Each chapter in MICAD has a unique PMID assigned to it so that it can be easily accessed through the PubMed database, via search filters on the MICAD home page or by using appropriate key words on Google or similar search engines. Only literature published in peer-reviewed journals that is indexed in the public domain such as the PubMed and MEDLINE databases is compiled to form appropriate sections of a chapter. Scientific meeting or conference abstracts are not included for publication in MICAD because the abstracts generally lack sufficient information regarding the synthesis, methods used in the study and the imaging characteristics of an agent. If an agent is commercially available, the US government–managed patent database (www.uspto.gov
) may be searched to obtain information that is not available in published literature (e.g., synthesis or the final formulation of a compound or biomolecule). To obtain information about the current diagnostic or therapeutic indication(s) for which an agent is approved by the FDA the package (product) insert of a clinical agent may also be reviewed on line (www.fda.gov
web sites). Information regarding the ongoing clinical evaluation of a MI probes and CAs is available from the www.clinicaltrials.gov
web site. However, this site gives only a brief overview of the clinical studies, including those involving MI probes and CAs, and results obtained from an investigation are usually not posted on this web site. Information in MICAD regarding MI probes and CAs that have been evaluated in humans is obtained only from peer-reviewed clinical studies published in scientific journals. Because MICAD does not promote any commercial or only-for-research MI probes, CAs or related products all agents in the database are mentioned strictly by generic names.
Chapters in MICAD are available as an online textbook format developed at the National Library of Medicine for use through the Entrez database which is a part of the “Bookshelf” infrastructure developed by the NCBI to publish several online books, including MICAD [1
]. Agents published in MICAD are either novel or derivatives/modifications of already existing MI probes and CAs. Each entry in MICAD is listed as a chapter and published literature that is consulted to write the chapter is referenced at the end of each chapter. Cited references are numbered in the order in which they appear in the text and each reference is linked to PubMed for easy access to its abstract. Publications used to write a chapter in MICAD should report sufficient details regarding the agent’s chemical or biological synthesis and must contain data obtained from animal or human studies. Information essentially expected in a publication that can be used to write a chapter for MICAD has been discussed earlier by Chopra et al. [2
]. Once published online, the MICAD editors from time-to-time update the information when new information or application(s) of the agent is published. Each chapter has a “Created” and an “Updated” date located just below the chapter title to inform the reader of how current is the information. At the start of a chapter the characteristics of the MI probe or CA are summarized in a table (Summary Table) just below the title (). In the summary table the chemical name, its abbreviations and synonyms are presented. The table includes other important characteristics regarding an agent such as the category of an agent (compound, antibody, peptide or nucleotide etc.), target of the agent (epidermal growth factor receptor, amino acid transporter, non-targeted etc.), target category (receptor, antigen etc.), mode of detection (SPECT, PET, MRI etc.), source of signal or contrast (111
Tc, I, Gd etc.) and whether in vivo
activation is necessary to generate a signal from the imaging agent. The Summary Table also indicates the type of studies performed with the agent, such as in vitro
studies and investigations in animals (rodent, non-primate non-rodent mammals and non-human primates) and/or in humans. The chemical structure of the MI probe or CA, if available, it is displayed next to the Summary Table () and is linked to the PubChem database where the chemical formula and properties, molecular weight, bioassays etc. of the agent may be available. For MI probes and CAs that have a protein or nucleotide backbone, a web link to the protein and nucleotide sequences, if available through PubMed is provided because the chemical structure for such molecules is rather large and cannot be displayed. Each chapter is divided into several informative sections: Background, Synthesis, In Vitro
Studies, Animal Studies, and Human Studies (the final two sections reflect in vivo
studies) that are discussed below.
MICAD chapter summary table and chemical structure.
