800 (group B) differed significantly from those of control mice (group A). The values obtained from mice of group C were in the same range as the values from mice of group A. Determination of blood plasma parameters at day 180 showed the same result as found at day 130 (Table 2).

Table 2.

Plasma parameters of group A (PBS), group B (20 MBq of 177Lu-EC0800), and group C (20 MBq of 177Lu-EC0800 and 0.4 mg of PMX)

Group C
DayGroup A PBSGroup B 177Lu-EC0800177Lu-EC0800 and PMXsubther
CRE (μmol/L) 50 21 ± 3.4 19 ± 2.1 <18 
 130 28 ± 12 58 ± 43 <18 
 180a 19 ± 0.7 114 ± 62b 21 ± 0.7 
BUN (mmol/L) 50 8.5 ± 0.9 11.8 ± 2.2b 10.0 ± 1.1 
 130 9.7 ± 0.6 35.0 ± 9.4c 8.61 ± 1.1 
 180a 8.8 ± 0.6 >49.98c 9.09 ± 1.4 
ALP (U/L) 50 78 ± 10 83 ± 20 70 ± 8 
 130 54 ± 16 132 ± 42c 71 ± 18 
 180a 58 ± 14 123 ± 55b 67 ± 23 
TBIL (μmol/L) 50 10 ± 1.3 8 ± 1.0 11 ± 1.7 
 130 12 ± 1.9 24 ± 11b 12 ± 4.5 
 180a 12 ± 3.2 36 ± 1.9c 12 ± 5.0 
Group C
DayGroup A PBSGroup B 177Lu-EC0800177Lu-EC0800 and PMXsubther
CRE (μmol/L) 50 21 ± 3.4 19 ± 2.1 <18 
 130 28 ± 12 58 ± 43 <18 
 180a 19 ± 0.7 114 ± 62b 21 ± 0.7 
BUN (mmol/L) 50 8.5 ± 0.9 11.8 ± 2.2b 10.0 ± 1.1 
 130 9.7 ± 0.6 35.0 ± 9.4c 8.61 ± 1.1 
 180a 8.8 ± 0.6 >49.98c 9.09 ± 1.4 
ALP (U/L) 50 78 ± 10 83 ± 20 70 ± 8 
 130 54 ± 16 132 ± 42c 71 ± 18 
 180a 58 ± 14 123 ± 55b 67 ± 23 
TBIL (μmol/L) 50 10 ± 1.3 8 ± 1.0 11 ± 1.7 
 130 12 ± 1.9 24 ± 11b 12 ± 4.5 
 180a 12 ± 3.2 36 ± 1.9c 12 ± 5.0 

Abbreviations: CRE, creatinine; BUN, blood urea nitrogen; ALP, alkaline phosphatase; TBIL, total bilirubin.

aGroup B terminal blood sampling.

bP < 0.05.

cP < 0.005.

The extent of accumulated 99mTc-DMSA in the kidneys is a measure for tubular function (43). It has previously been used as a valuable in vivo tool for monitoring kidney function during radionuclide therapy (39). In week 3, baseline measurements of 99mTc-DMSA uptake in the kidneys showed no significant difference among groups B and C from control mice of group A (Fig. 2A). However, in week 15 the average % ID per kidney of mice treated with 177Lu-EC0800 only (group B: 4.37 ± 2.6% ID/kidney, 2 hours p.i., P < 0.005) was significantly lower than in control mice (group A: 11.74 ± 0.7% ID/kidney, 2 hours p.i.). Renal uptake of 99mTc-DMSA in mice that received PMXsubther in addition to 177Lu-EC0800 was comparable (group C: 10.58 ± 1.8% ID/kidney, 2 hours p.i., P = 0.2) with the value obtained from control animals (Fig. 2B). In week 23 of follow-up, renal accumulation of 99mTc-DMSA in mice of group B had dropped to 1.38 ± 0.16% ID/kidney 2 hours p.i. (P < 0.005), whereas in group C the uptake was still in same range as found for control animals of group A (P = 0.6; Fig. 2C).

Figure 2.

