demonstrated consistently lower levels of background radioactivity in non-target tissues, such as the liver and the gastrointestinal tract. In addition to aiding in their prompt elimination *via* the renal-urinary pathway, the small size and hydrophilic nature of the DPR derivatives containing amino acid linkers allowed for the rapid penetration of these conjugates into GRPr-positive target tissues, as evidenced by their comparatively high levels of uptake by both GRPr-expressing pancreatic and tumor tissues. Tumor uptake and retention in T-47D xenografted breast cancer tumors were 2.3-2.4 ± 0.4-0.8% ID/g (Y = GSG) and from 2.4-3.7 ± 0.7-1.8% ID/g (Y = SSS) at 1 hour p.i., respectively.

Fig. 2. 99mTc-(CO)3-(H2O)-DPR-GSG-BBN(7-14)NH2 (A) and 99mTc-(CO)3-(H2O)-DPR-SSS-BBN(7-14)NH2 (B) conjugates.

Fig. 3. microSPECT/MRI/CT imaging of 99mTc-(H2O)3(CO)3-DPR-SSS-BBN(7-14)NH2 after 24 h p.i.

Molecular Imaging of Breast Cancer Tissue *via* Site-Directed Radiopharmaceuticals 291

conditions is very important (Wadas et al., 2007). Increasing the stability of copper complexes *in vivo* has been a major focus in the field of nuclear medicine for some time (Di Bartolo et al., 2001 and 2006; Gasser et al., 2008; Kukis et al., 1993; Voss et al., 2007; Pippin et al., 1991; Geraldes et al., 2000; Wieghardt et al., 1982; van der Merwe et al., 1985; Prasanphanich et al., 2007; Soluri et al., 2003; Maina et al., 2005; Monstein et al., 2006; Sun & Chen, 2007) as the development of stable copper complexes will allow for a variety of

Recent work in the field of copper radiochemistry has produced very promising results. A majority of the work has focused on synthesis of new ligand frameworks to chelate copper in a multi-dentate fashion to prevent loss of the Cu2+ ion under *in vivo* conditions. Crossbridged TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetracetic acid) derivatives, such as 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CB-TE2A) and 1-N-(4 aminobenzyl)-3,6,10,13,16,19-hexa-aza-bicyclo-[6.6.6]eichosane-1,8-diamine (SarAr), have shown increased copper-complex stability *in vivo* versus the TETA and DOTA (1,4,7,10 tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelators (Garrison et al., 2007; Di Bartolo et al., 2001 and 2006). Additionally, derivatives of the triazacyclononane macrocycle have produced improved stability of copper complexes (Prasanphanich et al., 2007). Pippin et al., have shown that complexes of Cu(II) with NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) are more inert towards isotopic exchange with 67Cu than DOTA or TETA (Pippin et al., 1991). Recent work by Gasser, et al., demonstrated a 2-[4,7-bis(2-pyridylmethyl)-1,4,7 triazacyclononan-1-yl] acetic acid (PMCN) bifunctional chelator containing the 64Cu radionuclide (Gasser et al., 2008). Recently, we have reported on the superior microPET imaging quality of NOTA bifunctional chelator over that of the DOTA using 64Cu in a GRPR-positive human prostate cancer mouse model (Prasanphanich et al., 2007). Previous investigations have reported a highly stable complex consisting of NOTA and divalent copper (Kukis et al., 1993; Geraldes et al., 2000; Wieghardt et al., 1982). Also reported are biomolecules conjugated with NOTA and chelating 64Cu. Interestingly, one investigation

NOTA in which one carboxylate arm of the triazacyclononane macrocycle is not involved in the coordination of copper(II) (van der Merwe et al., 1985). This implies that occupation of this non-chelating carboxylate of NOTA, as in our bifunctional NO2A, may not alter the

 We have recently evaluated 64Cu-NO2A-8-Aoc-BBN(7-14)NH2 radiopharmaceutical (Figure 4) to be used as a PET targeting agent for primary or metastatic breast cancer disease (Prasanphanich et al., 2009). In this study, we were able to prepare high specific activity 64Cu-NO2A-8-Aoc-BBN(7-14)NH2 conjugate in very high radiochemical yield and to evaluate the 1) targeting capacity of 64Cu-NO2A-8-Aoc-BBN(7-14)NH2 for GRPr-positive tissues *in vivo*; 2) particular routes of clearance; and 3) extent of retention of

A number of *in vitro* assays were used to evaluate the uptake and affinity of 64Cu-NO2A-8- Aoc-BBN(7-14)NH2 for the GRPr in T-47D human breast cancer cells. Competitive binding displacement assays using 125I-[Tyr4]-BBN(7-14)NH2, as the radioligand were used to quantify the relative binding affinity of Cu-NO2A-8-Aoc-BBN(7-14)NH2 for GRPrs located on the surface of T-47D cells. The concentration of Cu-NO2A-8-Aoc-BBN(7-14)NH2 needed to inhibit 125I-[Tyr4]-BBN(7-14)NH2 binding by 50% was determined to be 7.56 2.23 nM. The rate at which cell-associated radioactivity was internalized within the T-47D cells was also measured. After incubating T-47D cells in media containing 64Cu-NO2A-8-Aoc-BBN(7-

], or [CuCl(NOTA)-], with a pentadentate

copper-based diagnostic and therapeutic strategies.

reported a crystal structure of [CuCl(TACNTA)-

native structure of the Cu-NOTA complex.

radiopharmaceutical in radiosensitive tissues.

Due to each rapid elimination from non-target tissues *via* the renal-urinary excretion pathway and moderate accumulation and retention in tumor tissue, 99mTc-(H2O)(CO)3-DPR-Y-BBN(7-14)NH2 conjugates were evaluated for microSPECT imaging in T-47D tumorbearing SCID mice at 24 hours p.i.(Figure 3). These conjugates produced favorable tumor-tobackground ratios, which allowed for clear visualization of tumor tissue, despite some level of background radioactivity. Although the location and intensity of this background radioactivity fluctuated slightly among these conjugates, the kidneys and the gastrointestinal tract were consistently the predominant sources of radioactivity at the time of imaging. Despite these subtle differences in biodistribution, clear identification of tumor tissue was readily achieved with both derivates, supporting the hypothesis that radiolabeled 99mTc(I)-BBN conjugates may be employed to diagnose GRPr-expressing breast cancers.

#### **5. microPET imaging with copper-64-BBN conjugates**

While a variety of diagnostic imaging techniques exist for identifying breast cancer, lowdose mammography is still considered to be the most accurate and dependable procedure (Prasad & Houserkova, 2007). Still, digital mammography is most accurate in women under 50, with low-density breast, who have either not entered or recently passed through menopause (Pisano et al., 2005). PET imaging using FDG (18F-fluorodeoxyglucose) has been an effective technique for diagnosis of primary breast cancer and benefited from PET imaging devices designed specifically for breast imaging. While FDG-PET was able to identify tumors in the breast, a high false-positive rate, 87%, limits its wide-spread use. (Kaida et al., 2008). Thus there is a need to develop diagnostic modalities with increased specificity for malignant breast tissues and capable of overcoming these limitations. Targeting a unique physiological manifestation of breast malignancy, such as overexpression of an extracellular receptor with a diagnostic radionuclide, might be a procedure capable of imaging tumors of the breast with higher resolution and lower false-positive rates.

Copper-64 is one of few radionuclides possessing physical properties that allow for medical imaging and therapy (Nijsen et al., 2007). The utility of 64Cu (*t*1/2 = 12.7 h, cyclotron generated as 64CuCl2) results from its diverse decay profile. Electron capture, with corresponding gamma emission at 1346 keV, accounts for 41% of the decay profile for 64Cu. This is accompanied by emission of a β particle (40%, 190 keV) and a positron (β+, 19%, 278 keV). The degree of β+ decay is sufficient for PET imaging *in vivo* and has been extensively investigated. Moreover, 67Cu-containing radiopharmaceuticals possess therapeutic potential and provide *in vivo* pharmacokinetic profiles nearly identical to 64Cu-containing drugs (Blower et al., 1996). 67Cu has ideal physical characteristics that are well-suited for radionuclide therapy (β- , 100%, 121 keV, *t*1/2 = 62 h) (Voss, et al., 2007). As such, 64Cu can be used as a dose-determinant indicator in 67Cu-based therapeutic regimens.

While the physical properties of copper are well-suited for use in radiopharmaceuticals, the metabolism and physiological properties of copper in the human body present a challenge to the development and wide-spread use of copper-containing radiopharmaceuticals. Ideally, site-directed radiopharmaceuticals accumulate at the target site quickly with little accumulation in non-target tissues and rapidly clear non-target tissues after administration. Hence, hepatobiliary clearance of radiopharmaceuticals is much less desirable to renal/urinary clearance. Since the liver is the anatomical location of copper metabolism, the stability of the copper chelate in a 64Cu-containing radiopharmaceutical under physiological

Due to each rapid elimination from non-target tissues *via* the renal-urinary excretion pathway and moderate accumulation and retention in tumor tissue, 99mTc-(H2O)(CO)3-DPR-Y-BBN(7-14)NH2 conjugates were evaluated for microSPECT imaging in T-47D tumorbearing SCID mice at 24 hours p.i.(Figure 3). These conjugates produced favorable tumor-tobackground ratios, which allowed for clear visualization of tumor tissue, despite some level of background radioactivity. Although the location and intensity of this background radioactivity fluctuated slightly among these conjugates, the kidneys and the gastrointestinal tract were consistently the predominant sources of radioactivity at the time of imaging. Despite these subtle differences in biodistribution, clear identification of tumor tissue was readily achieved with both derivates, supporting the hypothesis that radiolabeled 99mTc(I)-BBN conjugates may be employed to diagnose GRPr-expressing breast cancers.

While a variety of diagnostic imaging techniques exist for identifying breast cancer, lowdose mammography is still considered to be the most accurate and dependable procedure (Prasad & Houserkova, 2007). Still, digital mammography is most accurate in women under 50, with low-density breast, who have either not entered or recently passed through menopause (Pisano et al., 2005). PET imaging using FDG (18F-fluorodeoxyglucose) has been an effective technique for diagnosis of primary breast cancer and benefited from PET imaging devices designed specifically for breast imaging. While FDG-PET was able to identify tumors in the breast, a high false-positive rate, 87%, limits its wide-spread use. (Kaida et al., 2008). Thus there is a need to develop diagnostic modalities with increased specificity for malignant breast tissues and capable of overcoming these limitations. Targeting a unique physiological manifestation of breast malignancy, such as overexpression of an extracellular receptor with a diagnostic radionuclide, might be a procedure capable of imaging tumors of the breast with higher resolution and lower false-positive

Copper-64 is one of few radionuclides possessing physical properties that allow for medical imaging and therapy (Nijsen et al., 2007). The utility of 64Cu (*t*1/2 = 12.7 h, cyclotron generated as 64CuCl2) results from its diverse decay profile. Electron capture, with corresponding gamma emission at 1346 keV, accounts for 41% of the decay profile for 64Cu.

keV). The degree of β+ decay is sufficient for PET imaging *in vivo* and has been extensively investigated. Moreover, 67Cu-containing radiopharmaceuticals possess therapeutic potential and provide *in vivo* pharmacokinetic profiles nearly identical to 64Cu-containing drugs (Blower et al., 1996). 67Cu has ideal physical characteristics that are well-suited for

While the physical properties of copper are well-suited for use in radiopharmaceuticals, the metabolism and physiological properties of copper in the human body present a challenge to the development and wide-spread use of copper-containing radiopharmaceuticals. Ideally, site-directed radiopharmaceuticals accumulate at the target site quickly with little accumulation in non-target tissues and rapidly clear non-target tissues after administration. Hence, hepatobiliary clearance of radiopharmaceuticals is much less desirable to renal/urinary clearance. Since the liver is the anatomical location of copper metabolism, the stability of the copper chelate in a 64Cu-containing radiopharmaceutical under physiological

used as a dose-determinant indicator in 67Cu-based therapeutic regimens.

particle (40%, 190 keV) and a positron (β+, 19%, 278

, 100%, 121 keV, *t*1/2 = 62 h) (Voss, et al., 2007). As such, 64Cu can be

**5. microPET imaging with copper-64-BBN conjugates**

rates.

