August 1, 2013 (Vol. 33, No. 14)

Sridhar Nadamuni

Pharmacoimaging combines traditional pharmacology with broad imaging capabilities, enabling better decision making, improved clinical trial design, and more confident lead candidate selection.

Imaging technologies are also being used for drug safety assessment, both to screen drugs in the discovery stage and to provide supporting data later on.

Increasingly, imaging will be incorporated into preclinical and clinical studies to interrogate and quantify both drug efficacy and safety in the same animals or patients, according to presentations given at World Pharma Congress’ recent “Molecular Imaging Conference in Drug Discovery and Development.”

“The uniqueness of our approach is to focus more on the drug target and disease biology up front, as we develop a picture of whether and how in vivo imaging can enhance intended decision making,” says Patrick McConville, Ph.D., CSO and COO at Molecular Imaging.

“Our approach drives successful pharmacoimaging by ensuring inherent relevance of the data output to the intended biological or molecular question, and how this relates to the clinical path and related decision making,” Dr. McConville explained.

In vivo imaging facilitates access to information that is more directly relevant to drug mechanism or that cannot be obtained through other means. Further, imaging biomarkers are being used to provide readout of a broad range of relevant physiological and functional parameters, including metabolism, cellularity, proliferation, hypoxia, and inflammation. Such data enables measurement of disease progression and response to targeted therapies, at a mechanistic level.

Imaging has become a standard tool for characterizing biologic molecule targeting and biodistribution using isotopes for PET, SPECT, and fluorophores. Labeling of antibodies, antibody-drug conjugates, proteins and peptides, and nanoparticles for image-based detection, is a powerful approach to drug discovery and development.

New imaging modalities including photoacoustic technology and clinical fluorescence will provide powerful approaches to translational medicine.

High-Throughput Toxicity Screening

The CMOS-based microelectrode array (MEA) system combines the advantages of both MEA systems and patch clamp systems, enabling noninvasive recording from complete cellular monolayers. It can record intracellular action potentials. With more than 16,000 sensor sites that can be addressed individually, the chip increases data output at single-cell resolution.

The new silicon microelectrode array platform developed by Dries Braeken, Ph.D., R&D team leader, and his colleagues at the Interuniversitair Micro-Electronica Centrum (IMEC) in Belgium, is equipped with thousands of sensors on a single silicon chip, to target single cells growing on the surface. The standard silicon technology integrating all amplifiers, filters, stimulation and impedance circuitry into the chip, facilitates massive parallelization and increasingly efficient and cheaper systems. Additional ultra-small electrodes enable recording and stimulation of single cells.

As Dr. Braeken explained, “We developed a new assay to record signals that are much larger in amplitude than extracellular signals—up to 20 mV versus one to two mV.”

These signals closely resemble intracellular signals recorded with patch clamp techniques. The assay makes use of the integrated stimulation circuitry to create transient nanopores by electroporation of the membrane patch. This creates a low-resistance path to the intracellular milieu, enabling observation of the full shape of the action potential. The intracellular signals are available from a single cell over five consecutive days.

For example, cardiac cellular signals recorded by this chip allowed for the accurate measurement of cardiac action potential duration, upstroke velocity, and different phases of the downstroke.

The CMOS MEA system is poised for a multiwell format, allowing ultra-high throughput without compromising signal quality. Software design and automation should enable extraction of specific ion channel parameters for drug screening. The technology can be integrated in organ-on-a-chip systems for more advanced predictive toxicology screening of cardiac or neuronal cells.

Dr. Braeken also presented a lens-free imaging method that is cheaper than conventional microscopy, while providing detailed information and a large field of view.

“Lens-free imaging makes it possible to perform bright-field microscopy of cells with high resolution—to 1.4 µm—and a large field of view—up to 20 mm,” he elaborates.

With this approach, the sample under investigation is illuminated by a coherent light source, and diffraction patterns are captured on a CMOS imager chip positioned underneath the sample. Images from cell cultures are obtained using dedicated image reconstruction algorithms.

“The lens-free imaging system is an optical component-free, compact, and cheap imaging system with high resolution and a large field of view,” Dr. Braeken claimed.

The resulting images are comparable to those taken with a conventional phase contrast microscope. The technique also enables reconstruction of a holographic image of cells. It is portable inside a cell incubator for direct time-lapse imaging.

Integrating the lens-free imaging system into cell bioreactors helps to monitor the kinetics of cell growth and differentiation, for example, in human stem cell cultures.

Scanning electron micrograph of a cardiac cell grown on the IMEC CMOS multi-electrode array. The chip is able to record intracellular action potentials from individual cells in a noninvasive manner, opening the door to novel drug screening platforms based on silicon technology.

Determining PK/PD

Different imaging modalities are being used to monitor disease progression and to analyze therapeutic efficacy in preclinical stages of drug development.