A–C, the average %ID per kidney in week 3, 15, and 23 after injection of 99mTc-DMSA in untreated control mice (group A), mice treated with 177Lu-EC0800 (group B), and mice treated with 177Lu-EC0800 and PMXsubther (group C; **, P < 0.005). D, graphs of the relative body weights of mice from each group over the whole time of 6 months. The lowest tolerable body weight is indicated at a relative value of 0.85. E, SPECT images of kidneys after injection of 99mTc-DMSA of one representative mouse of each group in week 23.

Figure 2.

A–C, the average %ID per kidney in week 3, 15, and 23 after injection of 99mTc-DMSA in untreated control mice (group A), mice treated with 177Lu-EC0800 (group B), and mice treated with 177Lu-EC0800 and PMXsubther (group C; **, P < 0.005). D, graphs of the relative body weights of mice from each group over the whole time of 6 months. The lowest tolerable body weight is indicated at a relative value of 0.85. E, SPECT images of kidneys after injection of 99mTc-DMSA of one representative mouse of each group in week 23.

Close modal

Constant body weight loss was observed in mice treated with 177Lu-EC0800 (group B) from about day 70 and thereafter. In all cases of group B, the endpoint criterion that required euthanasia was reached before day 180. However, mice that had received PMXsubther before the injection of 177Lu-EC0800 (group C) showed body weight gain similar to the untreated controls (group A).

In vivo tumor therapy studies

The injection protocol of the therapy study with KB and IGROV-1 tumor-bearing mice is shown in Fig. 3A. PMX was applied either in a subtherapeutic dose of 0.4 mg (PMXsubther) or at a therapeutic dose of 2 × 0.8 mg (PMXther) corresponding to 80% of the maximal tolerated dose (MTD). The MTD of PMX was previously determined in mice under the experimental conditions of a folate-free diet and revealed a dose of 2 × 1 mg per mouse (body weight ∼ 25 g) with a time lag of 1 week (Supplementary Methods and Fig. S6; ref. 16).

Figure 3.

A, injection scheme of the in vivo therapy studies conducted in KB (model I) and IGROV-1 tumor-bearing mice (model II). B, graphs of the mean relative tumor volumes of KB tumor xenografts (model I). C, graphs of mean relative tumor volumes of IGROV-1 xenografts (model II). D, graphs of the mean relative body weights of KB tumor-bearing mice (model I). E, graphs of the mean relative body weight of IGROV-1 tumor-bearing mice (model II).

Figure 3.

A, injection scheme of the in vivo therapy studies conducted in KB (model I) and IGROV-1 tumor-bearing mice (model II). B, graphs of the mean relative tumor volumes of KB tumor xenografts (model I). C, graphs of mean relative tumor volumes of IGROV-1 xenografts (model II). D, graphs of the mean relative body weights of KB tumor-bearing mice (model I). E, graphs of the mean relative body weight of IGROV-1 tumor-bearing mice (model II).

Close modal

In both tumor models (I and II), constant tumor growth was observed in control mice (group A) where the first mouse reached the endpoint criterion at day 17. For model I, the average RTV in mice treated with PMXther (group B: 9.8 ± 4.1; P = 0.90) and in mice treated with 177Lu-EC0800 and PMXsubther (group C: 8.3 ± 3.5, P = 0.21) were not significantly different from the average RTV of control mice (group A: 10.0 ± 2.7) at day 17 (Table 3). However, the combined application of 177Lu-EC0800 with PMXther resulted in a significant decrease of the average RTV (group D: 5.0 ± 2.2; P < 0.0001; Fig. 3B). For tumor model II, the average RTV of PMX-treated mice (group B: 27.9 ± 11.8; P = 0.73) was not significantly reduced compared to control mice (group A: 30.0 ± 14.9) at the same time point (Table 3). However, a significant reduction of the average RTV was observed in both groups of mice treated with 177Lu-EC0800 (group C: 7.3 ± 3.9, P = 0.0006; group D: 2.0 ± 1.7, P = 0.0002; Fig. 3C). Monitoring of the body weight revealed slight weight gain over time in model I and a largely constant body weight in model II (Fig. 3D and E). In contrast, pronounced loss of body weight was observed in group B mice of both tumor models (which received PMXther only). Importantly, the average survival time was increased 75–100% in group D mice from both tumor models compared to group A mice (Table 3).

Table 3.

