Mohan R. Aruva

PET Imaging of VPAC1 Expression in Experimental and Spontaneous Prostate Cancer

Among U.S. men, prostate cancer (PC) accounts for 29% of all newly diagnosed cancers. A reliable scintigraphic agent to image PC and its metastatic or recurrent lesions and to determine the effectiveness of its treatment will contribute to the management of this disease. All PC overexpresses VPAC1 receptors. This investigation evaluated a probe specific for a 64Cu-labeled receptor for PET imaging of experimental human PC in athymic nude mice and spontaneously grown PC in transgenic mice. Methods: The probe, TP3939, was synthesized, purified, and labeled with 64Cu and 99mTc. Using a muscle relaxivity assay, biologic activity was assessed and inhibitory concentrations of 50% calculated. Receptor affinity (Kd) for human PC3 cells was determined using 99mTc-TP3939 and 64CuCl2. Blood clearance and in vivo stability were studied. After intravenous administration of either 64Cu-TP3939 or 64CuCl2 in PC3 xenografts and in transgenic mice, PET/CT images were acquired. Prostate histology served as the gold standard. Organ distribution studies (percentage injected dose per gram [%ID/g]) in normal prostate were performed. The ratios of tumor to muscle, tumor to blood, normal prostate to muscle, and tumor to normal prostate were determined. Results: Chemical and radiochemical purities of TP3939 were 96.8% and 98% ± 2%, respectively. Inhibitory concentrations of 50% and affinity constants were 4.4 × 10−8 M and 0.77 × 10−9 M, respectively, for TP3939 and 9.1 × 10−8 M and 15 × 10−9 M, respectively, for vasoactive intestinal peptide 28. Binding of 64CuCl2 to PC3 was nonspecific. Blood clearance was rapid. In vivo transchelation of 64Cu-TP3939 to plasma proteins was less than 15%. 64Cu-TP3939 uptake in PC was 7.48 ± 3.63 %ID/g at 4 h and 5.78 ± 0.66 %ID/g at 24 h after injection and was significantly (P < 0.05) greater than with 64CuCl2 (4.79 ± 0.34 %ID/g and 4.03 ± 0.83 %ID/g at 4 and 24 h, respectively). The ratios of PC to normal prostate at 4 and 24 h were 4 and 2.7, respectively. 64Cu-TP3939 distinctly imaged histologic grade IV prostate intraepithelial neoplasia in transgenic mice, but 18F-FDG and CT did not. Conclusion: Data indicate that TP3939, with its uncompromised biologic activity, delineated xenografts and cases of occult PC that were not detectable with 18F-FDG. 64Cu-TP3939 is a promising probe for PET imaging of PC. It may also be useful for localizing recurrent lesions and for determining the effectiveness of its treatment.

PET Imaging of CCND1 mRNA in Human MCF7 Estrogen Receptor–Positive Breast Cancer Xenografts with Oncogene-Specific [64Cu]Chelator-Peptide Nucleic Acid-IGF1 Analog Radiohybridization Probes

Treatment of breast cancer is hampered by a large unmet need for rapid, sensitive, specific staging and stratification of palpable and nonpalpable abnormalities. Mammography and physical examination miss many early breast cancers, yet detect many benign lesions. Cyclin D1, encoded by CCND1 messenger RNA (mRNA), and insulin-like growth factor 1 receptor (IGF1R) are key regulators of cell proliferation that are overexpressed in most breast cancers. Therefore, we hypothesized that malignant breast masses could be imaged and quantitated externally by PET with a dual-specificity probe that targets both CCND1 mRNA and IGF1R. Methods: We designed a CCND1-specific peptide nucleic acid (PNA) hybridization sequence (CTGGTGTTCCAT), separated by a C-terminal spacer to a cyclized IGF1 peptide analog (d-Cys-Ser-Lys-Cys), for IGF1R-mediated endocytosis. On the N-terminus we attached a chelator (1,4,7-tris(carboxymethylaza)cyclododecane-10-azaacetyl [DO3A]) for the positron-emitting nuclide 64Cu. We administered the [64Cu]CCND1-IGF1 analog radiohybridization probes, as well as sequence controls, by tail vein to immunocompromised female NCr mice bearing human MCF7 estrogen-dependent, receptor-positive xenografts. We imaged the mice by PET and CT 4 and 24 h later, and measured tissue distribution of the radiohybridization probes. Results: We observed 8 ± 2-fold higher PET intensity in the center of the breast cancer xenografts than in the contralateral tissues at 24 h after injection of the [64Cu]CCND1-IGF1 analog radiohybridization probe. IGF1 blocking yielded significantly weaker images (P < 0.05) relative to the tumor-free side at 24 h after injection, as did a PNA mismatch probe, a peptide mismatch probe, and free 64CuCl2. Conclusion: These results are consistent with our hypothesis for radiohybridization PET of overexpressed CCND1 mRNA, dependent on IGF1R-mediated endocytosis, in suspect masses. Early noninvasive detection of initial cancerous transformation, as well as invasive or recurrent breast cancer, with dual-specificity radiohybridization probes, might enable molecularly targeted staging, stratification, and choice of therapy.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) receptor specific peptide analogues for PET imaging of breast cancer: In vitro/in vivo evaluation.

