from Leadership Medica n. 3/2004

Introduction
Molecular imaging is a novel field involving both basic medical research and diagnostic applications, that concerns the measurement of biologic processes (functional activities, metabolism, expression of specific molecules) at cellular level utilizing imaging approaches.
Molecular imaging can be considered a branch and a development of “functional imaging”. Functional imaging has been an active field of diagnostic imaging for several decades.
Different imaging techniques and methodologic approaches have been used for decades for functional assessment using Nuclear Medicine , and also Ultrasound, Computed Tomography and several applications of Magnetic Resonance and, to a lesser extent , even conventional diagnostic radiology.
Compared to “conventional“ diagnostic imaging, molecular imaging is focused on the detection of molecular changes using approaches that will eventually permit demonstration and measurement of functional abnormalities.
In biomedical research molecular imaging techniques can be applied in all areas , however oncology, neurodegenerative disorders and heart disorders are the areas of major interest in this field.

The development of new molecular imaging applications
To obtain specific molecular images in vivo, high affinity probes with appropriate pharmacokinetics are required, capable of crossing the biological barriers (capillary wall, interstitial spaces, cell membranes) and of reaching the desired targets. With molecular probes a crucial issue is the often limited density of target cells. Available signals are frequently not sufficient and signal amplification is necessary both on the probe side on the technique side. Furthermore receptor ligands, enzymatic substrates and monoclonal antibodies of potential value must have high affinity and favorable pharmakokinetics, permitting successful in vivo visualization . In a systematic approach to the development of novel molecular imaging applications careful planning is required. An accurate definition of targets and goals (based on available knowledge of epidemiologic, bioinformatic, biochemistry and genetic data) is necessary. Molecules to be synthesized and labeleled (using appropriate organic chemistry and radiochemistry methods) must be carefully selected. Assessement of interactions of synthesized molecules with plasma molecules as well as cells and tissues (receptor binding, internalization , intracellular trafficking, and assessment of possible approcahes for signal amplification) should follow.
After these initial steps, in vivo studies must be carried out including assessment of biodistribution in normal subjects and then testing in animal models of human diseases. During the initial in vivo studies, the characterization of delivery mechanism of the probe and testing of appropriate labeling tools are critical issues. Different labels can be used for comparative assessment of different imaging techniques.
Experimental imaging studies can be performed using different techniques including Nuclear Medicine (planar scintigraphy as well as PET and SPECT), high field MRI and MRS, ultra high frequency US imaging and Computed Tomography. In addition to these traditional imaging tools novel imaging techniques (optical imaging) using on visible or near visible (fluorescence, near infrared) radiation are increasingly used.
Furthemore imaging studies can be supplemented with direct exploration using endoscopy and intravital microscopy.
If successful, the laboratory studies on molecular probes are followed by preclinical safety studies according to the general rules for investigative new drugs and finally by clinical studies; finally assessment of efficacy and cost-benefit criteria prior to widespread will have to be carried out.
Even if the above described approach is appropriate and universally accepted within the scientific community, in the past approaches that opened the way toward molecular imaging have been far less accurate and systematic.
Until now research in diagnostic imaging has been largely focused on the development of new imaging techniques and equipment. More recently research concerning image processing and management has received major inputs. However, together with technological research, for over fifty years different approaches have been pursued to augment the diagnostic information through the detection ands measurement of functional changes in addition to the evaluation of morphostructural changes.
Nuclear Medicine applications historically provided the first possibility to explore and assess quantitatively functional processes, combining the scintigraphic distribution maps of radioactive tracers with the measurement of tracer uptake, and with the measurement of more complex functions such as glomerular filtration and cardiac output, using dynamic acquisition protocols and appropriate tracer distribution models.
At approximately the same time, conventional diagnostic radiology applications were being expanded thanks to the development of contrast media, substances capable of modifyng the density of anatomic structures poorly visible or not visible in direct radiograms. These contrast media have been increasingly used with a “functional approch”, particularly in the case of organotropic media, but also inert contrast media (such as barium sulphate) have been used for morphofunctional studies to assess the esophageal, gastric and intestinal peristaltic activity. These approches are indeed functional ,although still far from “molecules”.

