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In Vitro Cytotoxicity of ²¹¹At-Astatide and ¹³¹I-Iodide to Glioma Tumor Cells Expressing the Sodium/Iodide Symporter

Department of Radiology, Duke University Medical Center, Durham, North Carolina

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION APPENDIX A APPENDIX B REFERENCES

 
The sodium/iodide symporter (NIS) has been identified as an attractive target for cancer therapy. The efficacy of ¹³¹I-iodide for NIS-expressing tumor therapy may be limited by a combination of poor cellular retention and unfavorable physical characteristics (long physical half-life and low linear-energy-transfer [LET] radiative emissions). On the other hand, ²¹¹At-astatide is also transported by NIS and offers several therapeutic advantages over ¹³¹I-iodide due to its physical characteristics (short half-life, high LET -particle emissions). The objective of this study was to directly compare the radiotoxicity of both radionuclides using a NIS-transfected cultured cell model. Methods: Cytotoxicity was determined by colony-forming assays. Also, a first-order pharmacokinetic model was used to simulate the closed compartmental system between the medium and cells. Experimental data were then fitted to this model and used to estimate the transfer coefficients between medium and cells, k_m^c, and between cells and medium, k_c^m. Using the pharmacokinetic model, the cumulated activity concentrations in the medium and cells were calculated. Monte Carlo transport methods were then used to assess absorbed doses from ¹³¹I and ²¹¹At. Results: ²¹¹At-Astatide was significantly more cytotoxic than ¹³¹I-iodide in this closed compartmental system. For ²¹¹At-astatide, absorbed doses per unit administered activity were 54- to 65-fold higher than for ¹³¹I-iodide. Both NIS-expressing and control cells showed increased sensitivity to ²¹¹At over ¹³¹I, with significantly lower D₀ (absorbed dose required to reduce the survival fraction to e^-1) and SF₂ (2-Gy survival fraction) values, highlighting the higher intrinsic cytotoxicity of -particles. However, NIS-independent (nonspecific) binding of ²¹¹At-astatide was higher than that of ¹³¹I-iodide, therefore, yielding a lower absorbed dose ratio between NIS-transfected and -nontransfected cells. Conclusion: Treatment of NIS-expressing cells with ²¹¹At-astatide resulted in higher absorbed doses and increased cytotoxicity per unit administered activity than that observed with ¹³¹I-iodide. These results suggest that ²¹¹At-astatide may be a promising treatment strategy for the therapy of NIS-expressing tumors.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION APPENDIX A APPENDIX B REFERENCES

 
It is now well established that the accumulation of iodide by the thyroid gland is mediated by the sodium/iodide symporter (NIS), an integral membrane protein expressed on the basolateral surface of the thyrocyte. Iodide is transported across the cell membrane against a concentration gradient in a sodium-dependent manner, in a process electrochemically coupled to the energy-dependent Na⁺/K⁺-adenosinetriphosphatase (1). The cloning of the NIS gene has allowed investigations into the effect of plasmid- or viral-mediated NIS expression on the iodide-concentrating capacity in a variety of thyroid and nonthyroid cell types (2). These studies have indicated that NIS transgene expression, under the control of a variety of promoters, confers iodide uptake capacity in a wide range of cell lines, allowing the possibility of radioiodide therapy for many tumor types. However, several of these studies also report that the efflux of iodide from NIS-transfected cells, including anaplastic thyroid cells, is extremely rapid (3–6). This observation is of fundamental importance for any endoradiotherapeutic strategy, as radiation-absorbed dose (and thus treatment efficacy) is a function of the cellular residence time and physical half-life (t_1/2) of the radionuclide (7), with optimal conditions reached when the radiologic t_1/2 matches the biologic t_1/2 (i.e., cellular retention). Dosimetric calculations and subsequent in vivo studies, using a murine xenograft model, have indicated that successful treatment of low-retention NIS-expressing tumor deposits with ¹³¹I-iodide (t_1/2 = 8.04 d) may not possible without the administration of prohibitively high activities of radioiodide (8–10). In addition, the effectiveness of ¹³¹I in curing micrometastatic disease decreases with tumor size, due to the relatively long path length of the ß-particle (11), resulting in an increased probability of disease recurrence.

Astatine (astatide), the heaviest of the halogens, exhibits a pronounced accumulation in the thyroid gland in halide form and in the stomach (12). We have previously demonstrated NIS-mediated accumulation of astatide, with uptake and efflux characteristics almost identical to those of iodide (4). Fortunately, ²¹¹At has a physical t_1/2 of 7.214 h, which closely matches the biologic t_1/2 reported for iodide in a variety of NIS-expressing xenograft models (9,13,14), unlike the physical t_1/2 of ¹³¹I. In addition, the decay of ²¹¹At results in the emission of -particles with a mean linear energy transfer (LET) of 97 keV/µm. This value, representing the ionizing energy deposited by the particulate emission per unit distance, is approximately 500 times that of the therapeutic ß-emitting radionuclides ⁹⁰Y and ¹³¹I. Upon interaction with DNA, this high density of ionization events result in a high incidence of generally irreparable double-strand DNA breaks, which accounts for the extreme radiotoxicity of this radionuclide (15). Also, the short range of ²¹¹At -particles in tissue (maximum range, 70 µm) makes such emissions more effective for the sterilization of micrometastatic tumor clusters than ß-particles. Another advantage of -particle therapy is that cell survival is independent of dose rate, which is of particular significance in relation to the rapid uptake and efflux kinetics of radiohalides in NIS-expressing cells lacking an organification mechanism.