Vasoactive intestinal peptide and pituitary adenylate cyclase activating peptide have high affinity for VPAC1, VPAC2 and PAC1 receptors overexpressed on human cancer cells. Four potent analogues of these peptides, TP3939, TP3982, TP4200 and TP3805 were labeled with (64)Cu and evaluated ex vivo and in vivo to asses their biological activity and receptor specificity. The ultimate goal is to utilize (64)Cu analogues for positron emission tomography (PET) imaging of breast cancers in humans. Radiochemical purity of each analogue was >92%. The muscle relaxivity assay revealed IC(50) to be 5.3x10(-8) M, 4.4x10(-8) M, 8.1x10(-8) M, 8.1x10(-9) M and Kd values determined by receptor specific cell binding assays were 3.3 nM, 0.33 nM, 0.2 nM and 0.72 nM for TP3805, TP3939, TP3982, and TP4200 respectively. The receptor affinity, using human breast cancer tissues, was 10.93 times greater than normal breast tissues. RT-PCR confirmed increased VPAC1 receptor expression on human breast tumor cells over normal cells and corroborated with autoradiography data. The blood clearance was rapid and in vivo translocation of (64)Cu to plasma protein was <15%. Data demonstrate that these analogues are potent, have uncompromised biological activity and are worthy of further evaluation for accurate PET imaging of human breast cancers and in determining malignant and benign lesions.

99mTc-peptide-peptide nucleic acid probes for imaging oncogene mRNAs in tumours.

Summary Imaging oncogene mRNA in tumours would provide a powerful tool for the early detection of occult malignant lesions. The goal was to prepare a chimera consisting of a dodecamer antisense peptide nucleic acid (PNA) specific for c‐MYC oncogene overexpressed in human breast cancer cells and a chelating moiety that facilitates quantitative radiolabelling with 99mTc and evaluate it for hybridization and tissue distribution in laboratory animals. The pentapeptide chelator‐PNA dodecamer specific for c‐MYC mRNA was extended from a solid support by 9‐fluorenylmethyloxycarbonyl (Fmoc) coupling. Similarly, a chelator‐PNA chimera with four central mismatches was also prepared which served as a control. The chimeras were purified, characterized and evaluated for hybridization to c‐MYC mRNA by fluorescent, real‐time polymerase chain reaction (RT‐PCR). The chimeras were labelled with 99mTc and their tissue distribution was examined in athymic nude mice bearing experimental human breast tumours. 99mTc radiolabelling was quantitative and presented a single peak in reversed phase liquid chromatography. Fluorescent real‐time polymerase chain reactions using primer and fluorescent probe sets previously calculated for c‐MYC mRNA demonstrated inhibition of reverse transcription by the c‐MYC specific chimera as compared to that of the control. Tissue distribution studies of antisense and mismatch chimeras at 4 h and 24 h after administration displayed modest accumulation in the liver, and appreciable levels in tumours. These observations suggest that 99mTc‐peptide‐PNA probes might be useful for imaging gene expression in tumours, and the approach is worthy of further investigation.

External imaging of CCND1, MYC, and KRAS oncogene mRNAs with tumor-targeted radionuclide-PNA-peptide chimeras.