Molecular imaging: oncologic applications
Two imaging approaches are possible with molecular imaging: direct visualization of the metabolic process or of the examined molecule, or indirect visualization. Direct visualization of metabolic activities, metabolites, membrane receptors, proteins, genes requires the synthesis of molecular probes that can be subsequently labeled for external detection and measurement.
Available labels include radionuclides, for nuclear medicine applications, high magnetic relaxivity complexes for MRI studies, visible radiation labels for optical imaging , and even labelled microbubbles for ultrasound applications are being developed. A number of direct molecular imaging approaches are already available for clinical application. Indirect visualization also attainable with labeled probes is particularly helpful for imaging gene expression and is still predominantly used as an experimental tool.
In oncology the discovery of the molecular mechanisms involved in tumor growth permitted the detection of a series of molecular targets that can be used for visualization of biologic processes such as glucose metabolism and other specific metabolic activities, as well as neoangiogenesis and apoptosis.

Receptor imaging
In Nuclear Medicine a number of diagnostic applications of receptor ligands, enzymatic substrates, monoclonal antibodies have been introduced Molecular approaches targeted to membrane receptors have been particularly popular . An example of such processes is represented by somatostatin and the somatostatin receptor.
Somatostatin is a polypeptide formed by 14 aminoacids synthesized by widely distributed cells, including neurons , pancreatic and GI cells (D cells) The somatostain receptor is a member of the G-protein coupled receptors: with a widespread distribution (in different brain regions, spinal cord, anterior pituitary , GI tract, adrenal gland, pancreas, thyroid and kidneys with five subtypes. Tumors originating from the above structures may express the somatostatin receptor. Direct receptor mediated response to somatostatin in tumor cells is responsible for growth arrest and apoptosis through the inhibition of the mithogenic signal, while an indirect action on nonneoplastic cells is obtained through the inhibition of Growth hormone secretion and of Growth factors, stimulation of vasoconstriction and inhibition of neoangiogenesis. Therefore clinical imaging trials have been set up using a somatostatin analog, Octreotide (an octapeptide labelled with Indium-DTPA) In fact somatostatin has a very short 3 minute halflife in plasma due to rapid enzymatic degradatio while octreotide labeled with 111-In has a longer half life and a a high affinity for R1 and R5 somatostain receptors The presence of somatostatin receptors demonstrated with imaging techniques could represent the basis for therapeutic utilization of somatostatin analogs in selected tumor types. And somatostatin receptor expression could be enhanced using transfection approaches. The induced expression of the somatostatin receptor could be used a trojan horse to target the tumor cells with somatostatin analogs to counteract tumor cell proliferation.

PET and glucose metabolism
Together with studies focusing on ligand interaction, in the development of molecular imaging the diffusion of PET (Positron Emission Tomography) has had a crucial role. Thyrty years after ist introduction in the diagnostic imaging armamentarium, PET is most powerful, versatile and sophisticated tool for the development of molecular imaging metods, thanks to the highly accurate measurement of tissue radioactivity and to the possibility of using a wide variaty of tracers labeled with positron emitting radionuclides. Together with the technical advances, a crucial step toward diagnostic molecular imaging has been represented by the development of the deoxyglucose method for the measurement tissue glucose utilization by Sokoloff and coworkers back in 70's. With the subsequent synthesis of the positron emitter Fluorine-18 deoxyglucose (FDG) the road to metabolic mapping or imaging in the present form was officialy opened. PET and the PET-FDG were initially looked at with some skepticism in the clinical world, partly caused by the organizational problems for production and distribution of FDG. However, in the past ten years PET-FDG studies have officially become part of the diagnostic imaging armamentarium with a large space in diagnostic oncology and more limited, although important role in neurology, psychiatry and cardiology While in the clinical environment PET is gaining growing acceptance, in the PET research labs, ongoing intense activities are pursuing the production of new tracers for the assessment of other metabolic aspects, protein and lipid metabolism, neurotransmission, DNA synthesis and gene expression. These activities will progressively widen the area of clinical applications of molecular imaging through PET.