This is accompanied by emission of a β-

radionuclide therapy (β-

conditions is very important (Wadas et al., 2007). Increasing the stability of copper complexes *in vivo* has been a major focus in the field of nuclear medicine for some time (Di Bartolo et al., 2001 and 2006; Gasser et al., 2008; Kukis et al., 1993; Voss et al., 2007; Pippin et al., 1991; Geraldes et al., 2000; Wieghardt et al., 1982; van der Merwe et al., 1985; Prasanphanich et al., 2007; Soluri et al., 2003; Maina et al., 2005; Monstein et al., 2006; Sun & Chen, 2007) as the development of stable copper complexes will allow for a variety of copper-based diagnostic and therapeutic strategies.

Recent work in the field of copper radiochemistry has produced very promising results. A majority of the work has focused on synthesis of new ligand frameworks to chelate copper in a multi-dentate fashion to prevent loss of the Cu2+ ion under *in vivo* conditions. Crossbridged TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetracetic acid) derivatives, such as 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CB-TE2A) and 1-N-(4 aminobenzyl)-3,6,10,13,16,19-hexa-aza-bicyclo-[6.6.6]eichosane-1,8-diamine (SarAr), have shown increased copper-complex stability *in vivo* versus the TETA and DOTA (1,4,7,10 tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelators (Garrison et al., 2007; Di Bartolo et al., 2001 and 2006). Additionally, derivatives of the triazacyclononane macrocycle have produced improved stability of copper complexes (Prasanphanich et al., 2007). Pippin et al., have shown that complexes of Cu(II) with NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) are more inert towards isotopic exchange with 67Cu than DOTA or TETA (Pippin et al., 1991). Recent work by Gasser, et al., demonstrated a 2-[4,7-bis(2-pyridylmethyl)-1,4,7 triazacyclononan-1-yl] acetic acid (PMCN) bifunctional chelator containing the 64Cu radionuclide (Gasser et al., 2008). Recently, we have reported on the superior microPET imaging quality of NOTA bifunctional chelator over that of the DOTA using 64Cu in a GRPR-positive human prostate cancer mouse model (Prasanphanich et al., 2007). Previous investigations have reported a highly stable complex consisting of NOTA and divalent copper (Kukis et al., 1993; Geraldes et al., 2000; Wieghardt et al., 1982). Also reported are biomolecules conjugated with NOTA and chelating 64Cu. Interestingly, one investigation reported a crystal structure of [CuCl(TACNTA)-], or [CuCl(NOTA)-], with a pentadentate NOTA in which one carboxylate arm of the triazacyclononane macrocycle is not involved in the coordination of copper(II) (van der Merwe et al., 1985). This implies that occupation of this non-chelating carboxylate of NOTA, as in our bifunctional NO2A, may not alter the native structure of the Cu-NOTA complex.

 We have recently evaluated 64Cu-NO2A-8-Aoc-BBN(7-14)NH2 radiopharmaceutical (Figure 4) to be used as a PET targeting agent for primary or metastatic breast cancer disease (Prasanphanich et al., 2009). In this study, we were able to prepare high specific activity 64Cu-NO2A-8-Aoc-BBN(7-14)NH2 conjugate in very high radiochemical yield and to evaluate the 1) targeting capacity of 64Cu-NO2A-8-Aoc-BBN(7-14)NH2 for GRPr-positive tissues *in vivo*; 2) particular routes of clearance; and 3) extent of retention of radiopharmaceutical in radiosensitive tissues.

A number of *in vitro* assays were used to evaluate the uptake and affinity of 64Cu-NO2A-8- Aoc-BBN(7-14)NH2 for the GRPr in T-47D human breast cancer cells. Competitive binding displacement assays using 125I-[Tyr4]-BBN(7-14)NH2, as the radioligand were used to quantify the relative binding affinity of Cu-NO2A-8-Aoc-BBN(7-14)NH2 for GRPrs located on the surface of T-47D cells. The concentration of Cu-NO2A-8-Aoc-BBN(7-14)NH2 needed to inhibit 125I-[Tyr4]-BBN(7-14)NH2 binding by 50% was determined to be 7.56 2.23 nM. The rate at which cell-associated radioactivity was internalized within the T-47D cells was also measured. After incubating T-47D cells in media containing 64Cu-NO2A-8-Aoc-BBN(7-

Molecular Imaging of Breast Cancer Tissue *via* Site-Directed Radiopharmaceuticals 293

Fig. 5. *In vivo* microPET/CT and microMRI images of a T-47D tumor-bering mouse 24 h after 5.1 mCi tail vein injection of 64Cu-NO2A-8-Aoc-BBN(7-14)NH2 conjugate. (A) MicroPET/MRI fusion coronal image. (B) MicroPET/CT fused coronal image showing specific uptake of 64Cu-NO2A-8-Aoc-BBN(7-14)NH2 conjugate. (C) Proton density-weghted microMRI coronal image. (D) An axial cross section of microPET/CT images showing functional image of tumor uptake. (E) The corresponding high-resolution T2-weighted microMRI axial image correlating anatomical features of the xenografted tumors to (D).

**6. Fluorescence molecular imaging using alexa fluor 680-BBN conjugates**

vectors for dynamic optical imaging of human cancers holds some significance.

Alternative optical imaging technologies such as fluorescence molecular tomography are emerging as new and exciting molecular imaging tools for diagnosis of human disease (Montet et al., 2006; Young & Rozengurt, 2005; Pu et al., 2005). Studies show that the highthroughput signal afforded by fluorescence imaging might offer an alternative to traditional tomographic imaging by alleviating many of the imaging artifacts seen in imaging systems of this general type (Weissleder & Ntziachristos, 2003; Zacharakis et al., 2005; Kwon et al., 2005). Therefore, design and development of new site-directed, fluorescent, targeting

Monet and co-workers have described a new peptide-nanoparticle conjugate based upon bombesin that may be potentially useful for imaging pancreatic ductal adenocarcinoma. They reported the ability of this new conjugate to specifically target GRPrs expressed on normal pancreatic tissue with minimal accumulation in normal and surrounding tissues. Furthermore, the utility of this new conjugate to be used as a dual modality MRI contrast agent was shown *in vivo* in T2 weighted MR images of rodents bearing MIA-PaCa2

14)NH2 for 45 min, nearly 90% of all cell-associated radioactivity had internalized. This remained nearly constant for subsequent time points. The externalizaton of 64Cu-NO2A-8- Aoc-BBN(7-14)NH2 from T-47D cells was measured by removing cells from conjugatecontaining media after 45 min and washing them to remove all surface-bound radioactivity. No significant efflux of radiotracer was observed over the 90 min period.

Fig. 4. 64Cu-NO2A-8-Aoc-BBN(7-14)NH2 conjugate.

Results obtained from biodistribution studies in SCID mice bearing human T-47D xenografted tumors suggested uptake in GRPr expressing tissues was receptor mediated. The average uptake and retention of conjugate in T-47D xenografted tumors at 1, 4, and 24 h p.i. were 2.27 0.08, 1.35 0.14, and 0.28 0.07% ID/g. The primary mode of excretion for this conjugate was the renal-urinary excretion pathway. A receptor blocking assay in which BBN(1-14)NH2 was administered 15 min prior to targeting vector in T-47D tumor-bearing mice showed a significant decrease in the accumulation of 64Cu-NO2A-8-Aoc-BBN(7-14)NH2 in tumor tissue. For example, uptake/accumulation of 64Cu-NO2A-8-Aoc-BBN(7-14)NH2 conjugate was reduced to 0.580.10% ID/g (1 h p.i.) with the addition of the blocking agent to the study. Furthermore, in the blocking assay, the degree of urinary excretion increased to 82 18% ID and suggests that the radioactivity detected in the intestines were at least partially the result of a receptor-mediated process located along the lower gastrointestinal tract and not solely a function of hepatobiliary excretion. Additionally, accumulation of radiotracer in pancreatic tissue and tumors was significantly decreased in this blocking experiment, suggesting that the 64Cu-NO2A-8-Aoc-BBN(7-14)NH2 conjugate is intact and that GRPr-mediated binding is facilitating uptake in the tissues.

As a result of optimal uptake and retention of 64Cu-NO2A-8-Aoc-BBN(7-14)NH2 in T-47D xenografted tumor, microPET/MicroCT/microMRI multimodal imaging experiments were conducted in tumor-bearing mice (Figure 5). A SCID mouse bearing T-47D xenografted tumors on the left and right hind flanks underwent microMR and microPET/CT imaging 24 h following injection with 5.1 mCi of 64Cu-NO2A-8-Aoc-BBN(7-14)NH2. Anatomical structures observed *via* microPET imaging were consistent with pharmacokinetic data obtained from *in vivo* experiments. For example, tumors, pancreas, liver and kidneys, to a lesser extent, were clearly identifiable *via* microPET imaging analysis. Analysis of MRI data indicated small necrotic regions in the xenografted tumors as evident by increased water diffusivity. Observable in the microPET data were regions of reduced intensity that correlated well to these necrotic regions of the tumors. Additionally, the liver is observed to lack a uniform intensity. The drop in intensity correlates to the separation between the median and left lobes of the liver. The identification of such fine structures in the microPET data is a goal of diagnostic nuclear medicine. High-resolution imaging may aid in identifying structural boundaries as well as possible malignant sites.

14)NH2 for 45 min, nearly 90% of all cell-associated radioactivity had internalized. This remained nearly constant for subsequent time points. The externalizaton of 64Cu-NO2A-8- Aoc-BBN(7-14)NH2 from T-47D cells was measured by removing cells from conjugatecontaining media after 45 min and washing them to remove all surface-bound radioactivity.

Results obtained from biodistribution studies in SCID mice bearing human T-47D xenografted tumors suggested uptake in GRPr expressing tissues was receptor mediated. The average uptake and retention of conjugate in T-47D xenografted tumors at 1, 4, and 24 h p.i. were 2.27 0.08, 1.35 0.14, and 0.28 0.07% ID/g. The primary mode of excretion for this conjugate was the renal-urinary excretion pathway. A receptor blocking assay in which BBN(1-14)NH2 was administered 15 min prior to targeting vector in T-47D tumor-bearing mice showed a significant decrease in the accumulation of 64Cu-NO2A-8-Aoc-BBN(7-14)NH2 in tumor tissue. For example, uptake/accumulation of 64Cu-NO2A-8-Aoc-BBN(7-14)NH2 conjugate was reduced to 0.580.10% ID/g (1 h p.i.) with the addition of the blocking agent to the study. Furthermore, in the blocking assay, the degree of urinary excretion increased to 82 18% ID and suggests that the radioactivity detected in the intestines were at least partially the result of a receptor-mediated process located along the lower gastrointestinal tract and not solely a function of hepatobiliary excretion. Additionally, accumulation of radiotracer in pancreatic tissue and tumors was significantly decreased in this blocking experiment, suggesting that the 64Cu-NO2A-8-Aoc-BBN(7-14)NH2 conjugate is intact and

As a result of optimal uptake and retention of 64Cu-NO2A-8-Aoc-BBN(7-14)NH2 in T-47D xenografted tumor, microPET/MicroCT/microMRI multimodal imaging experiments were conducted in tumor-bearing mice (Figure 5). A SCID mouse bearing T-47D xenografted tumors on the left and right hind flanks underwent microMR and microPET/CT imaging 24 h following injection with 5.1 mCi of 64Cu-NO2A-8-Aoc-BBN(7-14)NH2. Anatomical structures observed *via* microPET imaging were consistent with pharmacokinetic data obtained from *in vivo* experiments. For example, tumors, pancreas, liver and kidneys, to a lesser extent, were clearly identifiable *via* microPET imaging analysis. Analysis of MRI data indicated small necrotic regions in the xenografted tumors as evident by increased water diffusivity. Observable in the microPET data were regions of reduced intensity that correlated well to these necrotic regions of the tumors. Additionally, the liver is observed to lack a uniform intensity. The drop in intensity correlates to the separation between the median and left lobes of the liver. The identification of such fine structures in the microPET data is a goal of diagnostic nuclear medicine. High-resolution imaging may aid in

No significant efflux of radiotracer was observed over the 90 min period.