Abstract: In 2005, breast cancer will kill approximately 40,410 women in the U.S., and pancreatic cancer will kill approximately 31,800 men and women in the U.S. Clinical examination and mammography, the currently accepted breast cancer screening methods, miss almost half of breast cancers in women younger than 40 years, approximately one‐quarter of cancers in women aged 40‐49 years, and one‐fifth of cancers in women over 50 years old. Pancreatic cancer progresses rapidly, with only 1% of patients surviving more than 5 years after diagnosis. However, if the disease is diagnosed when it is localized, the 5‐year survival is approximately 20%. It would be beneficial to detect breast cancer and pancreatic cancer at the earliest possible stage, when multimodal therapy with surgery, radiotherapy, and chemotherapy have the greatest chance of prolonging survival. Human estrogen receptor‐positive breast cancer cells typically display elevated levels of Myc protein due to overexpression of MYC mRNA, elevated cyclin D1 protein due to overexpression of CCND1 mRNA, and elevated insulin‐like growth factor 1 receptor (IGF1R) due to overexpression of IGF1R mRNA. We hypothesized that scintigraphic detection of MYC or CCND1 peptide nucleic acid (PNA) probes with an IGF1 peptide loop on the C‐terminus, and a Tc‐99m‐chelator peptide on the N‐terminus, could measure levels of MYC or CCND1 mRNA noninvasively in human IGF1R‐overexpressing MCF7 breast cancer xenografts in immunocompromised mice. Similarly, human pancreatic cancer cells typically display elevated levels of KRAS mRNA and elevated IGF1R. Hence, we also hypothesized that a KRAS Tc‐99m‐chelator PNA‐peptide probe could detect overexpression of KRAS mRNA in pancreatic cancer xenografts by scintigraphic imaging, or by positron emission tomography (PET) with a KRAS Cu‐64‐chelator PNA‐peptide. Human MCF7 breast cancer xenografts in immunocompromised mice were imaged scintigraphically 4‐24 h after tail‐vein administration of MYC or CCND1 Tc‐99m‐chelator PNA‐peptides, but not after administration of mismatch controls. Similarly, human Panc‐1 pancreatic cancer cells xenografts were imaged scintigraphically 4 and 24 h after tail‐vein administration of a KRAS Tc‐99m‐chelator PNA‐peptide, and AsPC1 xenografts were imaged by PET 4 and 24 h after tail‐vein adminstration of a KRAS Cu‐64‐chelator PNA‐peptide. The radioprobes distributed normally to the kidneys, livers, tumors, and other tissues. External molecular imaging of oncogene mRNAs in solid tumors with radiolabel‐PNA‐peptide chimeras might in the future provide additional genetic characterization of pre‐invasive and invasive breast cancers.

Radiohybridization PET imaging of KRAS G12D mRNA expression in human pancreas cancer xenografts with [64Cu]DO3A-peptide nucleic acid-peptide nanoparticles

There is a compelling need to image pancreas cancer at an early stage. Human pancreas cancer cells display elevated levels of KRAS protein due to high copy numbers of KRAS mRNA, and elevated levels of insulin-like growth factor 1 receptor (IGF1R) due to overexpression of IGF1R mRNA. Therefore we hypothesized that pancreas cancer could be detected in vivo with a single probe that targets both KRAS mRNA and IGF1R. Because positron emission tomography (PET) is a sensitive imaging technique, we designed a probe incorporating the positron-emitting nuclide 64Cu. The KRAS-specific hybridization probe consisted of 1,4,7-tris(carboxymethylaza)cyclododecane-10-azaacetyl (DO3A) on the N-terminus of a peptide nucleic acid (PNA) hybridization sequence (GCCATCAGCTCC) linked to a cyclized IGF1 peptide analog (D-Cys-Ser-Lys-Cys) on the C-terminus, for IGF1R-mediated endocytosis. A series of such KRAS radiohybridization probes with 0, 1, 2, or 3 mismatches to KRAS G12D mRNA, including exact matches to wild type KRAS mRNA and KRAS G12V mRNA, along with a double D(Ala) replacement IGF1 peptide control, were assembled by continuous solid phase synthesis. To test the hypothesis that KRAS-IGF1 dual probes could specifically image KRAS mRNA expression noninvasively in human IGF1R-overexpressing AsPC1 pancreas cancer xenografts in immunocompromised mice, [64Cu]PNA radiohybridization probes and controls were administered by tail vein. The [64Cu]KRAS¬-IGF1 radiohybridization probe yielded strong tumor contrast in PET images, 8.6±1.4-fold more intense in the center of human pancreas cancer xenografts than in the contralateral muscle at 4 h post-injection. Control experiments with single base KRAS¬ mismatches, an IGF1 peptide mismatch, and a breast cancer xenograft lacking KRAS activation yielded weak tumor contrast images. These experiments are consistent with our hypothesis for noninvasive PET imaging of KRAS oncogene expression in pancreas cancer xenografts. Imaging oncogene mRNAs with radiolabel-PNA-peptide nanoparticles might provide specific genetic characterization of pre-invasive and invasive pancreas cancers for staging and choice of therapy.