Results of the therapy studies with KB and IGROV-1 tumor-bearing mice using 177Lu-EC0800 and/or PMX

Model I: KB tumor xenografts
GroupPMX (mg)Radioactivity (MBq)RTV day 17TGI (%)TGDI4Average survival time (%)
– – 10.0 ± 2.7 – 1.0 – 
2 × 0.8(ther) – 9.8 ± 4.1 2.1 1.2 +22.5% 
1 × 0.4(subther) 1 × 20 8.3 ± 3.5 16.9 1.4 +50.0% 
2 × 0.8(ther) 1 × 20 5.0 ± 2.2 50.4 1.9 +75.0% 
Model II: IGROV-1 tumor xenografts 
Group PMX (mg) Radioactivity(MBq) RTV day 17 TGI (%) TGDI4 Average survival time (%) 
– – 30.0 ± 14.9 0.0 1.0 – 
2 × 0.8(ther) – 27.9 ± 11.8 7.1 1.8 +0% 
1 × 0.4(subther) 1 × 20 7.3 ± 3.9 75.8 2.6 +63.0% 
2 × 0.8(ther) 1 × 20 2.0 ± 1.7 93.2 4.0 +100% 
Model I: KB tumor xenografts
GroupPMX (mg)Radioactivity (MBq)RTV day 17TGI (%)TGDI4Average survival time (%)
– – 10.0 ± 2.7 – 1.0 – 
2 × 0.8(ther) – 9.8 ± 4.1 2.1 1.2 +22.5% 
1 × 0.4(subther) 1 × 20 8.3 ± 3.5 16.9 1.4 +50.0% 
2 × 0.8(ther) 1 × 20 5.0 ± 2.2 50.4 1.9 +75.0% 
Model II: IGROV-1 tumor xenografts 
Group PMX (mg) Radioactivity(MBq) RTV day 17 TGI (%) TGDI4 Average survival time (%) 
– – 30.0 ± 14.9 0.0 1.0 – 
2 × 0.8(ther) – 27.9 ± 11.8 7.1 1.8 +0% 
1 × 0.4(subther) 1 × 20 7.3 ± 3.9 75.8 2.6 +63.0% 
2 × 0.8(ther) 1 × 20 2.0 ± 1.7 93.2 4.0 +100% 

Combining anticancer therapies is a strategy to broaden the therapeutic index by taking an advantage of additive or synergistic antitumor effects and by reducing undesired side effects. This study addressed the question of whether PMX contributes to the antitumor effect of radiofolates and prevents the risk of radionephropathy. In vitro the clonogenic potential of KB and IGROV-1 tumor cells was reduced upon exposure to 177Lu-EC0800 in a concentration-dependent manner (Fig. 1). Moreover it was proven that this effect was specifically folate receptor mediated. It was more pronounced in KB cells, which express the folate receptor at higher levels than IGROV-1 cells, and it was abolished if the cells were coincubated with excess folic acid to block folate receptor binding of 177Lu-EC0800. Inhibition of cell viability through application of 177Lu-EC0800 was enhanced if cells were coincubated with PMX (Fig. 1). Determination of the combination indices at different drug concentrations revealed that 177Lu-EC0800 and PMX provided synergistic inhibitory effects on the viability of both tumor cell lines. It was observed that incubation of the cancer cells with PMX resulted in an accumulation of the cells in the G1–S boundary or early S phase as previously reported (Supplementary Methods and Fig. S7; refs. 25 and 44). However, exposure of cells to 177Lu-EC0800 showed a cell-cycle arrest in the G2 –M phase, which is a common phenomenon in eukaryotic cells exposed to ionizing radiation (45). However, if the cancer cells were simultaneously exposed to 177Lu-EC0800 and PMX the cell-cycle arrest in G2–M phase was abrogated. Notably, the disruption of the radiation-induced G2-checkpoint by chemotherapeutic agents (e.g., protein kinase inhibitors) was previously shown to sensitize cancer cells to radiation-induced apoptosis and cell death (46–48). This mechanism might also have been responsible for PMX-induced radiosensitization of KB and IGROV-1 cells. An increased apoptotic cell fraction was measured if the cells were treated with 177Lu-EC0800 and PMX compared to the application of each of these therapeutics alone (Supplementary Methods and Fig. S8).