This article describes in vitro studies directed at evaluating the hypothesized cytotoxic advantages of ²¹¹At-astatide over ¹³¹I-iodide for the treatment of NIS-expressing cells lacking a halide organification mechanism. Experiments were performed using the human glioma cell line UVW, transfected with human NIS complementary DNA (cDNA) (called NIS6 hereafter), using the parental cell line (UVW) as a negative control. The sensitivity of each cell line to external beam irradiation was established by clonogenic assay, allowing comparison of intrinsic radiosensitivity in both cell lines and also of the cellular sensitivity to both low and high LET particle-emitting radionuclides. Sensitivity of both cell lines to varying activity concentrations of ¹³¹I-iodide and ²¹¹At-astatide was also established by colony-forming assay. A pharmacokinetic model was derived to describe the closed compartmental system between the medium and cells. Experimentally derived kinetic data were then fitted to this model and used to estimate transfer coefficients between medium and cells. Using the pharmacokinetic model, the cumulated activity concentrations in the medium and cells were calculated. Monte Carlo transport methods were then used to assess absorbed doses from ¹³¹I and ²¹¹At, which were correlated with the radionuclide sensitivity data, allowing the determination and comparison of radiobiologic parameters for both cell lines to each irradiation modality.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION APPENDIX A APPENDIX B REFERENCES

 
Cell Culture
The human glioma cell line UVW (European Collection of Cell Cultures no. 86022703) was transfected with cDNA encoding the human NIS gene to create the NIS6 cell line with a method that has previously been described (4). Cells were maintained in Eagle’s minimal essential medium (Gibco), supplemented with 10% fetal bovine serum (Hyclone), penicillin/streptomycin (100 U/mL), and fungizone (2 mg/mL) (Gibco). Cells were cultured at 37°C in a 5% CO₂ atmosphere. Transfected cells were maintained in geneticin G-418 sulfate (0.5 mg/mL) (Gibco) in addition to the conditions described above.

External Beam Irradiations
Cells were trypsinised, counted, and resuspended in sterile tubes containing 5 mL fresh medium to a final concentration of 5 x 10⁵ cells per milliliter. Irradiations (0–9 Gy) were performed using a 6-MV external photon beam accelerator at a dose rate of 0.5 Gy-min^-1 with the appropriate bolus to ensure full buildup of the radiation beam. Cells were plated for colony-forming assay in 6-well plates (growth area, 9.6 cm²) and grown at 37°C in a 5% CO₂ atmosphere for 7–10 d. Colonies were then stained and counted, with colonies comprising 50 or more cells scored as viable.

Radionuclides
The radionuclide ²¹¹At was produced at the Duke University Medical Center CS-30 cyclotron via the ²⁰⁹Bi(, 2n)²¹¹At reaction using the MIT-1 internal target system as previously described (12). ²¹¹At was distilled from the molten bismuth target and trapped in a cooled condenser. It was then isolated from the condenser by washing with approximately 1 mL phosphate-buffered saline (PBS), pH 7.4. After isolation, Na₂SO₃ (Mallinckrodt) was added to a final concentration of 2 x 10^-4 mol/L, to minimize the formation of higher oxidation state species. Sodium ¹³¹I-iodide (no carrier added) was purchased from Dupont–New England Nuclear.

Pharmacokinetics
Experiments were carried out to assess the uptake and efflux pharmacokinetics of radiohalides. Cells were plated in 24-well plates at approximately 5 x 10⁴ cells per well and allowed to attach overnight. For radionuclide uptake curves, cells were incubated for 1–30 min in wells with 0.5 mL PBS containing either ²¹¹At-astatide (100 kBq/mL) or ¹³¹I-iodide (50 kBq/mL). ²¹¹At-astatide uptake and ¹³¹I-iodide uptake were terminated by the removal of the radiohalide-containing incubation buffer, followed by 3 rapid washes in ice-cold PBS. Cells were then solubilized by the addition of 0.5 mL lysis buffer (0.1 mol/L NaOH, 1% sodium dodecyl sulfate, 2% Na₂CO₃) and assessed for radioactivity using an LKB 1282 -counter (LKB), in dual-channel mode. For radiohalide efflux studies, cells were plated as described above in PBS containing ²¹¹At-astatide (100 kBq/mL) and ¹³¹I-iodide (50 kBq/mL) for 30 min. Radioactive incubation buffer was then removed, and cells were incubated in PBS containing 1 x 10^-3 mol/L thiocyanate ion (SCN^-) to inhibit the reuptake of radiohalide for the various time intervals to ensure the same conditions used in the colony-forming assay. Cells were then solubilized in lysis buffer and assessed for radioactivity using a -counter.