Fig. 4. 64Cu-NO2A-8-Aoc-BBN(7-14)NH2 conjugate.

that GRPr-mediated binding is facilitating uptake in the tissues.

identifying structural boundaries as well as possible malignant sites.

Fig. 5. *In vivo* microPET/CT and microMRI images of a T-47D tumor-bering mouse 24 h after 5.1 mCi tail vein injection of 64Cu-NO2A-8-Aoc-BBN(7-14)NH2 conjugate. (A) MicroPET/MRI fusion coronal image. (B) MicroPET/CT fused coronal image showing specific uptake of 64Cu-NO2A-8-Aoc-BBN(7-14)NH2 conjugate. (C) Proton density-weghted microMRI coronal image. (D) An axial cross section of microPET/CT images showing functional image of tumor uptake. (E) The corresponding high-resolution T2-weighted microMRI axial image correlating anatomical features of the xenografted tumors to (D).

#### **6. Fluorescence molecular imaging using alexa fluor 680-BBN conjugates**

Alternative optical imaging technologies such as fluorescence molecular tomography are emerging as new and exciting molecular imaging tools for diagnosis of human disease (Montet et al., 2006; Young & Rozengurt, 2005; Pu et al., 2005). Studies show that the highthroughput signal afforded by fluorescence imaging might offer an alternative to traditional tomographic imaging by alleviating many of the imaging artifacts seen in imaging systems of this general type (Weissleder & Ntziachristos, 2003; Zacharakis et al., 2005; Kwon et al., 2005). Therefore, design and development of new site-directed, fluorescent, targeting vectors for dynamic optical imaging of human cancers holds some significance.

Monet and co-workers have described a new peptide-nanoparticle conjugate based upon bombesin that may be potentially useful for imaging pancreatic ductal adenocarcinoma. They reported the ability of this new conjugate to specifically target GRPrs expressed on normal pancreatic tissue with minimal accumulation in normal and surrounding tissues. Furthermore, the utility of this new conjugate to be used as a dual modality MRI contrast agent was shown *in vivo* in T2 weighted MR images of rodents bearing MIA-PaCa2

Molecular Imaging of Breast Cancer Tissue *via* Site-Directed Radiopharmaceuticals 295

Figure 7 summarizes the results of studies to assess the degree of uptake, internalization, and blocking of the Alexa Fluor-GGG-BBN(7-14)NH2 conjugate in T-47D breast cancer cells *via* confocal fluorescence microscopy. Assessment of the degree of AF 680-GGG-BBN(7- 14)NH2 conjugate associated with the cells after a 40 min incubation period was evaluated following a cell wash with pH 7.4 incubation media. Results of these studies clearly indicate the effectiveness of AF 680-GGG-BBN(7-14)NH2 to specifically target the GRPr. To assess receptor-mediated internalization of AF 680-GGG-BBN(7-14)NH2 conjugate, surface-bound conjugate was removed using pH 2.5 (0.2 M acetic acid and 0.5 M NaCl) buffer. After the acid wash, much of the conjugate remained internalized within the cells. *In vitro* blocking studies, in which high levels of BBN(1-14) were administered to the cells prior to the AF 680- GGG-BBN(7-14)NH2 conjugate, reduced the uptake/retention in normal GRPr-expressing T-47D cells. This illustrates the high affinity and selectivity of this conjugate for GRPrs overexpressed on T-47D breast cancer cells. In fact, there is little or no indication of fluorescent

Fig. 7. Confocal fluorescence photomicrograph of internalized uptake of Alexa Fluor 680- GGG-BBN(7-14)NH2 in human T-47D breast cancer cells (left). Confocal fluorescence photomicrograph of blocked uptake of Alexa Fluor 680-G-G-G-BBN(7-14)NH2 in human T-

To assess the *in vivo* uptake of the Alexa Fluor conjugate, we have evaluated AF 680-GGG-BBN(7-14)NH2 in rodents bearing human T-47D cancer cell xenografted tumors. In this study, dynamic optical imaging studies of T-47D breast cancer tumor xenografts in rodent models demonstrated the effectiveness of AF 680-GGG-BBN(7-14)NH2 to specifically target GRPr-expressing cells *in vivo*. Montet and co-workers have demonstrated the effectiveness of fluorescent bombesin conjugates to effectively target implanted tumors of the pancreas (Montet et al., 2006). However, the effectiveness of imaging was only demonstrated using excised pancreatic/tumor tissue. Our more recent investigations have indicated specific uptake of conjugate in human tumor tissue (Figure 8a) in xenografted rodent models. Some degree of uptake was also observed in collateral tissue of the abdomen. This, however, is not entirely unexpected due to the hydrophobic nature of the high molecular weight targeting vector. Blocking investigations (Figure 8b), in which BBN(1-14) was used to saturate the GRPr prior to administration of AF 680-GGG-BBN(7-14)NH2, showed little or no fluorescent signal associated with tumor tissue. To complement and verify the presence of GRPr-

signal associated with the cells following the blocking experiment.

47D breast cancer cells by wild-type BBN(1-14).

pancreatic tumors (Montet et al., 2006). This study well demonstrates the potential of dualmodality imaging for diagnosis of human cancers. Young and co-workers have recently described the use of quantum dots conjugated to bombesin to successfully image living mouse Swiss 3T3 and Rat-1 cells *in vitro* (Young & Rozengurt, 2005) and Pu et al., have described a new Cypate-bombesin Peptide Analogue Conjugate (Cybesin) that has potential use as a prostate tumor receptor-mediated contrast agent (Pu et al., 2005). All of these studies provide either *in vivo* or *in vitro* evidence for production of fluorescence-based targeting vectors of bombesin for early detection of human cancers. In addition, fluorescence molecular tomography shows advantages as a relatively low cost, noninvasive procedure that utilizes highly sensitive, non-ionizing probes for tissue targeting.

To complement our work using technetium-99m and copper-64 tagged bombesin tracers to image T-47D breast cancer cells, we developed a new non-radioactive, fluorescent probe based upon BBN having high tumor uptake and optimal pharmacokinetics for specific targeting and optical imaging of the same cell line. In this study, we have developed a conjugate of bombesin having very high affinity for the GRP-receptor holding an N-terminal fluorescent tag that might be useful in determining the diagnosis and disease progression of estrogen receptor positive (ER+) breast cancer. Targeting ER+ breast cancer cells *via* a targeting vector bearing a fluorescent label is a viable alternative to traditional radiolabeled conjugates of this general type.

The new Alexa Fluor 680-GGG-BBN(7-14)NH2 conjugate (Figure 6) was conveniently synthesized by solid phase peptide synthesis of the parent BBN ligand followed by Nterminal conjugation of the active succinimidyl ester of the Alexa Fluor molecule (Ma et al., 2007). This conjugation technology provides a mechanism for appending large molecular weight molecules having inherent fluorescent properties to either the N-terminal primary amine or the epsilon primary amines of lysine-containing peptides or antibodies to produce stable conjugates for dynamic *in vivo* optical imaging. Less-reactive amidated C-termini, however, do not readily react with succinimidyl esters, making this a very selective conjugation method. In our hands, AF 680-GGG-BBN(7-14)NH2 conjugate was stable at temperatures of -80 ºC for periods extending 6 months. Other Alexa Fluor® 680 compounds of this general type designed and developed in our laboratory have demonstrated similar stability profiles.

Fig. 6. AF 680-GGG-BBN(7-14)NH2 conjugate.

In order to assess the binding affinity of the Alexa Fluor 680-GGG-BBN(7-14)NH2 conjugate for the GRPr, *in vitro* competitive cell-binding assays were performed on T-47D breast cancer cells using 125I-[Tyr4]-BBN(7-14)NH2 as the displacement radioligand. This new peptide conjugate demonstrated very high affinity for the GRPr in T-47D breast cancer cells, exhibiting an IC50 value of 7.7 ± 1.4 nM.

pancreatic tumors (Montet et al., 2006). This study well demonstrates the potential of dualmodality imaging for diagnosis of human cancers. Young and co-workers have recently described the use of quantum dots conjugated to bombesin to successfully image living mouse Swiss 3T3 and Rat-1 cells *in vitro* (Young & Rozengurt, 2005) and Pu et al., have described a new Cypate-bombesin Peptide Analogue Conjugate (Cybesin) that has potential use as a prostate tumor receptor-mediated contrast agent (Pu et al., 2005). All of these studies provide either *in vivo* or *in vitro* evidence for production of fluorescence-based targeting vectors of bombesin for early detection of human cancers. In addition, fluorescence molecular tomography shows advantages as a relatively low cost, noninvasive procedure

To complement our work using technetium-99m and copper-64 tagged bombesin tracers to image T-47D breast cancer cells, we developed a new non-radioactive, fluorescent probe based upon BBN having high tumor uptake and optimal pharmacokinetics for specific targeting and optical imaging of the same cell line. In this study, we have developed a conjugate of bombesin having very high affinity for the GRP-receptor holding an N-terminal fluorescent tag that might be useful in determining the diagnosis and disease progression of estrogen receptor positive (ER+) breast cancer. Targeting ER+ breast cancer cells *via* a targeting vector bearing a fluorescent label is a viable alternative to traditional radiolabeled

The new Alexa Fluor 680-GGG-BBN(7-14)NH2 conjugate (Figure 6) was conveniently synthesized by solid phase peptide synthesis of the parent BBN ligand followed by Nterminal conjugation of the active succinimidyl ester of the Alexa Fluor molecule (Ma et al., 2007). This conjugation technology provides a mechanism for appending large molecular weight molecules having inherent fluorescent properties to either the N-terminal primary amine or the epsilon primary amines of lysine-containing peptides or antibodies to produce stable conjugates for dynamic *in vivo* optical imaging. Less-reactive amidated C-termini, however, do not readily react with succinimidyl esters, making this a very selective conjugation method. In our hands, AF 680-GGG-BBN(7-14)NH2 conjugate was stable at temperatures of -80 ºC for periods extending 6 months. Other Alexa Fluor® 680 compounds of this general type designed and developed in our laboratory have demonstrated similar

In order to assess the binding affinity of the Alexa Fluor 680-GGG-BBN(7-14)NH2 conjugate for the GRPr, *in vitro* competitive cell-binding assays were performed on T-47D breast cancer cells using 125I-[Tyr4]-BBN(7-14)NH2 as the displacement radioligand. This new peptide conjugate demonstrated very high affinity for the GRPr in T-47D breast cancer cells,

that utilizes highly sensitive, non-ionizing probes for tissue targeting.

conjugates of this general type.

Fig. 6. AF 680-GGG-BBN(7-14)NH2 conjugate.

exhibiting an IC50 value of 7.7 ± 1.4 nM.

stability profiles.

Figure 7 summarizes the results of studies to assess the degree of uptake, internalization, and blocking of the Alexa Fluor-GGG-BBN(7-14)NH2 conjugate in T-47D breast cancer cells *via* confocal fluorescence microscopy. Assessment of the degree of AF 680-GGG-BBN(7- 14)NH2 conjugate associated with the cells after a 40 min incubation period was evaluated following a cell wash with pH 7.4 incubation media. Results of these studies clearly indicate the effectiveness of AF 680-GGG-BBN(7-14)NH2 to specifically target the GRPr. To assess receptor-mediated internalization of AF 680-GGG-BBN(7-14)NH2 conjugate, surface-bound conjugate was removed using pH 2.5 (0.2 M acetic acid and 0.5 M NaCl) buffer. After the acid wash, much of the conjugate remained internalized within the cells. *In vitro* blocking studies, in which high levels of BBN(1-14) were administered to the cells prior to the AF 680- GGG-BBN(7-14)NH2 conjugate, reduced the uptake/retention in normal GRPr-expressing T-47D cells. This illustrates the high affinity and selectivity of this conjugate for GRPrs overexpressed on T-47D breast cancer cells. In fact, there is little or no indication of fluorescent signal associated with the cells following the blocking experiment.

