Current Pharmaceutical Design - Volume 10, Issue 24, 2004

Radioactive tracers have made an immense contribution to the understanding of human physiology and pathology. At the start of the 21st century nuclear imaging has emerged as the main metabolic imaging modality which is of growing importance in drug development and clinical pharmacology. Using techniques adapted from those undertaken in clinical radiopharmacy and nuclear medicine facilities drug molecules and carrier systems may be radiolabelled and their release, biodistribution and uptake may be visualized in human subjects. Imaging studies are capable of locating the uptake of specific receptors in the brain, the site of disintegration of a tablet in the GI tract, the penetration of a nebulized solution into the lung and the residence time of an eye drop on the cornea. The technology uses suitable gamma emitting radionuclides such as 99mTc, 111In, 123I and 153Sm, which may be imaged with a gamma camera or positron emitters such as 11C, 13N, 15O and 18F for positron emission tomography (PET). Positron emitters are more appropriate for the direct labeling of drug molecules rather than metals such a 99mTc or 111In. A particular asset of these techniques is that the in vivo distribution and kinetics of a radiolabelled pharmaceutical formulation may be quantified. In this way correlation between the observed pharmacological effects and the precise site of delivery may be made. A powerful feature of nuclear molecular imaging is the evaluation of drug delivery systems in patient groups for whom the treatment is intended. Such studies not only provide data on the nature and characteristics of a product, such as reliability and reproducibility, but can demonstrate proof of principle for the new generation of targeted therapeutics. Imaging data are increasingly being used in product registration dossiers for submission to Regulatory Authorities.

With the help of radiolabeled compounds, drug development can be made faster; especially with microdosing and radiopharmacokinetics, some elements of phase I and II trials necessary for conventional cancer drug development can be avoided. Imaging may proof the principle of actual targeting. However, radiopharmacokinetics is dependent on the radionuclide, the radionuclide linker with the drug and the size of the drug molecule. Optimally, some of the drug molecule atoms may be replaced with a radionuclide that can be visualized. In this article drug development utilizing radionuclides both in PET and SPET has been reviewed.

Nuclear medicine offers powerful noninvasive techniques for visualization of infectious and inflammatory disorders using whole body imaging enabling the determination of both localization and number of inflammatory foci. A wide variety of approaches depicting the different stages of the inflammatory response have been developed. Non-specific radiolabeled compounds, such as 67Ga-citrate and radiolabeled polyclonal human immunoglobulin accumulate in inflammatory foci due to enhanced vascular permeability. Specific accumulation of radiolabeled compounds in inflammatory lesions results from binding to activated endothelium (e.g. radiolabeled anti-E-selectin), the enhanced influx of leukocytes (e.g. radiolabeled autologous leukocytes, anti-granulocyte antibodies or cytokines), the enhanced glucoseuptake by activated leukocytes (18F-fluorodeoxyglucose) or direct binding to micro-organisms (e.g. radiolabeled ciprofloxacin or antimicrobial peptides). Scintigraphy using autologous leukocytes, labeled with 111In or 99mTc, is still considered the “gold standard” nuclear medicine technique for the imaging of infection and inflammation, but the range of radiolabeled compounds available for this indication is still expanding. Recently, positron emission tomography with 18Ffluorodeoxyglucose has been shown to delineate various infectious and inflammatory disorders with high sensitivity. New developments in peptide chemistry and in radiochemistry will result in specific agents with high specific activity. A gradual shift from non-specific, cumbersome or even hazardous approaches to more sophisticated, specific approaches is ongoing. In this review, the different approaches to scintigraphic imaging of infection and inflammation, already in use or under investigation, are discussed.

Peptide-based radiopharmaceuticals have been introduced into clinical work more than a decade ago. The first and most successful imaging agent to date is the somatostatin analog octreotide. It is used for somatostatin receptor scintigraphy and also receptor-mediated peptide-radiotherapy of neuroendocrine tumors. For in vivo use as radiopharmaceutical, the natural peptide is modified in order to enhance the metabolic stability and to allow stable labeling with a so-called residualizing label. This means, that a radiometal chelator complex bound to a modified peptide stable in serum is internalized into the target cells via a specific receptor. The peptide then undergoes lysosomal degradation leaving the radiometal-chelator complex trapped inside the cell, leading to a high target to background ratio. The successful development of new radiopeptides is thus dependent on modifications of a given natural peptide while preserving the binding affinity for the target receptor(s) at the same time. Other peptides than somatostatin are under development for use as radiopeptides such as Minigastrin, GLP-1, VIP, Substance P, or Neurotensin. Some show very favorable results in clinical trials, like Minigastrin for example. Furthermore, there is increasing interest in peptidebinding sites other than the “classical” receptors for regulatory peptides specifically over-expressed by (neuroendocrine) tumors. In this paper, we provide an overview of the biochemical and radiochemical aspects of radiopeptide development, the current state of clinical use of radiopeptides for diagnosis and therapy of tumors, the current state of development of new compounds, and future developments.