To assess variation in total uptake as a function of cell density (number of cells per unit volume d_c), cells were plated at various cell numbers between 1 x 10⁴ and 5 x 10⁵ per well and incubated as described above in 0.5 mL PBS containing either ²¹¹At-astatide (100 kBq/mL) or ¹³¹I-iodide (50 kBq/mL) for 30 min. Cells were then washed and counted for radioactivity with exact cell numbers determined with a hemocytometer. In this manner, we were able to estimate the uptake fraction as a function of cell density.

Cell Survival Fraction Assay
UVW and NIS6 cells were seeded in 24-well plates and allowed to attach for 24 h. Culture medium was then aspirated and replaced with fresh medium containing ¹³¹I-iodide (0–5 MBq/mL) or ²¹¹At-astatide (0–50 kBq/mL). For the cell survival fraction assay, cells were incubated for 30 min at 37°C in a 5% CO₂ atmosphere. The total number of cells per well was determined by hemocytometer counting to assess the cell density d_c. Cells were then given 3 rapid washes in PBS, trypsinized, and plated for colony-forming assay as described above. After 10–14 d, colonies were stained and counted, with colonies comprising 50 or more cells scored as viable. Absorbed dose calculations were then carried out to assess the survival fraction as a function of absorbed dose and estimate the radiotoxicity of ²¹¹At-astatide and ¹³¹I-iodide.

Pharmacokinetic Model of Halide Distribution
Figure 1 illustrates a closed system describing the distribution of halide anion between medium and cells, and the differential equations that describe this closed system, following Michaelis–Menten kinetics, are given by:

(Eq. 1)

where n_m is the average molar concentration of the nuclide in the medium; n_c is the average molar concentration in a cell (mol/L); V_max is the theoretic maximum transfer rate (mol/L s^-1); and K_m is the Michaelis–Menten constant for NIS in this system (mol/L); v_c is the average volume of an adherent cell (cm³); d_c = N_c/V_T is the cell density (cell cm^-3); and N_c is the total number of cells. We have previously estimated the K_m for NIS in this system as 35 µmol/L with respect to iodide (4) but were unable to obtain a similar estimate using astatide due to the absence of a stable astatine isotope. However, we have previously demonstrated that astatide competes equally with iodide in this system (16) and, therefore, assume that the K_m value for astatide is similar to that of iodide.

View larger version (9K): [in this window] [in a new window]   FIGURE 1. Compartmental model describing distribution of NIS substrate anions between medium and cell.

(Eq. 2)

where k_m^c = V_max/K_m is the transfer constant from the medium to a cell (s^-1) where the net transfer rate is given by V_maxd_cv_c/K_m, and k_c^m is the transfer constant from a cell to the medium (s^-1); N_c is the total number of cells; and V_c = N_cv_c is the volume of all cells (cm³); V_T = V_m + V_c where V_T is the total volume of the system (cm³); and V_m is the volume of the incubation medium (cm³). During uptake (u), the initial conditions are n_m⁰ = cte, n_c^u = 0, and by conservation of mass V_mn_m⁰ = V_mn_m^u + N_cv_cn_c^u, where d_c^u = N_c/V_T^u is the cell density and V_T^u is the volume of the system during the uptake period; thus, the solutions for n_m^u and n_c^u are:

(Eq. 3)

where the decay-corrected fractional cell uptake F(d_c) as a function of cell density d_c for an incubation period t_u is given by:

(Eq. 4)

which tends to equilibrium conditions when t thus, F(d_c) d_cv_ck_m^c/(d_cv_ck_m^c + k_c^m). This expression is used to estimate the values of k_m^c and k_c^m by means of nonlinear regression analysis from experimental observations. The solutions during efflux (e) for n_m^e and n_c^e after an incubation period t_u and removal of the medium and dilution to a new cell density d_c^e = N_c/V_T^e are:

(Eq. 5)

where V_T^e is the total volume of system during efflux; n_c⁰ = n_c^u(t_u) is the initial molar concentration of cells; and by conservation of mass N_cv_cn_c⁰ = V_mn_m^e + N_cv_cn_c^e any time afterwards. The activity concentration in the medium a_m and in cells a_c are then given as:

(Eq. 6)

where N_A is Avogadro’s number; _m and _c are the densities of the medium and cells, respectively; and is the physical decay constant for the radionuclide in consideration. The cumulated activity concentration in the medium ã_m and in the cells ã_c are then given as:

(Eq. 7)

where ã_m^u and ã_c^u are the cumulated activity concentration in the medium and a cell during uptake, respectively; and ã_m^e and ã_c^e are the corresponding cumulated activity concentrations for the medium and cells during efflux, respectively. The expressions for ã_m^u, ã_m^e, ã_c^u, and ã_c^e are given in Appendix A. The total cumulated activity per cell is then given as:

(Eq. 8)

where v_c is the average volume of a cell. Because this compartmental model is a closed system, the residence time of the radionuclide in the medium and cells during uptake is given by ^u = _m^u + _c^u = ^-1(1 - e^-t_u), and during efflux as ^e = _m^e + _c^e = ^-1d_cv_c(1 - d_cv_c)^-1a_c⁰/a_m⁰, where a_c⁰ is the cell activity concentration after an incubation period t_u. A glossary of the terminology used is given in Appendix B.