Fig. 7. Confocal fluorescence photomicrograph of internalized uptake of Alexa Fluor 680- GGG-BBN(7-14)NH2 in human T-47D breast cancer cells (left). Confocal fluorescence photomicrograph of blocked uptake of Alexa Fluor 680-G-G-G-BBN(7-14)NH2 in human T-47D breast cancer cells by wild-type BBN(1-14).

To assess the *in vivo* uptake of the Alexa Fluor conjugate, we have evaluated AF 680-GGG-BBN(7-14)NH2 in rodents bearing human T-47D cancer cell xenografted tumors. In this study, dynamic optical imaging studies of T-47D breast cancer tumor xenografts in rodent models demonstrated the effectiveness of AF 680-GGG-BBN(7-14)NH2 to specifically target GRPr-expressing cells *in vivo*. Montet and co-workers have demonstrated the effectiveness of fluorescent bombesin conjugates to effectively target implanted tumors of the pancreas (Montet et al., 2006). However, the effectiveness of imaging was only demonstrated using excised pancreatic/tumor tissue. Our more recent investigations have indicated specific uptake of conjugate in human tumor tissue (Figure 8a) in xenografted rodent models. Some degree of uptake was also observed in collateral tissue of the abdomen. This, however, is not entirely unexpected due to the hydrophobic nature of the high molecular weight targeting vector. Blocking investigations (Figure 8b), in which BBN(1-14) was used to saturate the GRPr prior to administration of AF 680-GGG-BBN(7-14)NH2, showed little or no fluorescent signal associated with tumor tissue. To complement and verify the presence of GRPr-

Molecular Imaging of Breast Cancer Tissue *via* Site-Directed Radiopharmaceuticals 297

Until now, the molecular basis for GRPr-targeted diagnosis or therapy of receptor-positive neoplastic disease has primarily focused upon targeting receptor over-expression using radiolabeled agonist ligands with inherent internalizing capability. However, recent reports by Nock et al*.*, show compelling evidence that radiopharmaceutical design and development based upon antagonist-type ligand frameworks bear reexamination (Nock et al., 2003 and 2005). Antagonist ligands are presumably not internalized, and therefore are not expected to residualize as effectively in tumor tissue when compared to agonist-based

NHEt13,des-Met14]BBN(6-14)) demonstrated very high affinity and selectivity for the GRPR with pronounced accumulation and retention of radioactivity in human tumor xenografts in rodents (Nock et al., 2003 and 2005). In addition, Maecke and co-workers have begun investigating another antagonist-like targeting vector, [DOTA-4-amino-1-carboxymethylpiperidine-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2], having high selectivity for the GRPr (Mansi et al., 2009 and 2011). Therefore, selective targeting of specific receptor subtypes expressed on the surfaces of primary or metastatic breast cancer tissues with new and improved radiolabeled antagonists might provide a new avenue for earlier diagnosis

In addition, we should point out that discussions herein have only considered monomeric peptide targeting vectors for molecular imaging of breast cancer tissue. It is important to note that the clinical utility of monomeric radiolabeled peptides can be limited by a number of factors including receptor density, binding affinity, and pharmacokinetics. For example, high-quality, high tumor-to-background PET or SPECT images require a high degree of receptor expression on tumor cells as compared to normal, collateral tissue. For these reasons, multimeric or multivalent peptide probes have recently become a new avenue for diagnostic molecular imaging tumors expressing either singly- or multitargetable receptors. Continued design and development of multimeric or multivalent peptides capable of targeting multiple receptor subtypes highly expressed on human cancers could do much to improve image resolution and contrast, all but eliminating the high false-positive rates and non-target uptake that oftentimes limits some of the clinically-approved radiopharmaceuticals from widespread usage for diagnosis of

This material is the result of work supported with resources and the use of facilities at the Harry S. Truman Memorial Veterans' Hospital and the University of Missouri School of Medicine. This work was funded in part by grants from the National Institutes of Health

American Cancer Society. *Cancer Facts and Figures 2010, Detailed Guide: Breast Cancer.* The

American Cancer Society, Atlanta: 2010. Available at www.cancer.org/acs/groups<EMAIL_ADDRESS>
and the United States Department of Veterans' Affairs VA Merit Award.

0-1,bzlg0,D-Phe6,Leu-

ligand frameworks. Studies using 99mTc-Demobesin1 ([99mTc-N4

and staging of patients presenting with disease.

**8. Future work**

malignant tissues.

**10. References**

**9. Acknowledgment**

expressing tumors, magnetic resonance imaging was performed (Figure 8c and 8d). These studies further demonstrate the high degree of selectivity and affinity of AF 680-GGG-BBN(7-14)NH2 conjugate for the GRPr.

Fig. 8. *In vivo* uptake and blocking experiment of Alexa Fluor 680-GGG-BBN(7-14)NH2 in SCID mice bearing human T-47D breast cancer cell xenografts. Xenogen fluorescence images of mice with (A) normal uptake and (B) blocking assay. Magnetic resonance images of cross sections through tumors for (C) normal uptake and (D) blocking assay.

#### **7. Conclusion**

The clinical successes of Octreoscan® have paved the way for exploration and radiolabeling of biologically-active peptides for targeted molecular imaging and peptide receptor scintigraphy of receptors that are highly expressed on specific human tumors. This book chapter has focused primarily upon those peptides that are potentially useful for molecular imaging of human breast cancer. Specifically, our research investigations have focused primarily upon targeting GRPrs with truncated analogues of BBN peptide, as these receptors tend to be expressed in very high numbers on the surface of breast cancer cells. However, there are other cancer types that also express regulatory peptide receptors in very high numbers, making possible early diagnosis and staging of primary and metastatic disease for these cancers as well. More importantly, continued research efforts toward development of thermodynamically stable, kinetically inert radiolabeled peptides for specific targeting of receptors that are highly expressed on the surfaces of human cancer cells creates the opportunity to use peptide receptor targeted radiotherapy as a highly selective treatment strategy for tumor targeting of breast cancer and many others, or as a mechanism for this treatment strategy to complement traditional, clinically-useful chemotherapeutic regimens of treatment.

### **8. Future work**

296 Breast Cancer – Recent Advances in Biology, Imaging and Therapeutics

expressing tumors, magnetic resonance imaging was performed (Figure 8c and 8d). These studies further demonstrate the high degree of selectivity and affinity of AF 680-GGG-

Fig. 8. *In vivo* uptake and blocking experiment of Alexa Fluor 680-GGG-BBN(7-14)NH2 in SCID mice bearing human T-47D breast cancer cell xenografts. Xenogen fluorescence images of mice with (A) normal uptake and (B) blocking assay. Magnetic resonance images of cross

The clinical successes of Octreoscan® have paved the way for exploration and radiolabeling of biologically-active peptides for targeted molecular imaging and peptide receptor scintigraphy of receptors that are highly expressed on specific human tumors. This book chapter has focused primarily upon those peptides that are potentially useful for molecular imaging of human breast cancer. Specifically, our research investigations have focused primarily upon targeting GRPrs with truncated analogues of BBN peptide, as these receptors tend to be expressed in very high numbers on the surface of breast cancer cells. However, there are other cancer types that also express regulatory peptide receptors in very high numbers, making possible early diagnosis and staging of primary and metastatic disease for these cancers as well. More importantly, continued research efforts toward development of thermodynamically stable, kinetically inert radiolabeled peptides for specific targeting of receptors that are highly expressed on the surfaces of human cancer cells creates the opportunity to use peptide receptor targeted radiotherapy as a highly selective treatment strategy for tumor targeting of breast cancer and many others, or as a mechanism for this treatment strategy to complement traditional, clinically-useful

sections through tumors for (C) normal uptake and (D) blocking assay.

BBN(7-14)NH2 conjugate for the GRPr.

**7. Conclusion**

chemotherapeutic regimens of treatment.

Until now, the molecular basis for GRPr-targeted diagnosis or therapy of receptor-positive neoplastic disease has primarily focused upon targeting receptor over-expression using radiolabeled agonist ligands with inherent internalizing capability. However, recent reports by Nock et al*.*, show compelling evidence that radiopharmaceutical design and development based upon antagonist-type ligand frameworks bear reexamination (Nock et al., 2003 and 2005). Antagonist ligands are presumably not internalized, and therefore are not expected to residualize as effectively in tumor tissue when compared to agonist-based ligand frameworks. Studies using 99mTc-Demobesin1 ([99mTc-N4 0-1,bzlg0,D-Phe6,Leu-NHEt13,des-Met14]BBN(6-14)) demonstrated very high affinity and selectivity for the GRPR with pronounced accumulation and retention of radioactivity in human tumor xenografts in rodents (Nock et al., 2003 and 2005). In addition, Maecke and co-workers have begun investigating another antagonist-like targeting vector, [DOTA-4-amino-1-carboxymethylpiperidine-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2], having high selectivity for the GRPr (Mansi et al., 2009 and 2011). Therefore, selective targeting of specific receptor subtypes expressed on the surfaces of primary or metastatic breast cancer tissues with new and improved radiolabeled antagonists might provide a new avenue for earlier diagnosis and staging of patients presenting with disease.

In addition, we should point out that discussions herein have only considered monomeric peptide targeting vectors for molecular imaging of breast cancer tissue. It is important to note that the clinical utility of monomeric radiolabeled peptides can be limited by a number of factors including receptor density, binding affinity, and pharmacokinetics. For example, high-quality, high tumor-to-background PET or SPECT images require a high degree of receptor expression on tumor cells as compared to normal, collateral tissue. For these reasons, multimeric or multivalent peptide probes have recently become a new avenue for diagnostic molecular imaging tumors expressing either singly- or multitargetable receptors. Continued design and development of multimeric or multivalent peptides capable of targeting multiple receptor subtypes highly expressed on human cancers could do much to improve image resolution and contrast, all but eliminating the high false-positive rates and non-target uptake that oftentimes limits some of the clinically-approved radiopharmaceuticals from widespread usage for diagnosis of malignant tissues.

#### **9. Acknowledgment**

This material is the result of work supported with resources and the use of facilities at the Harry S. Truman Memorial Veterans' Hospital and the University of Missouri School of Medicine. This work was funded in part by grants from the National Institutes of Health and the United States Department of Veterans' Affairs VA Merit Award.

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**16**

*USA*

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**Imaging the Sigma-2 Receptor for Diagnosis**

Sigma (σ) receptors represent a class of proteins that were initially classified as a subtype of the opiate receptors. Subsequent studies revealed that σ binding sites are a distinct class of receptors that are located in the central nervous system as well as in a variety of tissues and organs [1, 2]. Two σ binding site subtypes were distinguished based on differences in their drug-binding profiles and molecular weight. The two binding sites are known as σ1 and σ<sup>2</sup> receptors. σ1 receptors have a molecular weight of ~25 kDa, whereas σ2 receptors have a molecular weight of ~21.5 kDa [3]. The σ1 receptor has been cloned and displays a 30% sequence homology with the enzyme, yeast C8-C7 sterol isomerase [4, 5], but this receptor lacks C8-C7 isomerase activity. Recent studies have shown that neuroactive steroids bind with moderate affinity to σ1 sites, and suggest that σ1 receptors may modulate the activity of GABA and NMDA receptors in the CNS [6-8]. The σ2 receptor has not been cloned, and most of what is known regarding the σ2 receptor has been obtained through the use of in vitro receptor binding studies aimed at the pharmacological characterization of this

**2. Characterization of the σ2 receptor as a biomarker of the proliferative**

The first report suggesting that there is an overexpression of σ receptors in tumors cells was by Bem et al. in 1991[9]. In this study, σ binding in tumors was found to be greater than or equal to 2-fold higher than that of control nonmalignant tissue. Later, Vilner et al.[10] demonstrated that many murine and human tumor cells possess a high density of σ<sup>2</sup> receptors when grown under cell culture conditions. These studies clearly indicate that σ<sup>2</sup> receptors may serve as a biomarker for differentiating solid tumors from the surrounding normal tissues. The proliferative status of a solid tumor, which is defined as the ratio of proliferating (P) cells in a solid tumor to those driven into a quiescent (Q) state by nutrient deprivation and/or hypoxia (the P:Q ratio), is an important parameter in determining how to treat a tumor with either radiation or chemotherapy[11]. Tumors having a high

**1. Introduction**

receptor.