MRS and MRI
In addition to PET, another technology today predominantly used for morphologic and structural characterization, MR (Magnetic Resonance) has many potential applications for functional and molecular studies. The Nuclear Magnetic Resonance (NMR) phenomenon, by its intrinsic nature , depends upon the atomic species and the molecular environment where the atom belongs. Magnetic Resonance Spectroscopy (MRS) is a formidable non invasive tool for the assesment of chemical characteristics of biologic samples . MRS has been extensively used in biochemistry labs for over forty yeras and is now becoming a powerful tool for biochemical characterization and imaging in vivo. With in vivo MRS distribution maps ofd metabolites can be obtained in the brain, in the heart and in tumors. The crucial characteristic of MRS is specificity, ie the possibility to selectively detect molecular species; however in the current operational conditions in vivo, MRS still has limited sensitivity; therefore MRS studies can require long acquisition times . However, faster acquisition can be obtained by increasing the signal/noise ratio at higher magnetic fields. Therefore the diffusion of high field (3 Tesla) scanners will likely expand the clinical utilization of MRS maps, while even higher fields (7-9 Tesla) are already used for experimental in vivo studies.
In addition to MRS, with Magnetic Resonance techniques other functional approaches are possible, including studies of perfusion and diffusion of water molecules that are genereally referred to as “functional MRI” studies . FMRI permits the assessment of brain functions and response to different activation stimuli and can also be used for the evalution of angiogenesis, and therefore to quantify the effect of treatment capable of stimulating or decreasing angiogenesis. (respectively in vascular disease and in neoplastic disease).
Furthermore vascularization and a number of specific tissue characteristics can be assessed using contrast media for MR applications. Using contrast media MR imaging approaches are being targeted to imaging gene expression, a field until now apparently limited to radionuclide approaches.
The major advantage of MR imaging techniques for gene expression imaging is represented by high spatial resolution and the possibility to obtain quantitative measurements by modifying image contrast. Indeed contrast in MR images can be modified by a number of different factors and can be enhanced using contrast media to augmeent the diagnostic content. The large majority of presently available contrast media for MRI use paramagnetic complexes incorporating Gadolinium . The efficacy of MR contrast media depends on its relaxivity and therefore upon a number of structural and dynamic properties of the chelate. In particular at high field strength currently used for diagnostic imaging purposes relaxivity is mainly determined by reorientational correlation time of the complex and is therefore strictly dependent upon its size.
In vivo visualization of structures having a low concentration requires the development of molecules with high relaxivity properties . In fact it has been estimated that using currently commercially available molecules the visualization of cell surface receptors is only possible with the accumulation of 100 -1000 Gadolinium complexes per receptor molecules. However it has been recently demonstrated that Gadolinium complexes with a 20 fold increased relaxivity compared to currently available molecules is possible.
That means that using these novel molecules visualization of cell surface receptors could be successfully achieved once Gd complexes are coupled with appropriate ligands.