Small-Scale Dosimetry of ¹³¹I-Iodide
Dosimetry of ¹³¹I-iodide was carried out using the EGS4 Monte Carlo transport code, which was estimated using the geometry utilized in the in vitro assays (17–19). The cell nucleus was considered to be the target in these calculations. UVW and NIS6 cells were stained using Cyto16 (Molecular Probes), which binds to nucleic acids. Phase-contrast and fluorescent images of cells were obtained and superimposed and used as a model for these calculations (Fig. 2). In these dosimetric calculations, 2 sources were identified: the medium where the activity was diluted, and the attached cells themselves, which take up the activity from the medium. The images provided the basic cell morphology that was used in these calculations. The nucleus of adherent cells had a semiellipsoid geometry. However, for simplification purposes, we used a spheric model of the cell and nucleus, where the volume of the semiellipsoid nucleus was that of the sphere nucleus. Thus, the estimated average cell and nuclear diameter used in these studies were 16 and 10 µm, respectively.

View larger version (137K): [in this window] [in a new window]   FIGURE 2. Superimposition of phase-contrast and fluorescent images of adherent cells labeled with Cyto16 nuclear stain to show nuclear and cell morphology of UVW and NIS6 cell lines. Colony-forming assays were performed under similar conditions. Images were used to derive cellular and nuclear geometry, which subsequently was used in calculations to determine cumulated absorbed dose. Strongly green regions identify cell nuclei.

(Eq. 9)

where ã_m is the cumulated activity concentration in the medium and _c is the average cumulated activity in a cell (Bq s).

Microdosimetry of ²¹¹At-Astatide
Microdosimetry of ²¹¹At-astatide was carried out using an -particle Monte Carlo transport code as described elsewhere (20). The cell nucleus was considered to be the target in these calculations. The total absorbed dose is then given as:

(Eq. 10)

where D is average absorbed dose (Gy); ã_m is the total cumulated activity in the medium (Bq s g^-1); h_m^c is the average number of hits from medium to cells (hits g Bq^-1 s^-1); _1m^c is the average specific energy per event from medium to cells (Gy hit^-1); _c is the cumulated activity per cell (Bq s cell^-1); h_c^c is the average number of hits from cells to cells (hits Bq^-1 s^-1); and _1c^c is the average specific energy per event from cells to cells (Gy hit^-1). The values for h_m^c, h_c^c, _1m^c, and _1c^c were estimated as a function of cells per unit area to account for cross fire among cells (data not shown). As an example, a cylindric well (diameter, 16.5 mm) containing 1.4 x 10⁵ adherent cells resulted in a surface cell density of 655 cells mm^-2, and the corresponding conversion factors for _1m^c, _1c^c, h_m^c, and h_c^c were 0.224 Gy hit^-1, 0.114 Gy hit^-1, 4.456 x 10^-9 hit cm³ Bq^-1 s^-1, and 0.407 hit Bq^-1 s^-1, respectively.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION APPENDIX A APPENDIX B REFERENCES

 
External Beam Radiation
Data obtained from the external beam radiation dose/survival assay were fitted using the linear quadratic model of cell survival SF = exp(-D - ßD²), where SF is the surviving fraction and D is the absorbed dose (Fig. 3). Using the statistical analysis software SAS, we carried out the model fitting to assess the parameters and ß. The estimated 2-Gy survival fraction, SF₂, for UVW and NIS6 cell lines was 0.71 (0.62–0.83, 95% confidence interval [CI]) and 0.61 (0.53–0.70, 95% CI), respectively. There was no statistically significant difference for the SF₂ between the 2 cell lines based on a t test with equal variances (P = 0.3) (SAS).

View larger version (18K): [in this window] [in a new window]   FIGURE 3. Survival fraction of NIS6 and UVW as function of external beam radiation-absorbed dose. Points represent average of triplicate determinations and corresponding SD. External beam dose rate was 0.5 Gy min-1. The estimated 2-Gy SF (95% confidence interval [CI]) was 0.71 (0.62–0.83, 95% CI) and 0.61 (0.53–0.70, 95% CI) for UVW and NIS6, respectively. There was no statistically significant difference between NIS6 and UVW cells lines (P = 0.296).