**status of solid tumors**

**and Prediction of Therapeutic Response**

Chenbo Zeng1, Jinbin Xu1 and Robert H. Mach1,2,3

*3Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO,*

*2Department of Cell Biology and Physiology,*

### **Imaging the Sigma-2 Receptor for Diagnosis and Prediction of Therapeutic Response**

Chenbo Zeng1, Jinbin Xu1 and Robert H. Mach1,2,3 *1Department of Radiology, 2Department of Cell Biology and Physiology, 3Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, USA*

#### **1. Introduction**

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Sigma (σ) receptors represent a class of proteins that were initially classified as a subtype of the opiate receptors. Subsequent studies revealed that σ binding sites are a distinct class of receptors that are located in the central nervous system as well as in a variety of tissues and organs [1, 2]. Two σ binding site subtypes were distinguished based on differences in their drug-binding profiles and molecular weight. The two binding sites are known as σ1 and σ<sup>2</sup> receptors. σ1 receptors have a molecular weight of ~25 kDa, whereas σ2 receptors have a molecular weight of ~21.5 kDa [3]. The σ1 receptor has been cloned and displays a 30% sequence homology with the enzyme, yeast C8-C7 sterol isomerase [4, 5], but this receptor lacks C8-C7 isomerase activity. Recent studies have shown that neuroactive steroids bind with moderate affinity to σ1 sites, and suggest that σ1 receptors may modulate the activity of GABA and NMDA receptors in the CNS [6-8]. The σ2 receptor has not been cloned, and most of what is known regarding the σ2 receptor has been obtained through the use of in vitro receptor binding studies aimed at the pharmacological characterization of this receptor.

#### **2. Characterization of the σ2 receptor as a biomarker of the proliferative status of solid tumors**

The first report suggesting that there is an overexpression of σ receptors in tumors cells was by Bem et al. in 1991[9]. In this study, σ binding in tumors was found to be greater than or equal to 2-fold higher than that of control nonmalignant tissue. Later, Vilner et al.[10] demonstrated that many murine and human tumor cells possess a high density of σ<sup>2</sup> receptors when grown under cell culture conditions. These studies clearly indicate that σ<sup>2</sup> receptors may serve as a biomarker for differentiating solid tumors from the surrounding normal tissues. The proliferative status of a solid tumor, which is defined as the ratio of proliferating (P) cells in a solid tumor to those driven into a quiescent (Q) state by nutrient deprivation and/or hypoxia (the P:Q ratio), is an important parameter in determining how to treat a tumor with either radiation or chemotherapy[11]. Tumors having a high

Molecular Probes for Imaging the Sigma-2 Receptor 305

receptors. Other compounds that were found to have a higher affinity for σ2 versus σ<sup>1</sup> receptors are: 1) the hallucinogen, ibogaine [23, 24]; 2) the mixed serotonin 5-HT3 antagonist/5-HT4 agonist **BIMU-1**[25]; 3) the tropane analog **SM-21**, an acetylcholine releaser that has been utilized as an antinociceptic agent [26, 27]; 4) the trishomocubane

Fig. 1. The 2 receptor densities in proliferating and quiescent cells. A: Differences of the <sup>2</sup> receptor densities in 66P and 66Q cells. B: The 2 receptor expression kinetics in 66 cells during the Q to P transition and the downregulation of the 2 receptor densities during the

A number of SAR studies using **BIMU-1** as the lead compound have identified high affinity, high selectivity σ2 receptor ligands [30-32]. **BIMU-1** is an ideal lead compound for SAR studies since it provides a variety of regions where structural modifications can be made to

P to Q transition.

analog **ANSTO-19** [28]; and 5) the piperazine analog **PB28** [29].

proliferative status (i.e., high P:Q ratio) typically respond better to hyperfractionated radiation therapy versus conventional radiation therapy. Similarly, tumors having a high P:Q ratio will respond better to cell cycle specific agents such as Ara-C and gemcitabine, whereas tumors having a lower proliferative status will respond better to non-cell cycle specific agents such as cisplatin and BCNU [11]. Also, a change in the proliferative status of a tumor during or after treatment has the potential to serve as a predictor of response and allow further tailoring of therapy.

In order to investigate the relationship between the density of σ2 receptors and the proliferative status of tumors, Wheeler and colleagues used the mouse mammary adenocarcinoma cell line 66[12, 13] to determine if there was a difference in the density of σ<sup>2</sup> receptors in proliferating (66P) and quiescent (66Q) tumor cells in cell culture or in solid tumor xenografts. This group demonstrated that the density of σ2 receptors in 66P cells was about 10 times greater than the density observed in 66Q cells (Figure 1A)[14]. The density of σ2 receptors in the 66P cells was found to be quite high, ~900,000 copies/cell versus ~90,000 receptors/cell in the 66Q cells. This group also reported that the expression kinetics of σ<sup>2</sup> receptors follows the growth kinetics of the 66 cells (Figure 1B)[15]. A subsequent study in solid tumor xenografts of the same tumor cell lines demonstrated the identical P:Q ratio to that measured in the cell culture condition[16]. The agreement between the solid tumor and tissue culture data indicates that the σ2 receptor is a receptor-based biomarker of cell proliferation in breast tumors. Thus the 2 receptor possesses properties similar to Ki-67, a marker of proliferation [17]. Ki-67 expression level is determined by immunohistochemistry using Ki-67 antibody. This method requires biopsy or surgical specimens of tumor. In contrast, radiotracers having a high affinity and high selectivity for σ2 receptors have the potential to assess the proliferative status of human breast tumors using noninvasive imaging techniques such as Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT). In addition, it is likely that this approach can be extended to assess the proliferative status of other human tumors, such as head and neck, melanoma, and lung tumors, which are known to express a high density of σ2 receptors[10]. Imaging the proliferative status with σ2 receptors radiotracers can provide useful information regarding the prognosis and aggressiveness of tumors, and this information can be used to guide treatment of cancer in clinical practice. Rapidly proliferating tumors requires aggressive initial treatment. A reduction in the proliferative status of a tumor can serve as a predictor of the tumor's response to therapy.

### **3. Development of σ2 receptor selective ligands**

A number of structurally-diverse compounds have been shown to possess a high affinity to σ receptors [2]. However, most of these compounds bind selectively to σ1 receptor or have similar affinities to both σ1 and σ2 receptors. One of the first σ2 selective ligands reported was the benzomorphan-7-one analog, **CB-64D** [18]. This compound was identified as part of a structure-activity relationship (SAR) study aimed at refining the affinity of (−)-2-methyl-5- (3-hydroxyphenyl)morphan-7-one for μ versus κ opioid receptors [19]. A second series of compounds having a high affinity for σ2 receptors are the 3-(ω-aminoalkyl)-1H-indole analogs [20, 21]. SAR studies with compounds that were originally designed to be serotonin 5-HT1A agonists, resulted in the synthesis of **Lu 28-179** [22], also known as siramesine, which has a subnanomolar affinity for σ2 receptors and a 140-fold selectivity for σ2 versus σ<sup>1</sup>

proliferative status (i.e., high P:Q ratio) typically respond better to hyperfractionated radiation therapy versus conventional radiation therapy. Similarly, tumors having a high P:Q ratio will respond better to cell cycle specific agents such as Ara-C and gemcitabine, whereas tumors having a lower proliferative status will respond better to non-cell cycle specific agents such as cisplatin and BCNU [11]. Also, a change in the proliferative status of a tumor during or after treatment has the potential to serve as a predictor of response and

In order to investigate the relationship between the density of σ2 receptors and the proliferative status of tumors, Wheeler and colleagues used the mouse mammary adenocarcinoma cell line 66[12, 13] to determine if there was a difference in the density of σ<sup>2</sup> receptors in proliferating (66P) and quiescent (66Q) tumor cells in cell culture or in solid tumor xenografts. This group demonstrated that the density of σ2 receptors in 66P cells was about 10 times greater than the density observed in 66Q cells (Figure 1A)[14]. The density of σ2 receptors in the 66P cells was found to be quite high, ~900,000 copies/cell versus ~90,000 receptors/cell in the 66Q cells. This group also reported that the expression kinetics of σ<sup>2</sup> receptors follows the growth kinetics of the 66 cells (Figure 1B)[15]. A subsequent study in solid tumor xenografts of the same tumor cell lines demonstrated the identical P:Q ratio to that measured in the cell culture condition[16]. The agreement between the solid tumor and tissue culture data indicates that the σ2 receptor is a receptor-based biomarker of cell proliferation in breast tumors. Thus the 2 receptor possesses properties similar to Ki-67, a marker of proliferation [17]. Ki-67 expression level is determined by immunohistochemistry using Ki-67 antibody. This method requires biopsy or surgical specimens of tumor. In contrast, radiotracers having a high affinity and high selectivity for σ2 receptors have the potential to assess the proliferative status of human breast tumors using noninvasive imaging techniques such as Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT). In addition, it is likely that this approach can be extended to assess the proliferative status of other human tumors, such as head and neck, melanoma, and lung tumors, which are known to express a high density of σ2 receptors[10]. Imaging the proliferative status with σ2 receptors radiotracers can provide useful information regarding the prognosis and aggressiveness of tumors, and this information can be used to guide treatment of cancer in clinical practice. Rapidly proliferating tumors requires aggressive initial treatment. A reduction in the proliferative status of a tumor can

A number of structurally-diverse compounds have been shown to possess a high affinity to σ receptors [2]. However, most of these compounds bind selectively to σ1 receptor or have similar affinities to both σ1 and σ2 receptors. One of the first σ2 selective ligands reported was the benzomorphan-7-one analog, **CB-64D** [18]. This compound was identified as part of a structure-activity relationship (SAR) study aimed at refining the affinity of (−)-2-methyl-5- (3-hydroxyphenyl)morphan-7-one for μ versus κ opioid receptors [19]. A second series of compounds having a high affinity for σ2 receptors are the 3-(ω-aminoalkyl)-1H-indole analogs [20, 21]. SAR studies with compounds that were originally designed to be serotonin 5-HT1A agonists, resulted in the synthesis of **Lu 28-179** [22], also known as siramesine, which has a subnanomolar affinity for σ2 receptors and a 140-fold selectivity for σ2 versus σ<sup>1</sup>

allow further tailoring of therapy.

serve as a predictor of the tumor's response to therapy.

**3. Development of σ2 receptor selective ligands**

receptors. Other compounds that were found to have a higher affinity for σ2 versus σ<sup>1</sup> receptors are: 1) the hallucinogen, ibogaine [23, 24]; 2) the mixed serotonin 5-HT3 antagonist/5-HT4 agonist **BIMU-1**[25]; 3) the tropane analog **SM-21**, an acetylcholine releaser that has been utilized as an antinociceptic agent [26, 27]; 4) the trishomocubane analog **ANSTO-19** [28]; and 5) the piperazine analog **PB28** [29].

Fig. 1. The 2 receptor densities in proliferating and quiescent cells. A: Differences of the <sup>2</sup> receptor densities in 66P and 66Q cells. B: The 2 receptor expression kinetics in 66 cells during the Q to P transition and the downregulation of the 2 receptor densities during the P to Q transition.

A number of SAR studies using **BIMU-1** as the lead compound have identified high affinity, high selectivity σ2 receptor ligands [30-32]. **BIMU-1** is an ideal lead compound for SAR studies since it provides a variety of regions where structural modifications can be made to

Molecular Probes for Imaging the Sigma-2 Receptor 307

being active in behavioral studies. In order to reduce the lipophilicity of **YUN250,** the 4-(2,3 dichlorophenyl) piperazine moiety of **YUN250** was replaced with other aromatic amine groups. Although this strategy resulted in a number of useful dopamine D3 receptor ligands [36], it was also observed that replacement of the 4-phenylpiperazine moiety with a 6,7 dimethoxy-1,2,3,4-tetrahydroisoqinoline ring resulted in compounds having a high affinity and excellent selectivity for σ2 versus σ1 receptors, and a dramatic reduction in affinity for dopamine receptors [35]. As discussed in greater detail below, the conformationally-flexible benzamide analogs have proven to be an important class of σ2-selective compounds for the

preparation of radiolabeled probes to image this receptor in vitro and in vivo.

