Current Radiopharmaceuticals - Volume 5, Issue 2, 2012

This hot topic issue is about radiopharmaceuticals and pre-clinical evaluation, being the oncology the main application field. In fact, there are several radiotracers and radiopharmaceuticals that are used at pre-clinical stage taking advantage of potentialities of nuclear medicine. These potentialities are related with the fact that nuclear medicine is an important tool to evaluate, understand and explore what happens when malignant transformation of cells occurs. The several changes of biochemical pathways that happen during this process can be explored, giving information that can be useful not only for the treatment, but also for the follow-up, staging and re-staging of cancer. The preclinical studies in cancer are currently an important subject of research and development, covering a vast area of knowledge about the understanding of the mechanisms involved in disease development. Hanahan and Weinberg described in 2000 six hallmarks of cancer which correspond to distinctive and complementary capabilities that allow tumor growth and metastatic dissemination [1]. These hallmarks are self-sufficiency in growth signals, insensitivity to antigrowth signals, evading apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis. In 2011, they rewrote them adding two hallmarks, deregulation of cellular energetics and avoiding immune destruction and two consequential characteristics, the genome instability and the tumor-promoting inflammation [2].....

Radiopharmaceuticals are able to give functional information about systems, organs or cells. This functional information can be about different cell mechanisms or molecular pathways. In terms of systems or organs this information can be assessed through biodistribution studies while that in terms of cells can be evaluated through more detailed research, like calculation of influx and efflux indexes or binding studies. Moreover recent advances in understanding the molecular mechanisms underlying the disease, allow the diagnosis in early stages of the disease, sometimes several months before the onset of morphological changes that translate the disease. These approaches are especially important in oncology. The nuclear medicine allows to map and to quantify the local changes related to the metabolic pathways involved in malignant transformation or in the tumoral proliferation. We made a longitudinal approach of the development of the malignant transformation and we show how it is possible to evaluate some metabolic steps using the nuclear medicine to get information not only about the functional situation of tumoral tissue but also about its therapeutic response. Therefore, covering different issues like tumor proliferation, tumor metabolism, tumor angiogenesis, tumor hypoxia, apoptosis, tumor receptors and multidrug resistance it is possible to confirm the important role of nuclear medicine on the detection, treatment and cancer follow-up.

Molecular imaging tools such as Positron Emission Tomography (PET) are increasingly being used in the drug development process. The unrivaled sensitivity of PET coupled with a solid experience in developing highly targeted molecular probes makes this technique a very valuable tool at all stages from pre-clinical development to the clinical phases. Positron emitting tracers allow us to measure, quantitatively, molecular processes and interactions between a candidate drug and its molecular targets. This information can save time and money by directing development towards the most promising compounds and excluding molecules with unfavorable properties that would otherwise only be recognized as failures in latter stages of the process. In this paper we review the application of positron emitting tracers in the pre-clinical stages of the drug development process in the areas of oncology, cardiology, neurosciences and inflammatory diseases. PET tracers provide an important support for drug development in the areas of: discovery of new drug targets, clarification of pathophysiology, identification of potential drug candidates and validation of drug effectiveness, as well as the evaluation of pharmacokinetic and pharmacodynamic parameters in vivo.

Introduction: Hypoxia is a biochemical condition where reduced oxygen partial pressure at tissue level occurs. This metabolic situation can lead to resistance to radio and chemotherapy. In malignant solid tumours, hypoxia is a common characteristic, having a great impact at biological level, being of tremendous importance for complete understanding of tumour progression. Objectives: We studied the behavior of 99mTc-HL91 in vivo, using an animal model based on Balb-c nu/nu mice with a xenotransplant of the human colorectal adenocarcinoma cell line, WiDr. Material and Methods: In vivo studies using an animal model of xenograft on Balb/c nu/nu nude mice were carried out. This model, allowed us to evaluate the radiopharmaceutical biodistribution and to calculate tumour/muscle ratio, acquired after 99mTc-HL91 injection. We also performed ex vivo studies, using the excised tumours to access viability and to characterize the intracellular production of reactive oxygen species and the status of mitochondrial membrane potential through flow cytometry. Results and Discussion: The biodistribution after 99mTc-HL91 injection showed urinary and hepatobilliary excretion in similar proportions and tumour uptake around 4.4% of administered activity. This uptake was higher at the bigger tumours. Through flow cytometry we observed that larger tumours have a higher amount of reactive oxygen species and a decrease in mitochondrial membrane potential. Conclusions: 99mTc-HL91 allowed a non-invasive evaluation of the solid tumours oxidative state by nuclear medicine functional imaging. This information can be of high importance at the pre-treatment estimation of this type of tumours.