View larger version (21K): [in this window] [in a new window]   FIGURE 4. (A) Fractional cell-associated activity (FCAA) of 211At-astatide for UVW and NIS6 cell lines during efflux as function of time. Similar curves were generated using 131I-iodide (data not shown). Data represent means ± SD from at least 2 triplicate determinations. (B) Fractional cell uptake F(dc) as function of cell density for NIS6 and UVW cell lines for incubation period of 30 min for 131I-iodide and 211At-astatide. Data represent means of 3 determinations. Experimental data were fitted to theoretic expression for F(dc) to estimate transfer constants kmc and kcm shown in Table 1. Uptake of 131I-iodide by NIS6 cells was approximately 60-fold higher than that of UVW (nonspecific binding). Total uptake of 211At-astatide to NIS6 cells was approximately 27-fold higher than that of UVW. Nonspecific binding of 211At-astatide was approximately 4-fold higher than that of 131I-iodide.

View larger version (23K): [in this window] [in a new window]   FIGURE 5. Calculated fractional cell-associated activity for UVW and NIS6 cell lines for 211At. Cell density during uptake was 5 x 105. Cell density during efflux was reduced by factor of 1 x 103 cells cm-3, reflecting conditions used during colony-forming assay. However, when cell density was left constant, a different efflux profile is observed as indicated in Equation 5. Uptake period was 30 min. Calculations were based upon estimated kmc and kcm coefficients that were derived from our experimental studies (Table 1). Area under the curve was calculated to assess cumulated activity concentration in cells and corresponding absorbed dose.

View larger version (18K): [in this window] [in a new window]   FIGURE 6. Survival fraction as function of activity concentration in medium for incubation period of 30 min. Survival fraction of NIS6 and UVW for 131I (A) and for 211At (B), respectively. Data represent means ± SD of at least 3 triplicate determinations. Therapeutic efficiency and toxicity ratios are given in Table 2.

View larger version (19K): [in this window] [in a new window]   FIGURE 7. Survival fraction of UVW and NIS6 cell lines as function of absorbed dose from 211At-astatide (A) and 131I-iodide (B). Estimated D0 of UVW and NIS6 for 211At-astatide was 0.27 (0.19–0.46, 95% CI) Gy and 0.28 (0.26–0.30, 95% CI) Gy, respectively. 2-Gy SF was 6.3 x 10-4 (3.1 x 10-5 - 1.3 x 10-2, 95% CI) and 7.4 x 10-4 (4.2 x 10-4 - 1.3 x 10-3, 95% CI), respectively. D0 of UVW and NIS6 for 131I-iodide was 5.27 (2.5–6.4, 95% CI) Gy and 4.31 (3.0–7.6, 95% CI) Gy, respectively, and 2-Gy SF for UVW and NIS6 was 0.68 (0.02–20.4, 95% CI) and 0.66 (0.35–1.22, 95% CI), respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION APPENDIX A APPENDIX B REFERENCES

We have previously reported that plasmid-mediated cellular expression of the NIS confers the ability to accumulate ²¹¹At-astatide in addition to iodide (16). Here we describe clonogenic survival experiments designed to directly compare the radiotoxicity of ¹³¹I-iodide and ²¹¹At-astatide on a cultured cell model and a pharmacokinetic model, which was used to estimate absorbed dose for each radionuclide in UVW and NIS6 cell lines. To determine whether expression of the NIS protein, or selection of NIS-positive clones, resulted in an alteration of cellular radiosensitivity, a comparison of the radiation sensitivity of UVW and NIS6 cell lines was performed by examining the clonogenic survival of both cell lines in response to external photon beam irradiation. The SF₂ values obtained for both cell lines were not significantly different, 0.71 (0.62–0.83, 95% CI) and 0.61 (0.53–0.70, 95% CI) for UVW and NIS6, respectively (Fig. 3), indicating that expression of NIS has no effect on intrinsic radiosensitivity. The effect of activity concentration of both ¹³¹I-iodide and ²¹¹At-astatide on UVW and NIS6 cells was determined by colony-forming assay. An activity concentration-dependent decrease in clonogenic survival was observed in both cell lines with both radionuclides (Fig. 6), with NIS6 cells having increased sensitivity to each radionuclide when compared with control UVW cells. Using these data, values were calculated to indicate the activity concentration required to cause a reduction in survival by 1 natural logarithm (A₀), using the incubation conditions described above. The ratio of A₀ values between NIS-negative (UVW) and NIS-expressing (NIS6) cells (ratio 1, Table 2) was 13.2 for ¹³¹I-iodide and 11.5 for ²¹¹At-astatide (Table 2). The similarity of these values reflects the similar pharmacokinetics of cellular iodide and astatide association in this model system. The smaller therapeutic differential calculated for ²¹¹At may reflect the higher degree of nonspecific binding observed with aqueous astatide solutions (16,21). For this study, we define the terms specific binding as NIS-mediated, perchlorate-sensitive cellular uptake and nonspecific binding as non-NIS-mediated, perchlorate-insensitive cellular uptake of radiohalide. Detailed examination of the properties of specific and nonspecific uptake of both ¹³¹I-iodide and ²¹¹At-astatide in this model system have previously been reported (16). Comparison of A₀ values for each radionuclide indicated an enhanced sensitivity to ²¹¹At of 686-fold in UVW cells and 598-fold in NIS6 cells compared with ¹³¹I. Similar enhancements in sensitivity (up to 1,400-fold) were observed previously when comparing the cytotoxicity of ²¹¹At- and ¹³¹I-labeled benzylguanidine compounds in neuroblastoma cells (22). These data, and our own observations, are in accordance with calculations that predict a 1,200-fold increase in cytotoxicity for -particles on a single cell basis (23). It should be noted that A₀ values, expressed in units of activity per unit volume (Bq mL^-1) only indicate the relative toxicity of each radionuclide under the experimental conditions used for these studies. Calculation of cumulated absorbed dose from each radionuclide also requires that radiologic t_1/2 and system geometry be taken into account. Absorbed dose calculations indicate that administration of ²¹¹At-astatide results in an increase (53.9- to 64.9-fold) over ¹³¹I-iodide in absorbed dose per administered activity concentration (Table 3).