This section discusses the rationale to develop the agent, the mechanism of action or activity, the target(s), clinical application (if any) etc. of the probe. Many agents are under development and in the preclinical stage, whereas others have completed the development process and are under evaluation in humans. Some agents are non-targeted because they do not bind to a specific target or exhibit any specific biochemical activity in vivo
. Such agents are usually used for diagnostic imaging e.g. Iopamidol which is a non-radioactive iodine-based contrast agent that circulates in the blood pool and non-specifically fills the extracellular fluid spaces. This agent has been approved by the FDA for diagnostic x-ray/CT imaging of the vasculature in humans [10
]. Similarly, 1,3-bis-[7-(3-amino-2,4,6-triiodophenyl)-heptanoyl]-2-oleoyl glycerol, another iodine-based contrast agent approved by the FDA, is approved for use only in animals to image the liver or the circulatory system [11
]. Recently a “Related Resource Links” section was introduced within the Background section of the chapters. The main purpose of this sub-section is to provide the reader with other relevant information regarding the scaffold, target, imaging properties and applications of the MI probe or CA that are not within the scope of a chapter. Such information can be of much use and interest to imaging scientists for the development of new tracers that can have far superior imaging characteristics compared to the parent compound or macromolecule. An additional benefit is that biology scientists and students in general can access current information regarding the biological properties of a molecule of their interest. In this sub-section, web links are available to related chapters in MICAD, gene information for biological agents (gene identification number in the Genebank, chromosomal location etc.), cellular pathways through which the agent scaffold may mediate its activity (e.g. signal transduction pathway), clinical trials, information from the FDA website and Online Mendelian Inheritance in Man databases etc. The link to all aspects regarding an MI probe or CA from the gene to the protein expression product to clinical trials makes MICAD a unique and single comprehensive source of information on new MI probes and CAs that can be used with the various modalities of molecular imaging.
The chemical or biological processes used to synthesize an imaging probe or CA are described briefly in this section. If available, the yield of each intermediate compound leading to the synthesis and purification of the final product is presented. If a compound is produced using an automated method (e.g., a peptide, polypeptide, and short carbohydrate chain etc.), the equipment, purification column(s) and other procedures used for the synthesis are described briefly in this section. Genetically engineered proteins, monoclonal antibodies (mAbs) or compounds used for imaging purposes are often labeled with nuclides such as 111In, 99mTc, or iodine (123I or 125I) and the methods used to produce and label these biomolecules are detailed in this section. The specific activity, radiochemical purity, and yield of the final labeled product obtained from compounds or biomolecules labeled with radioisotopes such as 18F, 99mTc, 111In, etc. are also given in this section. In addition, the total time required to synthesize and purify probes labeled with radioisotopes that have short half-lives (e.g., 18F, 11C etc.) is reported here. Brief details regarding the chemical procedure used to conjugate a radionuclide to a biomolecule through a chelating agent or a linker may be described in this section as well. In such an agent the number of radionuclide atoms linked to a tracer will depend on the characteristics and number of chelator molecules conjugated to the biochemical. Therefore, investigators should determine and report the number of chelator molecules attached to a biomolecule used as a platform to generate an MI probe or CA. The number of chelator molecules also facilitates determination of the expected molecular weight (theoretical) and specific activity of the purified radiochemical.
If available, the storage conditions and stability of the radioactive or non-radioactive imaging agent (e.g., at different temperatures, in rodent or human serum, etc.) are also provided in this section. The final formulation of an imaging agent, especially for those reconstituted from a freeze-dried kit, may be detailed here as well. Almost all publications have a detailed description or provide relevant references related to the synthesis and labeling of an agent. However, the MICAD editors have noticed that many investigators do not report the specific activity or the stability of a radio-labeled probe [2
The analytical techniques used to characterize a CA used for MRI, x-ray/CT, OI or ultrasound imaging depends on the chemical nature of the compound or the scaffold used to prepare the agent. In general, MRI agents used in the clinic are non-targeted and have gadolinium (Gd), a paramagnetic metal, as the source of signal, but these agents are linked to nephrotoxicity in some patients, particularly those who have compromised kidney function [13
]. As an alternative to Gd-based CAs several MRI agents under development (non-targeted and targeted) have an organic (liposomes) or an inorganic (iron oxide particles) NP platform [14
]. A wide size range of NPs (from <100 nm to micron size) has been used for imaging studies under in vivo
]. Targeted NPs used for MRI usually bear biomolecules such as receptor ligands (peptides, polypeptides, or small proteins) or mAbs on the surface and are directed toward specific organs in the body or tumors that over-express possible diagnostic biomarkers (antigens or receptors). With such an agent, the image quality is affected by the extent of penetration into the target site, the expression level of the target in the tissue, and the binding characteristics and number of biomolecules attached to each NP. Agents used for x-ray/CT imaging are water- soluble small molecules that contain iodine as a source of contrast, and those having an osmolality higher than 800 –900 mosm kg−1
may produce side effects in humans [17
]. The chemical composition and the characteristics of a targeting biomolecule attached to the surface of a NP influences the circulation half-life, biodistribution and the quality of MRI or x-ray/CT images generated by it [16
]. Although the synthesis and chemical composition of NPs is important information to understand the biochemical and physiological characteristics of the NPs, few investigators provide detailed information regarding the synthesis and composition of these molecules in the publications.