Angiogenesis, Apoptosis and Multidrug resistance
Angiogenesis is a complex process entailing a series of steps that can occur both in normal and neoplastic tissues . In particular, angiogenesis is necessary for malignant tumors to growth and to metastasize to other organs. Molecular imaging research has addressed the study of a number of molecules involved in the regulation of angiogenesis. Among the molecules involved in angiogenesis , some molecules (Vascular Endothelial Growth factors, Ang1 and bFGF.) induce proliferation, migration and endothelial cell assembly. Molecules such as Ang2 and proteinases mediate basal membrane degradation; integrin avb3 and avb5 modulate migration of endothelial cells . Other molecules can interfere with different steps of the neoangiogenetic process, and therefore with tumor growth.
Specific surface receptors of endothelial cells belonging to newly formed vessels can be used as target for both imaging and development of treatment strategies. Among these molecules, avb3 integrin is overexpressed in activated endothelial cells and is currently considered a promising target for both imaging of angiogenesis and selective distribution of antitumor drugs.
LM609 an anti avb3 monoclonal antibody containing liposomes has been used for detection of neovessel formation in experimental tumor models using MRI coupled with Gadolinium, or nuclear imaging techniques after labelling with Iodine or Fluorine. Apoptosis is a physiologic process that entails selective programmed cell death . Abnormal control of apoptosis occurs in tumors, autoimmune diseases and some infectious diseases. Various stimuli such as drugs, radiation, ischemia can activate apoptosis through acticvation of proteolytic enzymes eventually leading to nuclear fragmentation and cell lysis.
A crucial step for apoptosis is the externalization on the outer side of cell membranes of phosphatydilserine, normally present on the internal side of the cell membrane. Phosphatydilserine has a high affinity for for a molecule called annexin V; therefore annexin V labelled with 99mTc has been used to image apoptosis in vivo.
Another metabolic process that can be characterized with molecular imaging procedures is Multi Drug Resistance (MDR) one of the major causes of failure chemotherapy. MDR is usually associate with presence of a transmembrane glycoprotein (P-glicoproteina), coded by the MDR1 gene. P-glycoprotein acts as a pump extruding antiblastic drugs from the intracellular compartment, therefore decreasing their lethal effect on tumor cells. A number of studies have been carried out both in animal models and in man to characterize MDR. One of the most valuable approaches has used 99mTc-sestamibi, a widely available radiopharmaceutical. 99m Tc-sestamibi accumulates within the cells in response to physiologic negative mithocondrial and plasma cell potential . In the presence of MDR1P-gp 99m Tc-sestamibi is transported out of the cells with subsequent reduiction of the intracellular concentration . MDR Pgp activity can therefore be studied using conventional nuclear medicine equipment and P-gp acitivity can be estimated and expressed in terms of sestamibi clearance from the tumor. As a practical result, the efflux rate of 99m Tc-sestamibi can be used for non invasive identification of MDR and prediction of tumor response to treatment in breast cancer. Higher tumor retention of sestamibi has been demonstrated to be predictive of good tumor response to chemotherapy. Experimental studies also showed that extrusion of sestamibi (an therefore of chemotherapeutic agents) can be blocked by modulators such as PSC 833 (Valspodar), Vx-710 (Biricodar) e XR-9576 (Tariquidar).

Reporter gene
With molecular imaging, in addition to direct imaging of the target process (such as imaging ligand receptor interaction or cell glucose metabolism with FDG) a number of approaches based on indirect demonstration of an enzymatic activity or of other cellular functions have been proposed.
Among these, Herpes simplex virus 1 thymidine kinase (HSV-Tk) has been used as a prodrug-converting enzyme for a series of gene therapy approaches in different cancer types.
To demonstrate the successful incorporation of th enzyme using different transfection schemes, produgs labelled with radioactive iodine or fluorine can be administered; since these prodrugs can freely cross the cell membrane, once they enter the cell in the presence of HSV-Tk they are phoshorylated and trapped, which permits imaging using PET o SPET. In the past years a number of different approaches using reporter systems have been proposed, including systems aimed at the expression of cell membrane receptors, and even of artificial fusion proteins that once expressed on the cell surface can bind to properly “labelled” molecules, permitting imaging using nuclear techniques or MR, or optical imaging.
Indeed imaging gene expression is among the present challenges of imaging and an area where Molecular imaging will be crucial for in vivo assessment of treatment success.

Conclusion
Molecular imaging is a diagnostic approach aimed at earlier detection of diseases through the visual detection of fine molecular changes associated with them, integrating the laboratory molecular findings by visualizing the pathologic processes in the anatomic locations where they occur.
Molecular imaging is a great opportunity to strenghten and stimulate the collaboration of specialists operating in different fields of diagnostic imaging and with basic science professionals (biotechnologist, chemists, physicists, informatics experts and engineers) and to stimulate cultural advances in Diagnostic imaging. A strong collaboration between academic research and pharmaceutical companies will be necessary to bring in the clinical arena a large number of molecular imaging procedures in the next years.
Given the increasing role in clinical applications, molecular imaging should become an important part in the residency program in Diagnostic Imaging.