The rapid efflux of iodide from NIS-expressing cells lacking an organification mechanism has been observed in several cell lines and rodent xenograft models (4,9,24). The biologic t_1/2 of iodide in xenograft tumors is generally longer than that observed in 2-dimensional cultured cell models, presumably due to the continual reuptake of iodide by the tumor. Although this has allowed effective demonstrations of the potential of NIS expression for tumor imaging using NIS substrate molecules such as ¹²³I^-, ¹²⁴I^-, and ^99mTcO₄^- (25–27), the successful sterilization of such tumors with ¹³¹I is likely to require the administration of prohibitively large quantities of radioactivity (9). However, studies performed using an in vivo prostate model have demonstrated, given sufficiently high administered radioactivity, that nonthyroid tumor xenografts with transgene-mediated NIS expression can be successfully treated with ¹³¹I (8). This demonstrates the principle that NIS-expressing tumor deposits can be successfully targeted and treated with an ionic radiohalide in vivo and that the therapeutic effect is dependent on delivering a sufficient radiation dose to tumor.

-Particle-emitting radionuclides have considerable potential as cytotoxic agents in the treatment of malignant disease. The short range and high LET -particle makes their use particularly suited to the treatment of micrometastatic and minimal residual disease, where the radiation dose to tumor is normally limited by the dose delivered to surrounding normal tissues. Tumor-specific targeting of -particles can result in highly focal irradiation, high tumor absorbed doses, and minimization of surrounding normal tissue toxicity. The first clinical trial with an ²¹¹At-labeled therapeutic antibody (antitenascin monoclonal antibody 81C6) is currently underway, with encouraging initial observations of high radiation doses to the target area and low doses to surrounding normal tissue (28).

The data presented in this article indicate that the administration of ²¹¹At-astatide results in a significant increase (55- to 65.6-fold) over ¹³¹I-iodide in absorbed dose per administered activity concentration (Table 3). However, the nonspecific association of ²¹¹At-astatide with cells lacking NIS expression also contributes to the dose to these cells, resulting in a lower therapeutic differential than that observed with ¹³¹I-iodide at the same administered activity concentration (12.0 and 14.5, respectively) (Table 3).

A previous study evaluating the uptake of ²¹¹At-astatide by NIS transgene-expressing thyroid tumor cells (21) presented several observations similar to our current and previously published data (16). Uptake and efflux kinetics of both ²¹¹At-astatide and ¹³¹I-iodide are similar in each cultured cell model system, and uptake of both radiohalides was sensitive to excess perchlorate (ClO₄^-) and iodide (I^-) concentration, demonstrating that ²¹¹At-astatide is transported by NIS with an efficiency approaching that of iodide. Using a murine xenograft model, the intraperitoneal administration of ²¹¹At-astatide resulted in a 14.4-fold increase in tumor absorbed dose over that predicted for ¹³¹I-iodide, with similar biologic half-lives observed for both anions (21). Although the difference in pharmacokinetics between in vitro and in vivo models precludes a direct comparison of absorbed dose, the conclusions of Petrich et al. (21), in accordance with this study, indicate that the administration of ²¹¹At-astatide results in a significantly higher absorbed dose to NIS-expressing cells than ¹³¹I-iodide per unit administered activity.

The pharmacokinetic model presented in this study describes the phenomenologic distribution of halide anions in a closed system, which accurately describes the conditions used in the cultured cell clonogenic assays described above. It is also possible to use this model to analyze the pharmacokinetics of other NIS substrate anions, such as ¹⁸⁸ReO₄^- or ^99mTcO₄^-, under similar experimental conditions. In addition, it can also be adapted, given appropriate experimental observations, to predict the effect of alteration of many system parameters (e.g., level of NIS expression, heterogeneous cell populations, pharmacologic inhibitors of halide efflux) on both pharmacokinetic distribution and cumulated absorbed dose. We intend to further develop this model to examine the pharmacokinetics of NIS-mediated anion transport in a 3-dimensional cultured cell system and, in combination with biodistribution data, to develop a model describing the pharmacokinetics in a dynamic extracellular system similar to that encountered in vivo, which will aid in the future design of NIS-based therapeutic strategies.