Depending on the nature of the study OI probes may be non-targeted, targeted or activatable as described by Sheth and Mahmood [20
]. Targeted OI probes (e.g. those containing a receptor ligand or Ab) may be conjugated to one or more types of fluorescent dyes and are used to detect or quantify the expression of an antigen or a receptor in tissues or on tumor cell surfaces. Chemical linking of the fluorophore to an OI probe can alter the conformational characteristics of the molecule, resulting in partial or complete loss of signal generated by the agent [21
]. In general, cells internalize most probes based on Abs or receptor ligands, and the signal generated by these agents can be attenuated because of metabolic degradation or pH changes in the cell compartments. Therefore, analytical characterization of an OI tracer exposed to various stress conditions in vitro
can provide a clue as to how suitable the probe will be for in vivo
imaging. Approximately 50% of investigators report the number of dye molecules conjugated to an OI agent, but most do not report the stability or analytical profile of the agent after its exposure to an unfavorable environment.
In Vitro Studies
This section summarizes the in vitro studies performed with an imaging agent. In general, these studies describe the cellular uptake, target or receptor binding and dissociation properties (with or without competing compounds) of an agent using either cultured mammalian cells or membranes derived from appropriate cells or tissues. Some of these studies provide information regarding the mechanism of action of an agent, the target specificity of the probe, or whether activation of the agent is necessary before it can generate a signal for imaging. In general, most MI probes and CAs do not require activation for imaging, but these studies help investigate the biochemical behavior of an agent under low background conditions. Studies performed to determine the stability and the partition coefficient of small molecule imaging probes are also reported in this section.
This section is divided into three subsections to provide relevant information about the agent obtained from studies performed in rodents, non-primate non-rodent mammals (such as rabbits, guinea pigs, dogs and pigs), or non-human primates (monkeys and baboons). The rodent subsection presents results obtained from preclinical studies with normal rats or mice, nude mice, or severe combined immunodeficient mice; these animals may or may not bear xenograft tumor implants derived from rodent or human tissue or cell lines. For some studies specific human disease models in rodents or knockout mice are used to characterize the MI probe or CA. Rodents are the most commonly used animals for preliminary investigations, and they are often used to confirm the systemic biodistribution, pharmacokinetics, biochemical characteristics, target specificity and the tumor detection efficiency of radiolabeled or optical imaging agents. A key experiment reported here is the proof of target specificity in the case of targeted probes which can be demonstrated with different experimental approaches (e.g., injecting excess ligand along with the probe, using knockout mice or the target protein, or by comparing the binding of an imaging agent to its target tissue versus the binding in tissue that do not express the target). If suitable results are obtained during preclinical studies in rodents, the molecules may be labeled with therapeutic isotopes (188
Lu, etc.) and evaluated for cancer therapy in other non-rodent non-primate mammals, followed by non-human primates and eventually in humans. Efficacy studies performed in non-human primates such as monkeys and baboons are presented in the Non-Human Primate subsection. Almost 95% of the chapters in MICAD contain information on studies performed in rodents, and ~10% have data from other non-human mammalian species. Although most investigators use 3 animals/group for the in vivo
characterization of an imaging agents the use of 5 animals/group is recommended by Eckelman et al. to obtain statistically meaningful results [22
Information available in this section is obtained only from clinical studies published in scientific journals. The Human Studies section describes results obtained from clinical trials performed with a MI probe or CA to determine the biodistribution, pharmacokinetics, dosimetry, and efficacy of an agent for diagnostic imaging of diseases or from imaging studies performed with radiolabeled therapeutic agents used to treat malignant or metastatic tumors. Some agents are used to detect infections and inflammation, quantify the over-expression of various receptors, or to detect bone growth in humans [23
]. Investigations to monitor the disease status or the biodistribution of radiolabeled small molecules in patients are also described in this section. Currently ~20% of the chapters in MICAD have information regarding human studies.
Any additional information regarding an agent that is available, but not related to the imaging characteristics of the agent (e.g., an alternate method of synthesis, toxicity, pharmacodynamics, metabolic products, etc.) is reported in this section. This information is submitted by members of the imaging community on a voluntary basis and is not reviewed by the MICAD editors for accuracy, reliability, completeness, currency, legality, quality, or appropriateness; the information is presented “as is” in the chapter.
The NIH Support section presents information regarding grants provided by the NIH or affiliated institutions that are used to conduct a part or all of the studies presented in a chapter. The last section of the chapter presents the various references including patents, books, reviews, and research papers consulted to write the chapter. Each reference is linked to PubMed to facilitate access to the abstract of the published paper. If a study is funded with a NIH grant(s), the complete journal article may be accessed free of charge through the PubMed database.