Imaging Human Pancreatic Cancer Xenografts by Targeting Mutant KRAS2 mRNA with [111In]DOTAn-Poly(diamidopropanoyl)m-KRAS2 PNA-d(Cys-Ser-Lys-Cys) Nanoparticles

95% of patients with ductal pancreatic cancer carry 12th codon activating mutations in their KRAS2 oncogenes. Early whole body imaging of mutant KRAS2 mRNA activation in pancreatic cancer would contribute to disease management. Scintigraphic hybridization probes to visualize gene activity in vivo constitute a new paradigm in molecular imaging. We have previously imaged mutant KRAS2 mRNA activation in pancreatic cancer xenografts by positron emission tomography (PET) based on a single radiometal, (64)Cu, chelated to a 1,4,7,10-tetra(carboxymethylaza)cyclododecane (DOTA) chelator, connected via a flexible, hydrophilic spacer, aminoethoxyethoxyacetate (AEEA), to the N-terminus of a mutant KRAS2 peptide nucleic acid (PNA) hybridization probe. A peptide analogue of insulin-like growth factor 1 (IGF1), connected to a C-terminal AEEA, enabled receptor-mediated endocytosis. We hypothesized that a polydiamidopropanoyl (PDAP) dendrimer (generation m), with increasing numbers (n) of DOTA chelators, extended via an N-terminal AEEA from a mutant KRAS2 PNA with a C-terminal AEEA and IGF1 analogue could enable more intense external imaging of pancreatic cancer xenografts that overexpress IGF1 receptor and mutant KRAS2 mRNA. ([(111)In]DOTA-AEEA)(n)-PDAP(m)-AEEA(2)-KRAS2 PNA-AEEA-IGF1 analogues were prepared and administered intravenously into immunocompromised mice bearing human AsPC1 (G12D) pancreatic cancer xenografts. CAPAN2 (G12 V) pancreatic cancer xenografts served as a cellular KRAS2 mismatch control. Scintigraphic tumor/muscle image intensity ratios for complementary [(111)In](n)-PDAP(m)-KRAS2 G12D probes increased from 3.1 +/- 0.2 at n = 2, m = 1, to 4.1 +/- 0.3 at n = 8, m = 3, to 6.2 +/- 0.4 at n = 16, m = 4, in AsPC1 (G12D) xenografts. Single mismatch [(111)In](n)-PDAP(m)-KRAS2 G12 V control probes showed lower tumor/muscle ratios (3.0 +/- 0.6 at n = 2, m = 1, 2.6 +/- 0.9 at n = 8, m = 3, and 3.7 +/- 0.3 at n = 16, m = 4). The mismatch results were comparable to the PNA-free [(111)In]DOTA control results. Simultaneous administration of nonradioactive Gd(n)-KRAS2 G12 V probes (n = 2 or 8) increased accumulation of [(111)In](8)KRAS2 G12 V probes 3-6-fold in pancreatic cancer CAPAN2 xenografts and other tissues, except for a 2-fold decrease in the kidneys. As a result, tissue distribution tumor/muscle ratios of (111)In uptake increased from 3.1 +/- 0.5 to 6.5 +/- 1.0, and the kidney/tumor ratio of (111)In uptake decreased by more than 5-fold from 174.8 +/- 17.5 to 30.8 +/- 3.1. Thus, PDAP dendrimers with up to 16 DOTA chelators attached to PNA-IGF1 analogs, as well as simultaneous administration of the elevated dose of nonradioactive Gd(n)-KRAS2 G12 V probes, enhanced tumor uptake of [(111)In](n)KRAS2 PNA probes. These results also imply that Gd(III) dendrimeric hybridization probes might be suitable for magnetic resonance imaging of gene expression in tumors, because the higher generations of the dendrimers, including the NMR contrast Gd(n)-KRAS2 G12 V probes, improved tumor accumulation of the probes and specificity of tumor imaging.

Imaging oncogene expression.