Given the similar pharmacokinetics of ¹³¹I-iodide and ²¹¹At-astatide in our model system, our observation of a higher absorbed dose using ²¹¹At-astatide can be explained by a combination of the higher energy -particle (5.87 MeV) versus ¹³¹I ß-particle emission (0.6 MeV) as well as the shorter physical t_1/2 of ²¹¹At (7.2 h vs. 192 h for ¹³¹I). Similarly, a study into the potential of ¹⁸⁸Re (in the form ¹⁸⁸ReO₄^-) to sterilize NIS-expressing cells calculated a 4.5-fold higher absorbed dose, when compared with ¹³¹I (14). This enhancement is likely due to a combination of factors: ¹⁸⁸Re emits ß-particles with a higher energy than ¹³¹I (2.12 vs. 0.61 MeV), and ReO₄^- had higher tumor uptake than I^- in a NIS-expressing xenograft model (14). However, much of the increase in absorbed dose results from the comparatively short physical t_1/2 (16.7 h) of ¹⁸⁸Re (14). These findings are of particular significance in relation to cell types lacking a peroxidase-based organification mechanism, as these cells generally exhibit a very short cellular retention of iodide (3,4), and matching of both the physical and biologic half-lives of the radioisotope is desirable for maximal radiobiologic effect.

In vivo, tumor cells may show a heterogeneous expression of NIS that can be predicted to result in a heterogeneous cumulated activity distribution with the tumor mass. However, depending on the range of the radiations emitted by the radionuclide used for therapy, there is a potential for cross fire among tumors cells. Although the range of -particles is significantly shorter than that of ß-particles, a degree of cross fire can be predicted. The proportion of cells receiving a highly toxic absorbed dose from ²¹¹At-astatide will therefore depend on a combination of the overall transfection efficiency, the spatial distribution of transgene expression and radionuclide within the tumor, and the degree of cross fire. The use of cultured cell models, such as the one described here, affords the opportunity for detailed analysis of these issues and may provide the basis for a clinical implementation of transgene-mediated radionuclide therapy in the future. It is also important to note that there are some tumor types, such as thyroid and invasive breast carcinoma, that may express sufficient NIS to facilitate ²¹¹At-astatide therapy without the need for transgene-mediated expression.

The use of ionic ²¹¹At-astatide for the treatment of thyroid carcinoma has previously been investigated in murine xenograft models, with high tumor uptakes reported for more differentiated thyroid tumor cells, which generally retain a higher level of NIS expression than anaplastic thyroid tumors (29–31). A study comparing the uptake of ¹²⁵I-iodide and ²¹¹At-astatide in nude mice bearing human fetal thyroid and human malignant thyroid xenografts observed that the human tissue accumulated both halides with similar kinetics, suggesting that ²¹¹At-astatide may be an alternative radionuclide to ¹³¹I-iodide for the treatment of thyroid carcinoma (29). Another study examining the comparative transport of ¹²⁵I-iodide and ²¹¹At-astatide using normal porcine thyrocytes in a polarized system also observed similarities in the basal membrane (i.e., NIS mediated) transport of both radiohalide anions (32). Interestingly, this study also reported the perchlorate- and ouabain-insensitive basal membrane transport of astatide, by normal thyrocytes, suggesting the possibility of another, NIS-independent astatide uptake mechanism in these cells. Further elucidation of this transport mechanism may provide some explanation as to the differences in biodistribution of astatide and iodide, in addition to providing another means of targeting astatine to tumor cells.

The biodistribution of systemically administered ²¹¹At-astatide, though similar to that of iodide, has several clinically relevant differences. In particular, a significantly higher uptake than iodide in both lung and spleen tissues is observed (12,33). At present, clinical trials of ²¹¹At-astatide for the treatment of thyroid carcinoma have not been initiated, possibly because of concerns over radiotoxicity in these tissues or due to the limited availability of the radionuclide. Previous investigations have revealed that pharmacologic intervention may modify the level of astatide uptake in normal tissues (12), and further studies into the possible mechanisms involved in the lung and spleen retention of astatide are currently underway. Although lung and spleen uptake currently precludes the systemic administration of ²¹¹At-astatide, combined gene transfer/radionuclide treatment of compartmentalized tumors such as those of brain, bladder, and ovary may be possible with minimal involvement of the systemic circulation. Further advances in the understanding of the mechanisms of astatine uptake and retention in lung and spleen will aid greatly in the clinical implementation of systemic ²¹¹At-astatide therapy for tumors with both endogenous and gene transfer–mediated NIS expression.

	CONCLUSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION APPENDIX A APPENDIX B REFERENCES

 
The data presented in this article demonstrate that ²¹¹At-astatide has potential as an alternative radionuclide to ¹³¹I-iodide for the treatment of NIS-expressing tumor cells. The direct comparison of ²¹¹At and ¹³¹I in this closed system highlights the enhancements in absorbed dose and radiotoxicity of high LET -particles over low LET ß-particle irradiation. ²¹¹At-Astatide administration results in increased cytotoxicity (686- to 598-fold) and higher absorbed doses (55- to 65.5-fold) per unit administered activity when compared with ¹³¹I-iodide. In addition, the physical t_1/2 of ²¹¹At is closely matched to the radionuclide residence time observed in nonthyroid tumor types, providing a further therapeutic advantage over ¹³¹I. These observations imply that some of the limitations of ¹³¹I administration for the therapy of endogenous or transgene-mediated NIS-expressing tumors can be overcome. The pharmacokinetic model presented here is a valuable tool in predicting the influence on absorbed dose resulting from modulation of many experimental system parameters. Further development of this model to describe a 3-dimensional tumor within a dynamic environment will aid in the elucidation of the relationship between administered radioactivity and resulting absorbed doses in NIS-expressing tumors with different retention characteristics.

	APPENDIX A

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION APPENDIX A APPENDIX B REFERENCES

 
Pharmacokinetics
System description:

The uptake solutions given conservation of mass V_mn_m⁰ = V_mn_m + N_cv_cn_c are:

The efflux solutions given conservation of mass N_cv_cn_c⁰ = V_mn_m + N_cv_cn_c are:

Residence times:

	APPENDIX B

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION APPENDIX A APPENDIX B REFERENCES

 
Glossary
n_m Average halide concentration in medium (mol/L)

n_m^u Average halide concentration in medium during uptake (mol/L)

n_m^e Average halide concentration in medium during efflux (mol/L)

n_c Average halide concentration in cells (mol/L)

n_c^u Average halide concentration in cells during uptake (mol/L)

n_c^e Average halide concentration in cells during efflux (mol/L)

V_max Theoretical maximum halide transfer rate (mol/L s^-1)

K_m Michaelis–Menten constant for NIS (mol/L)

v_c Average volume of a cell (cm^-3)

d_c Average cell density (cell cm^-3)

d_c^u Average cell density during uptake (cell cm^-3)

d_c^e Average cell density during efflux (cell cm^-3)

N_c Total number of cells (cell)

k_c^m Transfer rate between a cell to medium (s^-1)

k_m^c Transfer rate between medium to a cell (s^-1)

V_m Volume of medium (cm³)

V_c Volume of cells (cm³)

V_T Total volume of system (cm³)

V_T^u Total volume of system during uptake period (cm³)

V_T^e Total volume of system during efflux period (cm³)

F(d_c) Fractional cell uptake (unitless)

FCAA Fractional cell-associated activity (unitless)

a_m Average activity concentration in medium (Bq cm^-3)

ã_m^u Average cumulated activity concentration in medium during uptake (Bq s cm^-3)

ã_m^e Average cumulated activity concentration in medium during efflux (Bq s cm^-3)

a_c Average activity concentration in the cell (Bq cm^-3)

ã_c^u Average cumulated activity concentration in a cell during uptake (Bq s cm^-3)

ã_c^e Average cumulated activity concentration in a cell during efflux (Bq s cm^-3)

_c Average cumulated activity in a cell (Bq s)

^u Residence time of system during uptake (s)

^e Residence time of system during efflux (s)

_m^u Residence time of halide in medium during uptake (s)

_c^u Residence time of halide in cells during uptake (s)

_m^e Residence time of halide in medium during efflux (s)

_c^e Residence time of halide in cells during efflux (s)

D Average absorbed dose (Gy)

S(m c) Dose conversion factor from medium (source) to cells (target) (Gy g Bq^-1 s^-1)

S(c c) Dose conversion factor from cells (source) to cells (target) (Gy g Bq^-1 s^-1)

h_m^c Average number of hits from medium (source) to a cell (target) (hit)

h_c^c Average number of hits from cells (source) to a cell (target) (hit)

_1m^c Specific energy per event from medium (source) to a cell (target) (Gy hit^-1)

_1c^c Specific energy per event from cells (source) to a cell (target) (Gy hit^-1)

SF Survival fraction (unitless)

SF₂ 2-Gy survival fraction (unitless)

Physical decay constant of radionuclide (s^-1)

D₀ Absorbed dose required to reduce survival fraction to e^-1 (Gy)

A₀ Activity concentration required to reduce survival fraction to e^-1 (MBq cm^-3)

	ACKNOWLEDGMENTS

 
NIS cDNA was kindly provided by Dr. Sissy M. Jhiang (Ohio State University, Columbus, OH). This work was supported by grants CA42324, CA70164, and CA91927 from the National Institutes of Health and by an AstraZeneca Union Internationale Contre le Cancer Translational Cancer Research Fellowship (American Cancer Society Beginning Investigator 11/2001).

	FOOTNOTES

 
Received Apr. 11, 2003; revision accepted Jul. 22, 2003.

For correspondence or reprints contact: Michael R. Zalutsky, PhD, Department of Radiology, Box 3808, Duke University Medical Center, Durham, NC 27710.

E-mail: zalut001{at}mc.duke.edu

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION APPENDIX A APPENDIX B REFERENCES

 

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