Vitamin C exists in two forms: the reduced (ascorbic acid - AA) and oxidized form (dehydroascorbic acid - DHA). This is a nutrient whose benefits are long known and widely publicized, being most of them related to its antioxidant action. As an antioxidant, the main role of vitamin C is to neutralize free radicals, reducing oxidative stress. However, some controversial studies suggest that this nutrient may have a preventive and therapeutic role in cancer disease due to their possible pro-oxidant activity, promoting the formation of reactive oxygen species that can induce cell death in cancer cells. This factor, coupled with the decrease of antioxidant enzymes and increase of decompartmentalized transition metals in tumor cells may result in the selective cytotoxicity of vitamin C and the subsequent revelation of its therapeutic potential. In this way the first purpose of this work was radioactively label the reduced form of vitamin C with Tc-99m, its quality control by HPLC and the time stability. The second purpose was to use the radioactive complex 99mTc-AA in in vitro and in vivo studies in order to evaluate its uptake by colorectal cancer cells and biodistribution in mices, respectively. The results suggest that the pharmaceutical formulation developed, which was reproducible and stable over time, was residually taken up by colorectal cancer cells. Future studies are needed to deepen our understanding about the radioactive complex 99mTc-AA and clarify the mechanisms of action of vitamin C in oncologic disease.

The multidrug resistance (MDR) phenotype is frequently associated with the overexpression of transmembrane drug proteins such as the P-glycoprotein (Pgp) and/or multidrug resistance related protein-1 (MRP1). These proteins belong to the superfamily of the so-called ATP-binding cassette superfamily and act as drug efflux pumps of a broad range of chemotherapeutic agents commonly used in the treatment of malignancies. These proteins have been found to be overexpressed in both haematological and solid tumours and are considered as adverse prognostic factors. The ability to obtain in vivo and non-invasively information regarding the functional activity of MDR-related transporters, using probes that mimic the antineoplastic agents, provide a very useful tool in the clinical setting by determining the individual tumour susceptibility to chemotherapy. This previous knowledge could serve as a critical tool for optimizing chemotherapeutic protocols on a patient-specific basis. The emergence of non-invasive molecular imaging techniques using radiolabelled probes provides an interesting approach for functional assessment of the classical mechanism of MDR in cancer patients. Toward this objective, the clinically approved 99mTc-labelled cationic lipophilic complexes (sestamibi and tetrofosmin) have been characterized as transport substrates of Pgp and MRP1 and proposed as surrogate markers of chemotherapeutic agents for functional evaluation of MDR by single-photon emission tomography (SPECT). Here we review the potential applications of these agents in identifying drug resistance mechanisms based on functional assays and their potential as a tool for evaluating the efficacy of MDR inhibitors, using cellular and animal models of chemoresistance.

The design and development of radiolabelled estradiol derivatives has been an important area of research due to their recognized value in breast cancer management. The estrogen receptor (ER) is a relevant biomarker in the diagnosis, prognosis and prediction of the therapeutic response in estrogen receptor positive breast tumours. Hence, many radioligands based on estradiol derivatives have been proposed for targeted functional ER imaging. The main focus of this review is to survey the current knowledge on estradiol-based radioiodinated receptor ligands synthesis for breast tumour functional imaging. The main preclinical and clinical achievements in the field will also be briefly presented to make the manuscript more comprehensive.

