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Aug 1, 2011 (Vol. 31, No. 14)

Imaging Secures Development Role

Early Integration May Speed Progression of Drug Candidates through Pipeline

  • Background Check

    A typical molecular imaging probe, such as a fluorescently labeled antibody, is introduced into a subject (animal, patient) and washed out over time. Contrast is achieved by maximizing the amount of signal at the target while minimizing that remaining associated with nonspecific tissue. A host of factors, from uptake, metabolism, and clearance of the probe to interference by endogenous factors (e.g., autofluorescence) to sensitivity and selectivity of the probe to timing of the imaging, can impact the achievable signal to noise ratio (called tumor to background ratio or TBR in oncology).

    Most currently available probes are “always on”—that is, they continuously emit signal (or in the case of fluorescently labeled probes, they always fluoresce in response to excitation). The targeting moiety should direct most of it to the organ, cell, or receptor of interest, yet TBR often remains an issue.

    What if a probe only emitted a signal when associated with its target? asked Hisataka Kobayashi, M.D., Ph.D., chief scientist in the National Cancer Institute's molecular imaging program. Dr. Kobayashi has been working on “activatable” probes that respond to biological processes. In one example, quenched fluorophores are conjugated to tumor-specific antibodies. Once bound, the probe is internalized where it can be activated by the acidic environment of the endolysosome.

    Although fluorescent imaging is typically limited to very shallow depths, it can be used to guide open surgery—for example, in the brain—and most parts of the body (except the heart and brain) can be reached by using endoscopy as well. “We can go almost anywhere through the GI or urinary tract, or peritoneal or plural spaces,” Dr. Kobayashi pointed out.

    He also uses different wavelength fluorophores and other different energy probes, to simultaneously query several discrete entities. In addition, MRI, nuclear, PET/CT, and optical can also be multiplexed to generate more information from the body, the radiologist added, “to detect things much better than the single modality.”

  • It's Personal

    Dr. Gelovani, who also leads the development of molecular imaging agents at the Center for Advanced Biomedical Imaging Research at the University of Texas' MD Anderson Cancer Center, wants to see molecular imaging used more as a means of early detection of cancers, as well as a way to stratify patients based on unique mutations or genomic or phenotypic abnormalities.

    Take, for example, small-cell lung carcinoma patients whose tumor is dependent on a mutation that locks the tyrosine kinase part of the epidermal growth factor receptor (EGFR) into a particular dominant active configuration. Such patients respond positively to therapy with EGFR inhibitors.

    “We have explored the chemical space of that configuration and developed a particular molecular imaging agent that would have selectivity for that particular activating mutation and irreversibly bind to that dominant active EGFR kinase,” said Dr. Gelovani.

    “Therefore, we can now enable imaging of patients noninvasively using PET/CT to predict the responsiveness or resistance of primary and metastatic tumor lesions in individual patients. We are really getting closer to that notion of individualized therapy.”

    Molecular imaging can seek out its target wherever it lies, is quantitative, and (because it's noninvasive) can serially query the same target. A biopsy, on the other hand, only reveals a mutation where it is sought, which because of the tumor heterogeneity often does not represent the rest of the tumor and metastatic tumor lesions. In contrast, molecular imaging can aid the characterization of tumors in the whole body at once.

  • Do It My Way

    Yet, not all scans—not even all PET scans—are created equal, and this has implications for the interpretation of clinical trials. “The problem is not so much that the drug is different, but that each center does the scan in its own particular way. Their equipment is calibrated in their own way, and radiologists interpret the study in his or her own way,” observed Peter Conti, M.D., Ph.D., co-chair of the Society of Nuclear Medicine's Clinical Trials Network (CTN).

    At the same time, drug companies may be pretty naïve as to how to structure the protocols, the University of Southern California professor continued. “So there's a tug of war between the right way to do it as per the radiologists and the right way to do it as per the drug company,” leaving the FDA with the problem of not being able to compare the studies.

    Basic parameters need to be optimized and standardized. “We can argue about which way may be better than the other, but we have to agree at least on some baseline,” Dr. Conti said. From there, it's a matter of education and training.

    Progress, he said, has been good. Manufacturers are trying to come up with common analytical methods so that data is transparent as to which device was used to generate it. More than 300 imaging sites worldwide have begun the process to join the CTN imaging registry, and over 100 have been qualified. More than 60 scanners have been fully validated.

  • Evaluation of COX-2 Binding Property of Fluorocoxib A Probe

    Click Image To Enlarge +
    Figure 1. In vitro binding of the Fluorocoxib A probe to COX-2 positive HT29-luc2 cells was shown after imaging with NuanceFX. Staining of the cytoplasm by the probe was displayed in red. Nuclei staining with DAPI was shown in black. Control COX-2 negative cell line HCT116 lacked staining by the Fluorocoxib A probe.

    Cyclooxygenase 2 (COX-2) is an important inflammation marker that is also involved in tumor development and progression. Aberrant induction of COX-2 and upregulation of the prostaglandin cascade play a significant role in carcinogenesis. Blockade of the process has strong potential for cancer prevention and therapy. Blockage of COX-2 activity either by nonselective COX-2 inhibitors, such as aspirin and ibuprofen, or by selective COX-2 inhibitors, such as celecoxib, reportedly reduce the risk of human cancer and precancerous lesions at all anatomic sites thus far investigated.

    Fluorocoxib A is a recently developed fluorescent probe that can specifically target and inhibit COX-2. Scientists at Caliper Life Sciences (www.caliperls.com) recently evaluated the COX-2 binding property of this probe both in vitro and in vivo with fluorescence imaging. Multispecral tissue fluorescence microscopy from the company’s Nuance imaging system showed that the Fluorocoxib A probe exhibited specific binding to COX-2 expressed in HT29 colon cancer cells but not to HCT116 colon cancer cells, which lack COX-2 expression (Figure 1).

  • Click Image To Enlarge +
    Figure 2. Nu/Nu mice were s.c. injected with 2x106 of HCT116 (left flank) and HT29 (right flank) tumor cells. Most of tumors were approximately 100 mm3 at day 16, when mice were injected with either Fluorocoxib A probe (2 mg/kg, i.p.) or control 5-ROX dye. Mice were imaged at 3, 6, and 24 hours after injection with IVIS Spectrum (Ex570, Em640). Images shown were taken at three hours. COX-2 positive HT29 tumor showed preferential labeling by the Fluorocoxib A probe whereas the COX-2 negative HCT116 showed very little binding. Quantification of fluorescence signal from the tumors showed a steady decrease of the fluorescence signal at the 6 and 24 hour imaging time points. A twofold difference between the COX-2 positive HT29 and COX-2 negative HCT116 was observed in Fluorocoxib A injected mice at 3 and 6 hours.

    They further evaluated Fluorocoxib A in vivo. They established subcutaneous tumors with both HT29 and HCT116 colon cancer cells. Following tail vein injection of the probe, they were able to detect specific targeting of the HT29 tumors, but not the COX-2 negative HCT116 tumors (Figure 2). Quantification of fluorescence signal from tumors showed that fluorescence detection was the highest at 3 hours and declined substantially at 6 and 24 hours. The HT29/HCT116 ratio indicated that the signal from the HT29 tumor was twofold above the background signal in comparison to the HCT116 tumor at the three and six time points.


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