According to Werner Scheuer, Ph.D., group leader, preclinical optical imaging, pharmacology TR-PD, and pharmaceutical research at Roche Diagnostics, fluorescence and bioluminescence technologies are best because of their sheer simplicity, fast scanning times, nonhazardous radiation, and nonradioactive isotopes.

In pharmaceutical drug development, long acquisition times can hinder efforts to determine pharmacodynamics and pharmacokinetics in preclinical models. Speaking with GEN, Dr. Scheuer explained that optical imaging has very short acquisition times, ranging from one second to two minutes.

Using fluorescence-labeled antibodies targeting a tumor-associated surface antigen, accumulation in tumor tissue can be accomplished within six to 24 hours post-injection. As such, it is possible to monitor the biodistribution of the therapeutic antibodies in preclinical cancer xenografts. By using fluorophores that differ in their emission spectra, it is possible to examine a combination of antibodies. Such multiplexing studies cannot be performed using radioactive isotopes.

Further, in combination with luciferase-transfected tumor cells, it is possible to monitor binding kinetics and antitumoral efficacy noninvasively and simultaneously. Fluorescence-labeled antibodies are stable ranging from six to 12 months at -20°C, making them superior to radioactive isotopes. Furthermore, noninvasive fluorescence imaging in mice allows monitoring of blood peak levels, half-life, organ distribution and saturation kinetics. It improves the quality of data for pharmacokinetic and pharmacodynamic simulation. It also reduces the number of animals needed, reducing time and costs.

Combination of fluorescence with bioluminescence and subsequent examination of explanted organs by 3D multispectral fluorescence histology enables the monitoring of primary tumor growth, metastasis, and angiogenesis. Dr. Scheuer and his colleagues have demonstrated the advantages of optical imaging in the combined measurement of pharmacodynamics and pharmacokinetics in cancer xenografts.

Predicting Therapeutic Response

Christopher P. Leamon, Ph.D., vp, research at Endocyte, presented a novel and personalized approach to identify patients who were most likely to benefit from folate receptor (FR)-targeted therapy.

Dr. Leamon has invented small molecule drug conjugates to target receptors that are overexpressed in cancer or arthritis. Vintafolide is a small molecule targeting the folate receptor that is linked to a potent chemotherapy drug. Dr. Leamon also developed the companion imaging agent, etarfolatide, which consists of the same small molecule that targets the folate receptor, but is instead conjugated to a 99mTc-based imaging group.

Patients who are identified using etarfolatide as overexpressing the folate receptor are then treated with vintafolide. The companion imaging agent is used to identify patients that overexpress the specific receptor, so that only patients who are likely to respond to treatment will actually be given the drug.

Vintafolide (MK-8109/EC145) and etarfolatide (EC20) are currently being studied in a Phase III trial involving patients with platinum-resistant ovarian cancer, and a Phase IIb study on patients with non-small-cell lung cancer (NSCLC).

Dr. Leamon reported that analysis of Phase II data shows that etarfolatide can identify ovarian cancer and NSCLC patients who could benefit from vintafolide. Patients identified with 100% FR+ target tumor lesions showed substantial progression-free survival compared to patients with 10% to 90% FR+ target lesions, when treated with single-agent vintafolide.

“Due to the numerous benefits of companion imaging agents, including the ability to conduct a noninvasive, whole-body scan of a patient and the ability to decrease clinical risk through use early on in drug development, interest in developing companion imaging technologies will only continue to grow,” he said.

Targeted small molecules linked to chemotherapy drugs are used in conjunction with a companion imaging agent. Vintafolide consists of a small molecule targeting the folate receptor that is linked to a potent chemotherapy drug. The companion imaging agent, etarfolatide, consists of the same small molecule that targets the folate receptor, but is instead conjugated to a 99mTc-based imaging group. [Endocyte]

Translational R&D, Clinical Trials

The increasing use of PET imaging has led to a dramatic increase in the number of novel F-18 tracers in preclinical studies, advancing into clinical development. The utility of PET imaging can be enhanced by creating new F-18 PET tracers that can pair with the ever expanding armamentarium of target drugs, said Scott Edwards, Ph.D., vp and GM, R&D, SciFluor Life Sciences.

Researchers at SciFluor are developing new methodologies for synthesizing F-18 tracers including innovative chemistry to incorporate F-18 into a wider array of small molecule drugs.

“SciFluor has developed SF0034 as an improved potassium channel opener for use in the treatment of partial-onset seizures,” he explained. “To aid in the preclinical and potentially clinical development of SF0034, we have also developed an F-18 radiolabeled analog of SF0034 for noninvasive PET imaging.”

According to Dr. Edwards, “This approach allows registered medicines, compounds in clinical development that have established clinical proof of concept, to be further optimized to generate new pre-clinical candidates without an extensive drug discovery effort.”