In vivo the preinjection of PMX improved the tissue distribution of 177Lu-EC0800 dramatically by increasing the tumor-to-kidney ratio (Table 1). In an attempt to understand the underlying mechanism, the uptake of 3H-PMX was determined in KB tumors and kidneys of mice. In the kidneys, we found a relatively high uptake 1 hour p.i. of 3H-PMX, which was quickly cleared over time (Supplementary Fig. S5). These findings, together with the fact that PMX has a high affinity for the folate receptor (49), support our hypothesis of a competition among PMX and 177Lu-EC0800 for folate receptor binding sites in the kidneys if PMX was injected about 1 hour before the radiofolate. In the tumor tissue, uptake of 3H-PMX was low but retained over time. It has been reported previously that the tumor uptake of PMX is mediated primarily through carriers such as the reduced folate carrier and the proton-coupled folate transporter, whereas folate receptors play a minor role (50). This may explain the fact that PMX does not compete with the folate receptor–mediated accumulation of 177Lu-EC0800 in tumor xenografts.

For the first time we were able to show in this study that PMX prevents damage to the kidneys by reducing renal accumulation of 177Lu-EC0800. Analysis of plasma parameters and the results of SPECT studies using 99mTc-DMSA consistently confirmed normal kidney function in 177Lu-EC0800-treated mice that had received a subtherapeutic amount of PMX. These findings unambiguously confirmed the beneficial role of PMX to prevent radionephropathy of radiofolate therapy.

Based on the in vitro results showing a synergistic effect of PMX and 177Lu-EC0800 on the viability of tumor cells, it is likely that the anticancer effect of 177Lu-EC0800 would be enhanced by coapplication of therapeutic amounts of PMX (2 × 0.8 mg, corresponding to 80% of the MTD; ref. 16). Therapy studies were conducted with KB (model I) and IGROV-1 tumor-bearing mice (model II) using 177Lu-EC0800 combined with either subtherapeutic (0.4 mg/mouse) or therapeutic doses (e.g., 2 × 0.8 mg) of PMX. In both models, tumor growth delay was observed after application of 177Lu-EC0800. Application of PMXther alone showed only minor inhibitory effects on growth of these tumor types. However, PMXther was able to enhance the anticancer effect of 177Lu-EC0800 against both KB and IGROV-1 tumor xenografts. Also, an increased survival time was achieved if 177Lu-EC0800 and PMX were combined compared to the result obtained with each of these agents applied as monotherapy.

With this study we were able to show the proposed dual effect of PMX in combination with folate receptor targeted radionuclide therapy using 177Lu-EC0800. On one hand, PMX at subtherapeutic and therapeutic amounts effectively reduced renal uptake of 177Lu-EC0800 and therewith prevented long-term radionephropathy (Table 1, Fig. 2). On the other hand, the application of PMXther enhanced the tumor growth delay induced by 177Lu-EC0800. The interplay of the proposed drug combination is absolutely unique. Therefore, we believe that combining PMX with 177Lu-folate therapy warrants further preclinical investigations. Assuming the kidney-reducing effect of PMX could be confirmed also in man, the combined application of therapeutic radiofolates and PMX has a potential translational impact. Such a therapy protocol would be particularly interesting for the treatment of non–small cell lung cancer, which frequently shows folate receptor overexpression and for which PMX is a Food and Drug Administration-approved indication.

No potential conflicts of interest were disclosed.

Conception and design: J. Reber, C. Müller

Development of methodology: J. Reber, C. Müller

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J. Reber, S. Haller, C. Müller

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. Reber, C.P. Leamon, C. Müller

Writing, review, and/or revision of the manuscript: J. Reber, S. Haller, C.P. Leamon, C. Müller

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Reber, S. Haller

Study supervision: C. Müller

The authors thank Prof. R. Schibli for helpful advice with regard to the design of this study and for reviewing the manuscript. The authors are grateful for technical assistance of the experiments by N. Romano.

C. Müller was financially supported by the Swiss National Science Foundation (Ambizione, Grants PZ00P3_121772 and PZ00P3_138834), S. Haller was supported by the Swiss Cancer League (KLS-02762-02-2011), and J. Reber was supported by COST-BM0607 (C08.0026).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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