Abstract: In 2003, approximately 39,800 women in the US will die from breast cancer. Mammography and physical examination miss up to 40% of early breast cancers. Moreover, if an abnormality is found, an invasive diagnostic procedure must still be performed to determine if the breast contains atypia or cancer, even though approximately 85% of abnormalities are benign. Scintigraphic imaging of gene expression in vivo by noninvasive means could direct physicians to appropriate targets for intervention at the onset of disease and thereby significantly impact patient management. Until now, no method has been available to image specific overexpressed oncogene mRNAs in vivo by scintigraphic imaging. We hypothesize that gamma‐emitting Tc‐99m‐PNA‐peptides can be taken up by human ER+ and ER− breast cancer xenografts, hybridize to complementary mRNA targets in those cells, and concentrate sufficiently in tumor tissue to allow noninvasive imaging of oncogene overexpression. To prepare the probes, peptide analogs of insulin‐like growth factor 1 (IGF1) were extended from a solid support by Fmoc coupling. Peptide nucleic acid (PNA) dodecamers antisense to CCND1 and MYC mRNAs were then extended from the N‐terminus of IGF1, followed by a chelator peptide, using Fmoc coupling for all residues. The cysteine thiols were cyclized on the solid support, either before or after PNA extension. This simplified synthetic approach allows preparation of a variety of multipeptide disulfide‐bridged PNA chimeras. A chelating peptide‐PNA chimera antisense to MYC mRNA was then labeled efficiently with Tc‐99m, yielding a single product. Tissue distribution studies of antisense and mismatch chimeras at 4 h and 24 h after administration displayed modest accumulation in the liver and kidneys, with appreciable levels in tumors. This result enables testing of Tc‐99m‐peptide‐PNA probes to image gene expression in tumors.

Synthesis of novel peptide nucleic acid-peptide chimera for non-invasive imaging of cancer

A chelator-peptide-PNA-peptide chimera specific for KRAS has been prepared by continuous solid phase coupling with a C-terminal insulin-like growth factor 1 (IGF1) ligand, d(cys-ser-lys-cys), and N-terminal bis(s-benzoyl thioglycoloyl) diaminopropanoate chelator for radionuclide labeling. The probe was purified by RP-HPLC and characterized by MALDI-TOF mass spectroscopy. The probe was labeled with 99 mTc and 64Cu. Both labeled probes accumulated in human pancreatic cancer xenografts in immunocompromised mice. Control experiments with mismatch chimeras and control xenografts will be necessary to determine the specificity of this molecular diagnostic strategy.

Receptor-mediated internalization of chelator-PNA-peptide hybridization probes for radioimaging or magnetic resonance imaging of oncogene mRNAs in tumours

Early external detection of cancer gene activity might enable early treatment of cancer and might reduce cancer mortality. We hypothesized that oncogene mRNA overexpressed at thousands of copies per malignant cell in a zone of transformed cells could be imaged externally by scintigraphic imaging, PET (positron emission tomography) or MRI (magnetic resonance imaging) with PNA (peptide nucleic acid) hybridization probes that include chelators for metal cations and a cyclized peptide analogue of IGF-1 (insulin-like growth factor 1), D(Cys-Ser-Lys-Cys), to mediate internalization by IGF1R (IGF-1 receptor) overexpressed on cancer cells. We observed that human MCF7 breast cancer cells that overexpress IGF1R efficiently internalized fluorescein-chelator-PNA-D(Cys-Ser-Lys-Cys) to the cytoplasm, but not with D(Cys-Ala-Ala-Cys). Scintigraphic imaging of MCF7 xenografts in immunocompromised mice revealed that CCND1 and MYC [(99m)Tc]chelator-PNA-D(Cys-Ser-Lys-Cys) probes yielded xenograft. PET imaging with [(64)Cu]chelator-PNA-D(Cys-Ser-Lys-Cys) yielded stronger signals. Scintigraphic imaging of human AsPC1 pancreas cancer xenografts with [(99m)Tc]chelator-KRAS PNA-D(Cys-Ser-Lys-Cys) yielded strong xenograft signals. Stronger xenograft image intensities were obtained by PET imaging of [(64)Cu]chelator-KRAS PNA-D(Cys-Ser-Lys-Cys). MRI required extension of chelator-polydiamidopropanoate dendrimers from the N-termini of the PNA probes to increase the number of contrast paramagnetic gadolinium (III) cations per probe. These results provide a basis for detection of oncogene activity in tissues from outside the body by hybridization with metal-chelator-PNA-peptides that are selectively internalized by cancer cells.