The mechanistic and clinical development knowledge of the parent compounds is driving preclinical and clinical development of these new chemical entities.

Elsewhere, studies being conducted at Imaging Endpoints are focused on facilitating effective clinical trial design, based on the bidirectional translational approach involving clinician feedback and preclinical response, said Ronald L. Korn, M.D., Ph.D., the firm’s founder and CEO.

Dr. Korn said that despite successful preclinical studies, drugs often show dose-related toxicity. He referred to his experience studying vascular disrupting drugs, which were effective in patients with metastasis, but still showed potentially damaging effects on red blood cells.

“We then found the most appropriate preclinical model to test the hypothesis— an MRI using a special contrast agent that measures oxidation, to screen the preclinical agent, which showed the oxidizing effects of this agent,” he explained.

Dr. Korn also cited his experience with stromal disrupting agents, which were promising in early preclinical testing. “We used MRI perfusion imaging to test its mechanism of action,” he said. Interestingly enough, “we also noticed that PET scans were going cold in preclinical phase, but were more appropriate for human testing,” Dr. Korn added.

Working in collaboration with scientists at TexRAD, Dr. Korn and colleagues at Imaging Endpoints used advanced imaging technology and software to extract information from standard medical images, to observe the heterogeneity and morphology of lesions. Quantifying treatment-related changes through texture analysis helps in the detection and measurement of tumor complexity. The resulting images correlate with the underlying biological processes such as blood flow or hypoxia, as well as tumor microarchitecture.

“Once we know the clinical drivers, we communicate with our preclinical colleagues to identify the drug’s mechanism of action on specific signaling pathways,” Dr. Korn explained. The results also enable linkage with genomic and proteomic data.

Dr. Korn’s team is currently developing noninvasive tests to measure mutational status in lung cancer and using advanced imaging tools to distinguish K-ras from wild-type mutations in colorectal cancer, as well as ER+ from ER- breast cancer.

Imaging has yet to be fully integrated into drug discovery and development, but when applied successfully, it can unearth tremendous insights into the biology and effectiveness of a drug, probing important issues like driver mutations and tumor heterogeneity.

A detailed evaluation of treatment response shows an axial FATSAT T1W MRI image (A) of a mouse tumor (red outline) and corresponding speckled transformation of the pixels within the region of interest (B). For a human tumor treated with exper-imental therapy (C & D), the shape and amplitude of the histogram curve has little difference on precontrast CT scans but a different shape and amplitude with contrast following therapy suggesting a favorable treatment response and change in tumor morphology. [Imaging Endpoints]

The World’s Brightest Luminescent Protein

An Osaka University professor has taken up a common challenge with optogenetic imaging: How do you use light to both control and analyze the activity of individual neurons while avoiding interference between the two?

Fluorescent indicators offer a means to analyze what happens inside the cell after optogenetic manipulation. But because of their common dependence on light, problems can arise when optogenetics and fluorescent indicators are used together in the same cell at the same time. The excitation light that’s used to “see” the signal from the indicator might misactivate what’s under optogenetic control. For instance, the blue light that excites a fluorescent-based calcium indicator may also activate an optogenetically controlled photosensitive receptor.

To address this problem, Osaka University’s Takeharu Nagai set out to develop an optogenetically compatible indicator that does not require light illumination. His strategy was to re-engineer a chemiluminescent probe—which produces its own light through a chemical reaction but is too weak for use in optogenetic studies—to make it as bright as a fluorescent probe.

As reported in a recent issue of Nature Communications, Dr. Nagai’s lab fused a luminescent protein from a sea pansy with another fluorescent protein. The result is the “Nano-lantern,” the world’s brightest luminescent protein, with a brightness and spatial resolution on par with fluorescence.

To test the protein in an optogenetics scheme, Dr. Nagai’s group modified it into a calcium sensor and co-expressed it with a light-sensitive photoreceptor in rat neurons. To visualize Nano-lantern signals, they turned to Photometrics’ Evolve 512 EMCCD camera.

First, though, they had to solve an imaging problem: The light used to stimulate optogenetic processes is so strong, it can “contaminate” the camera and lead to unacceptable background noise. To reduce the noise, they conducted light stimulation and erased the charges during the camera’s “dead-time,” a feature that is easily accessible in the Evolve. Using these techniques, they were able to track photoreceptor excitation by imaging the Ca2+ increase as reported by the Nano-lantern Ca2+ indicator.

With the arrival of the Nano-lantern, imaging can now be performed in the absence of external light, enabling analysis of events that cascade from optogenetically controlled proteins.

Luminescence (left) and fluorescence (right) imaging of HeLa cells expressing Nano-lantern targeted to cytoplasm, mitochondria, and histone H2B. The reference fluorescence signal was captured by exciting Venus with light at 490 nm. Scale bars, 50 mm. [Photometrics]

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