Fig. 3. The conformationally-flexible benzamide-based σ2 receptor ligands for in vitro studies of the σ2 receptor. **A**: **YUN250** was a lead for generating the conformationallyflexible benzamide analogues which are selective for σ2 receptors. The tritium labeled ligand, [3H]**RHM-1** , has been used to conduct the receptor binding assay. **B**: Scatchard studies of σ2 receptors in EMT-6 mouse breast tumors and NMNU-induced rat breast tumors with **[3H]RHM-1.** This probe has high affinity to the σ2 receptors in the rodent breast

tumors and is useful for in vitro binding studies.

optimize the σ2 receptor affinity and reduce the affinity for serotonin 5-HT3 and 5-HT4 receptors (Figure 2). The structures of **BIMU-1** were altered in three different regions: 1) replacement of the urea linkage with a comformationally-flexible carbamate moiety; 2) replacement of the N-methyl group with an N-benzyl group to diminish serotonin receptor affinity; and 3) preparation of both tropane (i.e., [3.2.1]azabicyclonoctane) and granatane (i.e., [3.3.1]azabicyclononane) ring systems. The most interesting analog from this initial SAR study was the compound **ABN-1**, which had a σ2 receptor affinity of ~ 3 nM and a σ2: σ<sup>1</sup> selectivity of ~30 (Figure 2)[31]. Compound **ABN-1** was used as a secondary lead compound for a series of subsequent SAR studies aimed at producing second-generation granatane analogs having an improved σ2 receptor affinity and high σ2: σ1 selectivity ratio. Consequently, this led to the development of a number of fluorescent probes, **K05-138**, **SW120**, **SW107 and SW116**, that have proven useful in two photon and confocal microscopy studies of σ2 receptors in tumor cells growing under cell culture conditions[33, 34].

Fig. 2. Structures of **BIMU-1** and the granatane analogs. **ABN-1** was used as a lead for producing second-generation granatane-based σ2 receptor ligands, which led to the development of a number of fluorescent probes, **K05-138**, **SW120**, **SW107 and SW116**, for two photon and confocal microscopy studies of σ2 receptors in tumor cells.

A second class of compounds having a high affinity for σ2 receptors and excellent σ1 : σ<sup>2</sup> selectivity ratios are the conformationally-flexible benzamide analogs. These compounds were identified in an SAR study of the benzamide analog, **YUN250,** that was developed as a dopamine D3-selective ligand (Figure 3A) [35]. The relatively high lipophilicity of **YUN250** (log P = 5.76) suggests that it is not capable of readily crossing the blood-brain barrier and

optimize the σ2 receptor affinity and reduce the affinity for serotonin 5-HT3 and 5-HT4 receptors (Figure 2). The structures of **BIMU-1** were altered in three different regions: 1) replacement of the urea linkage with a comformationally-flexible carbamate moiety; 2) replacement of the N-methyl group with an N-benzyl group to diminish serotonin receptor affinity; and 3) preparation of both tropane (i.e., [3.2.1]azabicyclonoctane) and granatane (i.e., [3.3.1]azabicyclononane) ring systems. The most interesting analog from this initial SAR study was the compound **ABN-1**, which had a σ2 receptor affinity of ~ 3 nM and a σ2: σ<sup>1</sup> selectivity of ~30 (Figure 2)[31]. Compound **ABN-1** was used as a secondary lead compound for a series of subsequent SAR studies aimed at producing second-generation granatane analogs having an improved σ2 receptor affinity and high σ2: σ1 selectivity ratio. Consequently, this led to the development of a number of fluorescent probes, **K05-138**, **SW120**, **SW107 and SW116**, that have proven useful in two photon and confocal microscopy

studies of σ2 receptors in tumor cells growing under cell culture conditions[33, 34].

Fig. 2. Structures of **BIMU-1** and the granatane analogs. **ABN-1** was used as a lead for producing second-generation granatane-based σ2 receptor ligands, which led to the development of a number of fluorescent probes, **K05-138**, **SW120**, **SW107 and SW116**, for

A second class of compounds having a high affinity for σ2 receptors and excellent σ1 : σ<sup>2</sup> selectivity ratios are the conformationally-flexible benzamide analogs. These compounds were identified in an SAR study of the benzamide analog, **YUN250,** that was developed as a dopamine D3-selective ligand (Figure 3A) [35]. The relatively high lipophilicity of **YUN250** (log P = 5.76) suggests that it is not capable of readily crossing the blood-brain barrier and

two photon and confocal microscopy studies of σ2 receptors in tumor cells.

being active in behavioral studies. In order to reduce the lipophilicity of **YUN250,** the 4-(2,3 dichlorophenyl) piperazine moiety of **YUN250** was replaced with other aromatic amine groups. Although this strategy resulted in a number of useful dopamine D3 receptor ligands [36], it was also observed that replacement of the 4-phenylpiperazine moiety with a 6,7 dimethoxy-1,2,3,4-tetrahydroisoqinoline ring resulted in compounds having a high affinity and excellent selectivity for σ2 versus σ1 receptors, and a dramatic reduction in affinity for dopamine receptors [35]. As discussed in greater detail below, the conformationally-flexible benzamide analogs have proven to be an important class of σ2-selective compounds for the preparation of radiolabeled probes to image this receptor in vitro and in vivo.

Fig. 3. The conformationally-flexible benzamide-based σ2 receptor ligands for in vitro studies of the σ2 receptor. **A**: **YUN250** was a lead for generating the conformationallyflexible benzamide analogues which are selective for σ2 receptors. The tritium labeled ligand, [3H]**RHM-1** , has been used to conduct the receptor binding assay. **B**: Scatchard studies of σ2 receptors in EMT-6 mouse breast tumors and NMNU-induced rat breast tumors with **[3H]RHM-1.** This probe has high affinity to the σ2 receptors in the rodent breast tumors and is useful for in vitro binding studies.

Molecular Probes for Imaging the Sigma-2 Receptor 309

### **4. In vitro binding and in vivo imaging studies of σ2 receptors**

The conformationally-flexible benzamide analogs have been used to develop the σ<sup>2</sup> radioligands for in vitro binding studies. The tritium labeled σ2 selective ligand, [3H]**RHM-1**, has been used to conduct Scatchard studies of σ2 receptors in tumors and normal tissues (Figure 3A)[37, 38]. The in vitro receptor binding studies indicate that **[3H]RHM-1** binds with a high affinity to both EMT-6 mouse breast tumors (Kd=3.45 nM) and chemicallyinduced rat breast tumors (Kd=4.66 nM). The σ2 receptor densities of both tumors are found to be high (Bmax =2290 fmol/mg of protein for EMT-6 mouse breast tumors and Bmax=2410 fmol/mg of protein for *N*-methyl-*N*-nitrosourea (NMNU)-induced rat breast tumors) (Figure 3B). The pharmacologic profile of **[3H]RHM-1** is in agreement with that of [3H]di-otolylguanidine ([3H]DTG), a most commonly used radioligand for the σ2 receptor binding assay. These results indicate that **[3H]RHM-1** is a useful radioligand for studying <sup>2</sup> receptors in vitro.

The high correlation between the density of σ2 receptors and the proliferative status of solid tumors indicates that σ2 selective radiotracers can be used for imaging the proliferative status of solid tumors with PET and SPECT. The conformationally-flexible benzamide analogs have been used for the development of PET radiotracers for imaging the σ2 receptor status of solid tumors. The first PET radiotracers prepared were [11C]**1**-**4** (Figure 4A), which involves labeling the corresponding ortho-hydroxy group with [11C]methyl iodide [38]. MicroPET and tumor uptake studies were conducted with [11C]**1**-**4**; the most promising analog proved to be [11C]**4**. Although all four analogs had a high affinity for σ2 receptors, the optimal lipophilicity of [11C]**4** was responsible for the high tumor uptake and suitable signal: normal tissue ratios for imaging. These data indicate that both receptor affinity and lipophilicity are important properties for successful receptor-based tumor imaging agents. MicroPET and MicroCT imaging studies in a murine solid breast tumor EMT-6 clearly show the potential of [11C]**4** as a radiotracer for imaging the σ2 receptor status of breast tumors with PET.

Although [11C]**4** provides a clear image of breast tumors in microPET imaging studies, the short half life of carbon 11 (*t*1/2 = 20.4 min) is not ideal for the development of radiotracers which can be used in clinical PET imaging studies. The longer half-life of 18F (*t*1/2 =109.8 min) compared to 11C places fewer time constraints on tracer synthesis, allows imaging studies to be conducted up to 2 h after injection of the radiotracers, and usually results in higher tumor: normal tissue ratios. A number of 18F-labeled radiotracers [18F]**5**-**8** (Figure 4B) based on the conformationally-flexible benzamide analogs have been generated and evaluated in murine breast tumor models[39]. The strategy involved replacement of the 2 methoxy group in the benzamide ring with a 2-fluoroethoxy group. The 2-fluoroethoxy- for methoxy-substitution is a common strategy used in the development of 18F-labeled radiotracers. Biodistribution studies in female Balb/c mice bearing EMT-6 tumor allografts demonstrated that all four 18F -labeled compounds had a high tumor uptake (2.5-3.7% ID/g) and acceptable tumor/normal tissue ratios at 1 and 2 h post-i.v. injection (Figure 4C). The moderate to high tumor/normal tissue ratios and the rapid clearance from the blood for [18F]**5** and [18F]**8** suggests that these radiotracers are likely the best candidates for imaging of solid tumors with PET. MicroPET imaging studies indicate that [18F]**5** and [18F]**8** are suitable probes for imaging the σ2 receptor status of solid tumors with PET (Figure 4D). Clinical studies of [18F]**8** are currently in progress in the US.

The conformationally-flexible benzamide analogs have been used to develop the σ<sup>2</sup> radioligands for in vitro binding studies. The tritium labeled σ2 selective ligand, [3H]**RHM-1**, has been used to conduct Scatchard studies of σ2 receptors in tumors and normal tissues (Figure 3A)[37, 38]. The in vitro receptor binding studies indicate that **[3H]RHM-1** binds with a high affinity to both EMT-6 mouse breast tumors (Kd=3.45 nM) and chemicallyinduced rat breast tumors (Kd=4.66 nM). The σ2 receptor densities of both tumors are found to be high (Bmax =2290 fmol/mg of protein for EMT-6 mouse breast tumors and Bmax=2410 fmol/mg of protein for *N*-methyl-*N*-nitrosourea (NMNU)-induced rat breast tumors) (Figure 3B). The pharmacologic profile of **[3H]RHM-1** is in agreement with that of [3H]di-otolylguanidine ([3H]DTG), a most commonly used radioligand for the σ2 receptor binding assay. These results indicate that **[3H]RHM-1** is a useful radioligand for studying <sup>2</sup>

The high correlation between the density of σ2 receptors and the proliferative status of solid tumors indicates that σ2 selective radiotracers can be used for imaging the proliferative status of solid tumors with PET and SPECT. The conformationally-flexible benzamide analogs have been used for the development of PET radiotracers for imaging the σ2 receptor status of solid tumors. The first PET radiotracers prepared were [11C]**1**-**4** (Figure 4A), which involves labeling the corresponding ortho-hydroxy group with [11C]methyl iodide [38]. MicroPET and tumor uptake studies were conducted with [11C]**1**-**4**; the most promising analog proved to be [11C]**4**. Although all four analogs had a high affinity for σ2 receptors, the optimal lipophilicity of [11C]**4** was responsible for the high tumor uptake and suitable signal: normal tissue ratios for imaging. These data indicate that both receptor affinity and lipophilicity are important properties for successful receptor-based tumor imaging agents. MicroPET and MicroCT imaging studies in a murine solid breast tumor EMT-6 clearly show the potential of [11C]**4** as a radiotracer for imaging the σ2 receptor status of breast tumors

Although [11C]**4** provides a clear image of breast tumors in microPET imaging studies, the short half life of carbon 11 (*t*1/2 = 20.4 min) is not ideal for the development of radiotracers which can be used in clinical PET imaging studies. The longer half-life of 18F (*t*1/2 =109.8 min) compared to 11C places fewer time constraints on tracer synthesis, allows imaging studies to be conducted up to 2 h after injection of the radiotracers, and usually results in higher tumor: normal tissue ratios. A number of 18F-labeled radiotracers [18F]**5**-**8** (Figure 4B) based on the conformationally-flexible benzamide analogs have been generated and evaluated in murine breast tumor models[39]. The strategy involved replacement of the 2 methoxy group in the benzamide ring with a 2-fluoroethoxy group. The 2-fluoroethoxy- for methoxy-substitution is a common strategy used in the development of 18F-labeled radiotracers. Biodistribution studies in female Balb/c mice bearing EMT-6 tumor allografts demonstrated that all four 18F -labeled compounds had a high tumor uptake (2.5-3.7% ID/g) and acceptable tumor/normal tissue ratios at 1 and 2 h post-i.v. injection (Figure 4C). The moderate to high tumor/normal tissue ratios and the rapid clearance from the blood for [18F]**5** and [18F]**8** suggests that these radiotracers are likely the best candidates for imaging of solid tumors with PET. MicroPET imaging studies indicate that [18F]**5** and [18F]**8** are suitable probes for imaging the σ2 receptor status of solid tumors with PET (Figure 4D). Clinical

studies of [18F]**8** are currently in progress in the US.