The most common PET radioisotopes, both in the literature and in clinical practice, are the cyclotron produced 11C and 18F, giving rise to tracers with minimal chemical changes with respect to the original biological molecule. However, the short half-life of these two radioisotopes and the relatively complex chemistry of their incorporation into the molecules of interest limits the number of molecules that really can be labelled in a suitable length of time. 68Ga is a positron emitter, produced by a 68Ge/68Ga generator rending the production of its radiopharmaceuticals independent of an onsite cyclotron. This paper covers the main aspects of the Ga3+ coordination chemistry together with the state of art of its radiopharmacy.

The new dihydrobis(azolyl)borate ligand Na[H2B(timMe)(3,5-Me2pz)] (L1) was synthesized and used to prepare the complexes fac-[M(κ3-H(μ-H)B(timMe)(3,5-Me2-pz))(CO)3] (M = Re (4), 99mTc (4a)). L1 and 4 were characterized by common analytical techniques, including X-ray diffraction analysis for 4. The successful synthesis of complex 4a, obtained with high radiochemical purity, has shown for the first time that dihydrobis(azolyl)borate ligands combining 2- mercaptoimidazolyl and pyrazolyl rings are capable of stabilizing the fac-[99mTc(CO)3]+ unit. Complex 4a displays a high in vitro stability, in PBS (pH 7.4), indicating that the B-H…99mTc bond is retained even under physiological conditions. Biodistribution studies in mice have shown that 4a can cross the blood-brain barrier, emerging as a good alternative for the design of radiopharmaceuticals for brain imaging.

The advent of nanomedicine and increase knowledge on cellular and molecular biology has opened new opportunities on the clinical field. Selective drug targeting and protection of healthy tissues rules the rising interest that is being devoted to drug delivery system strategies, considering that the accurate choice of the carrier molecule will determine the pharmacokinetics and pharmacodynamics of drugs, yielding higher therapeutic efficacy. Despite the improvements in surgery and immunological approaches, tumor staging and cancer therapy remains a challenge, typically because they are ineffective in advanced stages of the disease, but also due to the conventional administration route (intravenous), and consequently the non-specificity of the potentially toxic drugs. The issue currently under the spotlight in drug targeting is the concept of drug delivery systems (DDS) and the impact that is inherent to their selectivity. Moreover, these particulate systems bring forth the possibility of using alternative routes to the conventional intravenous administration. This article reviews the applications of gamma-scintigraphic image technique to evaluate the advances and research on DDS engineering to the pulmonary administration, and the dependency of lung particle removal mechanism on both the administration route and the particulate system characteristic, based on literature data, as well as through the experimental studies performed in our group.

Liposomes, usually assembled from organic/synthetic lipidic compounds, are biocompatible, biodegradable, non-toxic, and do not induce immune response. Due to their structural versatility in terms of size, composition, surface charge, bilayer fluidity and ability to encapsulate drugs regardless of their solubility, liposomes enable the production of a vast number and type of formulations with potential clinical use. They can be administered through several routes of administration (e.g. i.v., i.m., oral, nasal, etc.). The use of liposomes enables the variation and control retention of drugs in biologic fluids, enhancing blood circulation and specific compartments residence. They can be tailored to target specific tissues and cells. They can play a very important role for imaging diagnosis and/or therapy. After an extensive literature review of the subject, we selected a particular area of potential clinical application: pulmonary oedema. This clinical entity has a variety of possible etiologies, conducing to two main types of edema: cardiogenic and non-cardiogenic. At the moment a dedicated technique for the early diagnosis/therapy of this pathology is lacking. We propose a new methodology using a specially designed GUV formulation, encapsulating chosen radiotracers labeled with 99mTc. The aim of the work has been successfully achieved in an experimental animal model of cardiogenic pulmonary oedema. Experiments using an animal model of non-cardiogenic pulmonary oedema are in course (simultaneous study with two different drugs), using the same GUV methodology. Preliminary results are very promising.