A major obstacle to successful cancer chemotherapy is drug resistance. Multidrug resistance (MDR) is often seen with chemotherapeutic agents such as anthracycline derivatives, vinca alkaloids and taxanes. Multiple aspects of cellular biochemistry have been implicated in the MDR process. Cellular mechanisms of resistance are due to the presence of efflux pumps, P-glycoprotein (P-gp) and multiple resistance-associated protein (MRP), which belong to the ATPbinding cassette (ABC) family of transporters. Another form of drug resistance is involved in the chemotherapy of cancers with alkylating agents such as nitrosourea derivatives and nitrogen mustards. The cytotoxicity of these agents is primarily due to alkylation of the DNA guanine residues at their O6-position, which leads, via a cascade of events, to DNA strand breaks. The DNA repair protein, alkylguanine-DNA alkyl transferase (AGT) removes the alkyl groups from the lesions stoichiometrically to a cysteine in its active site. This process is irreversible and results in the degradation of the protein and its recovery is entirely from de novo synthesis. Noninvasive methodologies for monitoring the transport activity of these efflux pumps and determining tumor content of AGT could serve as critical tools for optimizing chemotherapeutic protocols on a patient-specific basis and gaining an understanding of the dynamics of resistance in living patients. In this review, we will describe the efforts made to date to synthesize radioactive probes of chemotherapy resistance and their use to quantitate these transporters and DNA repair protein by radionuclide imaging.

Liposomes, which are biodegradable and essentially non-toxic vehicles, can encapsulate both hydrophilic and hydrophobic materials, and are utilized as drug carriers in drug delivery systems. In addition, liposomes can be used to carry radioactive compounds as radiotracers can be linked to multiple locations in liposomes. One option is the hydrated compartment inside the liposome, another the lipid core into which especially hydrophobic conjugates can be attached, and the third option is the outer lipid leaflet where molecules can be bound by covalent linkage. Delivery of agents to the reticuloendothelial system (RES) is easily achieved, since most conventional liposomes are trapped by the RES. For the purpose of delivery of agents to target organs other than RES, long-circulating liposomes have been developed by modifying the liposomal surface. Understanding of the in vivo dynamics of liposome-carried agents is required for the evaluation of the bioavailability of drugs encapsulated in liposomes. In this review, we focus on the in vivo trafficking of liposomes visualized by positron emission tomography (PET) and discuss the characteristics of liposomes that affect the targeting of drugs in vivo.

Radiolabeled compounds are excellent investigative tools and widely used to carry out ADME studies during drug discovery and development stages. The most commonly used radioisotopes are 14C and 3H. 3H materials are generally easier to synthesize than 14C materials. Therefore, a variety of probes and substrates used in in vitro assays are labeled with 3H. Since synthesis of 14C material requires intensive resources, it is usually not available until after a molecule is considered for potential development or after the molecule enters the development phase. Improvement in the technology in radiochemistry has enabled the use of radiolabeled compounds earlier in pre-clinical and clinical development to address mechanistic issues. For in vitro studies, radiolabeled probes are utilized to test affinity with various transporters, to perform metabolism comparison among species and to assess possible formation of reactive metabolites. For in vivo studies, radiolabeled compounds are employed to identify and elucidate metabolites formed, to investigate the extent of absorption, bioavailability, tissue distribution, mass balance, routes of excretion, and pre-systemic metabolism. Due to the significant impact of radiolabeled studies on drug development, these studies will be performed earlier than have been in the past and will continue to be an integral part of drug discovery and development.

Recent years have seen an increased effort in the development of peptide based radiopharmaceuticals for nuclear medicine applications in imaging and therapy. This field is of particular interest in the development of new cancer imaging and treatment strategies. Major developments in the molecular biology of cancer have brought forth the discovery of a number of receptor systems and other cell surface molecules that can be utilized as molecular targets for peptide based agents. Although such a strategy is very appealing and shows great potential for application in humans, there are as yet very few radiopharmaceuticals developed based on this scheme that have made it into clinical practice. Many different factors contribute to the generation of a successful radiopharmaceutical. Among these, a thorough and efficient preclinical evaluation process is necessary to single out those lead compounds that show more promise and are most likely to be winners in clinical applications. To maximize the efficacy of pre-clinical testing one must utilize currently available technology to the best extent possible. Expertise in different areas of drug development is indispensable for this type of research. This work will analyze currently available methods utilized to evaluate radiopharmaceuticals being developed, from compound design to evaluation in pre-clinical animal models.

Modern molecular targeting provides new opportunities for imaging, diagnosis and treatment of diseases. Small molecular weight peptides have the potential for enhancing targeting of compounds, and they may also have therapeutic effects by themselves. The limiting step for successful molecular targeting is the development of efficient peptide delivery systems. This review will focus on peptides developed by phage display and combinatorial chemistry for the delivery of pharmaceuticals, radioactive compounds and gene expression vectors. Target cell-specific delivery can be improved by peptides that penetrate the cell membrane or alternatively induce receptor-mediated endocytosis. In addition, peptides that contain endosomal escape signals or nuclear localization motifs may help trafficking of therapeutics to appropriate locations inside the cell. Small molecule radiolabelled peptides are the preferred agents for targeting and for diagnostic imaging of various organs as they are easily synthesized, effectively penetrate tissues, and are rapidly cleared from the circulation. Such peptides have been tested in animals and humans in the fields of cancer, cardiology, neurology, inflammation / infection, atherosclerosis and thrombosis.