**4. In vitro binding and in vivo imaging studies of σ2 receptors**

receptors in vitro.

with PET.

Molecular Probes for Imaging the Sigma-2 Receptor 311

suggesting that σ2 receptors may localize in mitochondria, endoplasmic reticulum, lysosomes and the plasma membrane (Figure 5). The data also showed that **SW120** did not co-localize with the nuclear marker, DAPI, suggesting that either the σ2 receptor does not exist in the nucleus or **SW120** does not enter the nucleus. The similar results were obtained

Fig. 5. Determination of the intracellular distribution of **SW120** in MDA-MB-435 cells with and without MitoTracker (A), ER-Tracker (B), LysoTracker (C), a nuclear marker, DAPI (D), or a membrane tracker, FM4-64FX (E), using confocal microscopy. MDA-MB-435 cells were incubated with 30 nM **SW120** and either 20 nM MitoTracker, 500 nM ER-Tracker, 50 nM LysoTracker, or 300 nM DAPI. After incubating for 2 h at 37C, live cells were imaged by confocal microscopy. MDA-MB-435 cells were also incubated with 50 nM **SW120** and 5 g/mL of the membrane tracker, FM4-64FX, for 15 min at 0C. The live cells were imaged

by confocal microscopy. Scale bar = 10 m.

for the other σ2 fluorescent probes (**SW116, K05-138** and **SW107**)[33, 34].

Fig. 4. The synthetic schemes for generating conformationally-flexible benzamide-based PET radiotracers. A: A number of 11C-labeled radiotracers, [11C]**1-4,** are prepared by labeling the corresponding ortho-hydroxy group with [11C]methyl iodide. B: A number of 18F-labeled radiotracers, [18F]**5**-**8,** are generated by replacing of the 2-methoxy group in the benzamide ring with a 2-fluoroethoxy group. C: Biodistribution studies in female Balb/c mice bearing EMT-6 tumor allografts demonstrating that all four 18F -labeled compounds had a high tumor uptake (2.5-3.7% ID/g) and acceptable tumor/normal tissue ratios at 2 h post-i.v. injection. D: MicroPET imaging studies indicate that [18F]**5** and [18F]**8** are suitable probes for imaging the σ2 receptor status of solid tumors with PET.

### **5. Confocal and two photon microscopy studies of σ2 receptors in tumor cells**

Since the σ2 receptor protein has not been cloned, current knowledge of this receptor is based predominantly on receptor binding studies with radiolabeled probes such as [3H]DTG. The function of the σ2 receptors has also been investigated by studying their effects on the biochemical and physiological properties of tumor cells. Several studies have shown that σ2 ligands induced cell death. The proposed mechanisms of cell death include caspase-independent apoptosis [40], lysosomal leakage [41], Ca2+ release [42, 43], oxidative stress [41], ceramide production [44] and autophagy [45]. However, the subcellular localizations where the σ2 ligands bind and function were not known. Using the fluorescent probes shown in Figure 2, Zeng and colleagues[33, 34] recently conducted a series of confocal and two photon microscopy studies that have provided the information regarding the localization of σ2 receptors in breast tumor cells.

MDA-MB-435 cells were incubated with 30 nM **SW120** and each of the five subcellular organelle markers: the mitochondria tracker, MitoTracker Red CMXRos (20 nM ), the endoplasmic reticulum tracker, ER-TrackerTM Red (500 nM), the lysosome tracker, LysoTracker Red DND-99 (50 nM), the nuclear marker, DAPI (300 nM), or the plasma membrane tracker, FM 4-64FX (5 g/mL). The results showed that **SW120** partially colocalized with MitoTracker, ER-Tracker, LysoTracker and the plasma membrane tracker,

**D**

Fig. 4. The synthetic schemes for generating conformationally-flexible benzamide-based PET radiotracers. A: A number of 11C-labeled radiotracers, [11C]**1-4,** are prepared by labeling the corresponding ortho-hydroxy group with [11C]methyl iodide. B: A number of 18F-labeled radiotracers, [18F]**5**-**8,** are generated by replacing of the 2-methoxy group in the benzamide ring with a 2-fluoroethoxy group. C: Biodistribution studies in female Balb/c mice bearing EMT-6 tumor allografts demonstrating that all four 18F -labeled compounds had a high tumor uptake (2.5-3.7% ID/g) and acceptable tumor/normal tissue ratios at 2 h post-i.v. injection. D: MicroPET imaging studies indicate that [18F]**5** and [18F]**8** are suitable probes for

**5. Confocal and two photon microscopy studies of σ2 receptors in tumor cells**  Since the σ2 receptor protein has not been cloned, current knowledge of this receptor is based predominantly on receptor binding studies with radiolabeled probes such as [3H]DTG. The function of the σ2 receptors has also been investigated by studying their effects on the biochemical and physiological properties of tumor cells. Several studies have shown that σ2 ligands induced cell death. The proposed mechanisms of cell death include caspase-independent apoptosis [40], lysosomal leakage [41], Ca2+ release [42, 43], oxidative stress [41], ceramide production [44] and autophagy [45]. However, the subcellular localizations where the σ2 ligands bind and function were not known. Using the fluorescent probes shown in Figure 2, Zeng and colleagues[33, 34] recently conducted a series of confocal and two photon microscopy studies that have provided the information regarding

MDA-MB-435 cells were incubated with 30 nM **SW120** and each of the five subcellular organelle markers: the mitochondria tracker, MitoTracker Red CMXRos (20 nM ), the endoplasmic reticulum tracker, ER-TrackerTM Red (500 nM), the lysosome tracker, LysoTracker Red DND-99 (50 nM), the nuclear marker, DAPI (300 nM), or the plasma membrane tracker, FM 4-64FX (5 g/mL). The results showed that **SW120** partially colocalized with MitoTracker, ER-Tracker, LysoTracker and the plasma membrane tracker,

imaging the σ2 receptor status of solid tumors with PET.

the localization of σ2 receptors in breast tumor cells.

**D**

suggesting that σ2 receptors may localize in mitochondria, endoplasmic reticulum, lysosomes and the plasma membrane (Figure 5). The data also showed that **SW120** did not co-localize with the nuclear marker, DAPI, suggesting that either the σ2 receptor does not exist in the nucleus or **SW120** does not enter the nucleus. The similar results were obtained for the other σ2 fluorescent probes (**SW116, K05-138** and **SW107**)[33, 34].

Fig. 5. Determination of the intracellular distribution of **SW120** in MDA-MB-435 cells with and without MitoTracker (A), ER-Tracker (B), LysoTracker (C), a nuclear marker, DAPI (D), or a membrane tracker, FM4-64FX (E), using confocal microscopy. MDA-MB-435 cells were incubated with 30 nM **SW120** and either 20 nM MitoTracker, 500 nM ER-Tracker, 50 nM LysoTracker, or 300 nM DAPI. After incubating for 2 h at 37C, live cells were imaged by confocal microscopy. MDA-MB-435 cells were also incubated with 50 nM **SW120** and 5 g/mL of the membrane tracker, FM4-64FX, for 15 min at 0C. The live cells were imaged by confocal microscopy. Scale bar = 10 m.

Molecular Probes for Imaging the Sigma-2 Receptor 313

Fig. 6. Characterization of σ2 fluorescent probes. A: The internalization kinetics of **SW120** in MDA-MB-435 cells by confocal microscopy studies show that **SW120** enters tumor cells rapidly. B: The internalization of **SW120** in MDA-MB-435 cells are partially blocked by σ<sup>2</sup> selective ligands, **SW43** and siramesine, but not by a σ1 selective ligand, (+)-pentazocine. C: The inhibition of **SW120** internalization by phenylarsine oxide (PAO). MDA-MB-435 cells were preincubated with 5 or 10 M PAO for 30 min at 37C, and then incubated with 10 nM

The σ2 receptor has been validated as a proliferation marker in cell culture and in solid tumors. Therefore it is possible that σ2 fluorescent probes could preferentially label proliferating cells versus non-proliferating cells and serve as agents to image cell proliferation in vivo. This hypothesis was tested in nude mice implanted with murine mammary adenocarcinoma 66 cells and BALB/C mice implanted with mouse mammary carcinoma cell line EMT6[34]. The mice were treated with **SW120 (**50 g in 100 L PBS) for one hour. The peripheral blood mononuclear cells (PBMC), which are commonly used as

**SW120** for an additional 30 min. The cells were analyzed by flow cytometry. The internalization of **SW120** was significantly reduced by 10 μM PAO (\* P < 0.05).

**B C**

The kinetic studies of internalization of σ2 fluorescent probes in MDA-MB-435 cells were conducted using confocal microscopy[33, 34]. σ2 fluorescent probes enter tumor cells rapidly. For example**, K05-138** and **SW120** reach 50% of the maximal fluorescent intensity (T1/2) in 16 seconds and 11 minutes, respectively (Figure 6A). The internalization of **SW120**  can be blocked by the σ2 selective ligands, **SW43** and siramesine, by 52% and 44%, respectively (Figure 6B). This internalization can not be blocked by the σ1 selective ligand, (+)-pentazocine. The data suggest that the internalization of the σ2 selective ligand is partially mediated by σ2 receptor. In order to study whether the internalization of the σ<sup>2</sup> ligand is mediated by endocytosis, Zeng et al examined the effect of phenylarsine oxide (PAO), a well-characterized endocytosis inhibitor [46], on the internalization of **SW120**. MDA-MB-435 cells were pretreated with 5 or 10 M PAO for 30 min, and then treated with 10 nM **SW120** in the absence or presence of PAO for an additional 30 min. Flow cytometric analysis (Figure 6C) showed that 10 M PAO significantly blocked internalization of **SW120**  by 30%. These data demonstrate that 30% of the 2 receptor ligand was internalized by an endocytosis-mediated mechanism, while the remaining 70 % was internalized by other mechanisms such as passive diffusion. The rapid internalization of σ2 receptors via endocytosis suggests that σ2 selective ligands may potentially serve as receptor-mediated probes for delivering cytotoxic agents to solid tumors.

The two photon and confocal microscopy studies conducted by Zeng et al.[33, 34] have provided useful information for the interpretation of studies demonstrating that σ2 receptor ligands may have a role as cancer chemotherapeutic agents. Mitochondria are a key organelle to regulate the intrinsic apoptotic pathway. Apoptotic signals such as UV irradiation or treatment with chemotherapeutic agents cause the release of cytochrome C from the mitochondria and the subsequent activation of caspase-3 leading to an apoptotic cell death[47]. The subcellular localization of σ2 ligands in mitochondria is consistent with the previous studies that 2 ligands trigger apoptosis in tumor cells by acting on mitochondria [48]. The data are also consistent with our observation by transmission electron microscopy that the 2 ligand siramesine induces distortion of mitochondria (unpublished data). The endoplasmic reticulum (ER) serves as a dynamic Ca2+ storage pool [49]. σ2 selective ligands have been reported to induce transient Ca2+ release from the ER, which may be responsible for σ2 ligand-induced cell death [42]. The presence of the σ<sup>2</sup> fluorescent probes in the ER is consistent with these results. Additional research will be required to determine how σ2 receptors regulate the Ca2+ release channels in the ER. Lysosomal proteases, such as cathepsins, calpains and granzymes, have been reported to contribute to apoptosis [50]. Under physiological conditions, these proteases are found within the lysosomes but are released into the cytoplasm upon exposure to cell damaging agents, thereby triggering a cascade of intracellular events leading to cell death. The σ<sup>2</sup> selective ligand siramesine has been reported to cause lysosomal leakage and induce cell death by caspase-independent mechanisms [41, 45]. The localization of fluorescent σ<sup>2</sup> receptor probes in the lysosomes is consistent with the hypothesis that siramesine induces cell death partially by targeting lysosomes to cause lysosomal damage, the release of proteases, and eventually cell death. Evidence has also been reported that σ2 receptors exist in lipid rafts which are mainly found in the plasma membrane [51]. Lipid rafts play an important role in the signaling associated with a variety of cellular events including adhesion, motility, and membrane trafficking [52, 53]. The observation that σ2 fluorescent ligands are co-localized with cytoplasmic membrane markers, and undergo receptor mediated endocytosis, is consistent with their localization in lipid rafts. How the σ2 receptor is involved in lipid raft function deserves further investigation.

The kinetic studies of internalization of σ2 fluorescent probes in MDA-MB-435 cells were conducted using confocal microscopy[33, 34]. σ2 fluorescent probes enter tumor cells rapidly. For example**, K05-138** and **SW120** reach 50% of the maximal fluorescent intensity (T1/2) in 16 seconds and 11 minutes, respectively (Figure 6A). The internalization of **SW120**  can be blocked by the σ2 selective ligands, **SW43** and siramesine, by 52% and 44%, respectively (Figure 6B). This internalization can not be blocked by the σ1 selective ligand, (+)-pentazocine. The data suggest that the internalization of the σ2 selective ligand is partially mediated by σ2 receptor. In order to study whether the internalization of the σ<sup>2</sup> ligand is mediated by endocytosis, Zeng et al examined the effect of phenylarsine oxide (PAO), a well-characterized endocytosis inhibitor [46], on the internalization of **SW120**. MDA-MB-435 cells were pretreated with 5 or 10 M PAO for 30 min, and then treated with 10 nM **SW120** in the absence or presence of PAO for an additional 30 min. Flow cytometric analysis (Figure 6C) showed that 10 M PAO significantly blocked internalization of **SW120**  by 30%. These data demonstrate that 30% of the 2 receptor ligand was internalized by an endocytosis-mediated mechanism, while the remaining 70 % was internalized by other mechanisms such as passive diffusion. The rapid internalization of σ2 receptors via endocytosis suggests that σ2 selective ligands may potentially serve as receptor-mediated

The two photon and confocal microscopy studies conducted by Zeng et al.[33, 34] have provided useful information for the interpretation of studies demonstrating that σ2 receptor ligands may have a role as cancer chemotherapeutic agents. Mitochondria are a key organelle to regulate the intrinsic apoptotic pathway. Apoptotic signals such as UV irradiation or treatment with chemotherapeutic agents cause the release of cytochrome C from the mitochondria and the subsequent activation of caspase-3 leading to an apoptotic cell death[47]. The subcellular localization of σ2 ligands in mitochondria is consistent with the previous studies that 2 ligands trigger apoptosis in tumor cells by acting on mitochondria [48]. The data are also consistent with our observation by transmission electron microscopy that the 2 ligand siramesine induces distortion of mitochondria (unpublished data). The endoplasmic reticulum (ER) serves as a dynamic Ca2+ storage pool [49]. σ2 selective ligands have been reported to induce transient Ca2+ release from the ER, which may be responsible for σ2 ligand-induced cell death [42]. The presence of the σ<sup>2</sup> fluorescent probes in the ER is consistent with these results. Additional research will be required to determine how σ2 receptors regulate the Ca2+ release channels in the ER. Lysosomal proteases, such as cathepsins, calpains and granzymes, have been reported to contribute to apoptosis [50]. Under physiological conditions, these proteases are found within the lysosomes but are released into the cytoplasm upon exposure to cell damaging agents, thereby triggering a cascade of intracellular events leading to cell death. The σ<sup>2</sup> selective ligand siramesine has been reported to cause lysosomal leakage and induce cell death by caspase-independent mechanisms [41, 45]. The localization of fluorescent σ<sup>2</sup> receptor probes in the lysosomes is consistent with the hypothesis that siramesine induces cell death partially by targeting lysosomes to cause lysosomal damage, the release of proteases, and eventually cell death. Evidence has also been reported that σ2 receptors exist in lipid rafts which are mainly found in the plasma membrane [51]. Lipid rafts play an important role in the signaling associated with a variety of cellular events including adhesion, motility, and membrane trafficking [52, 53]. The observation that σ2 fluorescent ligands are co-localized with cytoplasmic membrane markers, and undergo receptor mediated endocytosis, is consistent with their localization in lipid rafts. How the σ2 receptor

probes for delivering cytotoxic agents to solid tumors.

is involved in lipid raft function deserves further investigation.

Fig. 6. Characterization of σ2 fluorescent probes. A: The internalization kinetics of **SW120** in MDA-MB-435 cells by confocal microscopy studies show that **SW120** enters tumor cells rapidly. B: The internalization of **SW120** in MDA-MB-435 cells are partially blocked by σ<sup>2</sup> selective ligands, **SW43** and siramesine, but not by a σ1 selective ligand, (+)-pentazocine. C: The inhibition of **SW120** internalization by phenylarsine oxide (PAO). MDA-MB-435 cells were preincubated with 5 or 10 M PAO for 30 min at 37C, and then incubated with 10 nM **SW120** for an additional 30 min. The cells were analyzed by flow cytometry. The internalization of **SW120** was significantly reduced by 10 μM PAO (\* P < 0.05).

The σ2 receptor has been validated as a proliferation marker in cell culture and in solid tumors. Therefore it is possible that σ2 fluorescent probes could preferentially label proliferating cells versus non-proliferating cells and serve as agents to image cell proliferation in vivo. This hypothesis was tested in nude mice implanted with murine mammary adenocarcinoma 66 cells and BALB/C mice implanted with mouse mammary carcinoma cell line EMT6[34]. The mice were treated with **SW120 (**50 g in 100 L PBS) for one hour. The peripheral blood mononuclear cells (PBMC), which are commonly used as

Molecular Probes for Imaging the Sigma-2 Receptor 315

receptor in the diagnosis and prediction of therapeutic response will rely on the cloning of the 2 receptor and elucidation of its functional role in normal and tumor cell biology. Note: While this article is accepted, a paper from our group is published in Nature Communication[54]. In this paper we have identified progesterone receptor membrane component 1(PGRMC1) as the putative 2 receptor. This finding will greatly facilitate investigations of the functions of the 2 receptor

This research was supported by grants CA80452, CA81825, and CA102869 awarded by the National Cancer Institute, and grant DAMD17-01-1-0446 awarded by the Department of Defense Breast Cancer Research Program of the US Army Medical Research and Materiel

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**8. References**

controls for non-proliferative cells, and tumor cells were prepared. These cells were analyzed by flow cytometry for **SW120** uptake and for Ki67 expression, a commonly-used proliferation marker. Our data showed that PBMC were Ki67 negative, whereas a large portion of the tumor cells were Ki-67 positive (Figure 7). The data also showed that PBMC were not labeled by **SW120**, whereas a portion of the tumor cells were labeled with **SW120**. The trend of the positive correlation between Ki67 expression and **SW120** labeling implies that the fluorescent may possess in vivo selectivity toward proliferating cells versus nonproliferative cells. These data suggest that σ2 fluorescent probes could be used as imaging agents for monitoring cell proliferation in mice. The data also suggest that σ2 selective ligands hold a potential to serve as therapeutic agents to selectively target tumor cells in vivo.

Fig. 7. Ki67 expression and **SW120** fluorescent intensity in solid mouse breast tumors and peripheral blood mononuclear cells (PBMC) of mice by flow cytometric analysis. The female nude mice were implanted with murine mammary adenocarcinoma 66 cells. BALB/C mice were implanted with mouse mammary carcinoma cell line EMT6. The mice were i.v. injected with **SW120** (50 µg/ mouse) and tissues harvested after 1 hour. The 66 cells and EMT6 cells were dissociated from the solid tumors. PBMC were prepared from the blood of mice. The cells were analyzed for the fluorescent intensity of **SW120** and the Ki67 expression determined by Ki67 immunostaining using a flow cytometer. The data indicate the trend of the positive correlation between Ki67 expression and **SW120** labeling.

#### **6. Conclusions**

The 2 receptor continues to be an important molecular target in the field of tumor biology. The high expression of this receptor in proliferating versus quiescent breast tumors indicates that the 2 receptor is an important clinical biomarker for determining the proliferative status of solid tumors using the functional imaging techniques PET and SPECT. The <sup>2</sup> receptor fluorescent probes are useful to study the subcellular localization and the function of 2 receptors using confocal and two photon microscopy techniques, and may be used to image tumor proliferation in mice using optical imaging techniques. The full utility of the <sup>2</sup>

receptor in the diagnosis and prediction of therapeutic response will rely on the cloning of the 2 receptor and elucidation of its functional role in normal and tumor cell biology.

Note: While this article is accepted, a paper from our group is published in Nature Communication[54]. In this paper we have identified progesterone receptor membrane component 1(PGRMC1) as the putative 2 receptor. This finding will greatly facilitate investigations of the functions of the 2 receptor in normal and tumor cells.

#### **7. Acknowledgement**

This research was supported by grants CA80452, CA81825, and CA102869 awarded by the National Cancer Institute, and grant DAMD17-01-1-0446 awarded by the Department of Defense Breast Cancer Research Program of the US Army Medical Research and Materiel Command Office.

#### **8. References**

314 Breast Cancer – Recent Advances in Biology, Imaging and Therapeutics

controls for non-proliferative cells, and tumor cells were prepared. These cells were analyzed by flow cytometry for **SW120** uptake and for Ki67 expression, a commonly-used proliferation marker. Our data showed that PBMC were Ki67 negative, whereas a large portion of the tumor cells were Ki-67 positive (Figure 7). The data also showed that PBMC were not labeled by **SW120**, whereas a portion of the tumor cells were labeled with **SW120**. The trend of the positive correlation between Ki67 expression and **SW120** labeling implies that the fluorescent may possess in vivo selectivity toward proliferating cells versus nonproliferative cells. These data suggest that σ2 fluorescent probes could be used as imaging agents for monitoring cell proliferation in mice. The data also suggest that σ2 selective ligands hold a potential to serve as therapeutic agents to selectively target tumor cells in

Fig. 7. Ki67 expression and **SW120** fluorescent intensity in solid mouse breast tumors and peripheral blood mononuclear cells (PBMC) of mice by flow cytometric analysis. The female nude mice were implanted with murine mammary adenocarcinoma 66 cells. BALB/C mice were implanted with mouse mammary carcinoma cell line EMT6. The mice were i.v. injected with **SW120** (50 µg/ mouse) and tissues harvested after 1 hour. The 66 cells and EMT6 cells were dissociated from the solid tumors. PBMC were prepared from the blood of mice. The cells were analyzed for the fluorescent intensity of **SW120** and the Ki67 expression determined by Ki67 immunostaining using a flow cytometer. The data indicate the trend of

The 2 receptor continues to be an important molecular target in the field of tumor biology. The high expression of this receptor in proliferating versus quiescent breast tumors indicates that the 2 receptor is an important clinical biomarker for determining the proliferative status of solid tumors using the functional imaging techniques PET and SPECT. The <sup>2</sup> receptor fluorescent probes are useful to study the subcellular localization and the function of 2 receptors using confocal and two photon microscopy techniques, and may be used to image tumor proliferation in mice using optical imaging techniques. The full utility of the <sup>2</sup>

the positive correlation between Ki67 expression and **SW120** labeling.

vivo.

**6. Conclusions**

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