Enfoque integral del paciente con Cáncer Diferenciado de Tiroides: Un largo camino desde el diagnóstico al tratamiento con I131. Visión Bioética dirigida al paciente

El cáncer diferenciado de tiroides presenta en la mayoría de los pacientes un pronóstico favorable, con una baja tasa de mortalidad a largo plazo. Estos resultados dependen del correcto abordaje diagnóstico y terapéutico, el cual requiere un enfoque multidisciplinario e integral, con el objetivo de lograr no sólo los resultados terapéuticos, sino también una contención apropiada para el paciente y su entorno. En primera instancia, el paciente debe afrontar el diagnóstico de cáncer de tiroides, el cual a pesar de presentar en términos generales un buen pronóstico, implica en el que lo padece la necesidad de asumir una situación de enfermedad potencialmente maligna con todo lo que ello implica. Además debe someterse a una cirugía la cual no está exenta de riesgos y complicaciones, luego de la cual debe recibir tratamiento con iodo radioactivo, que genera en èl y en su entorno una serie de cambios y complicaciones que afectan seriamente su situación personal, familiar, laboral y social. El seguimiento de esta patología requiere controles periódicos comportándose como una patología crónica. El objetivo de este trabajo es analizar los aspectos sociales, éticos, culturales y económicos que se presentan ante un paciente que se enfrenta al diagnóstico y tratamiento del cáncer diferenciado de tiroides.(AU)

Differentiated thyroid cancer occurs in the majority of patients with a favorable prognosis, with a low rate of long-term mortality. These results depend on the correct diagnostic and therapeutic approach, which requires a multidisciplinary and comprehensive approach, in order to achieve results not only therapeutic but also appropriate containment for the patient and his environment. At first, the patient must meet the diagnosis of thyroid cancer, which despite having generally a good prognosis, involves those who suffer the need to assume a position of potentially malignant disease with all that that implies. In addition face a surgery which is not free of risks and complications, after which the patient should be treated with radioactive iodine, which generates in the patient and his environment a series of changes and complications that seriously affect his life, his family, and his social work. The monitoring of this disease requires regular checks, like a chronic disease. The aim of this paper is to analyze the social, ethical, cultural and economic factors that are presented to a patient facing the diagnosis and treatment of differentiated thyroid cancer.(AU)

Perfusión miocárdica SPECT con prueba de frío como predictor de desarrollo de isquemia de esfuerzo en el seguimiento de pacientes asintomáticos con riesgo cardiovascular moderado / Cold Pressor Test Myocardial Perfusion SPECT as a Predictor of the Development of Ischemia at Exercise in the Follow up of Asymptomatic Patients with Moderate Cardiovascular Risk

Introducción Estudios previos han publicado la correlación entre defectos de perfusión miocárdica (PM) SPECT durante la prueba de frío (PF) y la acetilcolina intracoronaria y su utilidad como marcador independiente de disfunción endotelial (DE). Objetivo Analizar la incidencia de positivización de los estudios de PM de esfuerzo en el seguimiento de pacientes asintomáticos con riesgo cardiovascular (CV) moderado y DE detectada con la PF. Material y métodos De 301 pacientes del Registro PARADIGMA (PM SPECT esfuerzo normal y probabilidad clínica < 20% de eventos a 10 años [riesgo moderado por índice de Framingham]), 55 tuvieron PF positiva (+) (18,3%). Se analizaron en forma prospectiva y consecutiva 15 pacientes asintomáticos con PF (+) y un grupo control (GC) de 15 pacientes con PF negativa (-), con apareamiento de sexo, edad y factores de riesgo coronario (FRC), que cumplieron un seguimiento de 12 ± 2 meses, en quienes se realizó una nueva PM SPECT de reposo y esfuerzo. Se utilizó un score de extensión de PM en un modelo de 17 segmentos. Se analizaron los FRC y la incidencia de isquemia en la PM de esfuerzo de seguimiento en cada grupo. Resultado Edad: PF (-) 57,3 ± 8,9 versus TF (+) 52,5 ± 7,5 (p = 0,09). Positivizaron la PM de esfuerzo: grupo PF (+) 5/15: 33,3% y 0 del GC (p = 0,04). Sin diferencias estadísticamente significativas en los FRC entre ambos grupos. Conclusiones Una PM SPECT anormal durante la PF en pacientes asintomáticos con riesgo CV moderado diferenció a aquellos pacientes que positivizaron los estudios de PM de esfuerzo a un año de seguimiento y no hubo estudios anormales en el grupo control.

Introduction Previous studies have published the correlation between myocardial perfusion SPECT (MP) during cold pressor test (CPT) and intracoronary acetylcholine and its usefulness as independent marker of endothelial dysfunction (ED). Objective To analyze the incidence of positivization of MP exercise studies in the follow up of asymptomatic patients with moderate cardiovascular risk (CV) and ED detected by PF. Material and Methods Of 301 patients of the PARADIGMA Registry (normal exercise MP SPECT and clinical probability < 20% of events at 10 years [moderate risk by Framingham index] 55 had positive PF (+) (18.3%). Prospectively and consecutively, 15 asymptomatic patients with PF (+), and a control group (CG) of 15 patients with negative PF, with paired sex, age and coronary risk factors (CRF), that accomplished a 12 ± 2 months follow up, and that underwent a new exercise and resting MP SPECT were analyzed. An MP extension score was used in a model of 17 segments. The CRF and the incidence of ischemia during follow up exercise MP of each group were assessed. Results Age: PF (-) 57.3 ± 8.9 versus TF (+) 52.5 ± 7.5 (p = 0,09). Positivized the exercise MP: PF group (+) 5/15: 33.3% and 0 in the CG (p=0.04). No statistically significant differences between CRF in both groups. Conclusions An abnormal MP SPECT during PF in asymptomatic patients with moderate CV risk differentiated those patients who positivized exercise MP studies at one year follow up and there were no abnormal studies in the control group.

Proton sensing of CLC-0 mutant E166D.

CLC Cl- channels are homodimers in which each subunit has a proper pore and a (fast) gate. An additional slow gate acts on both pores. A conserved glutamate (E166 in CLC-0) is a major determinant of gating in CLC-0 and is crucially involved in Cl-/H+ antiport of CLC-ec1, a CLC of known structure. We constructed tandem dimers with one wild-type (WT) and one mutant subunit (E166A or E166D) to show that these mutations of E166 specifically alter the fast gate of the pore to which they belong without effect on the fast gate of the neighboring pore. In addition both mutations activate the common slow gate. E166A pores have a large, voltage-independent open probability of the fast gate (popen), whereas popen of E166D pores is dramatically reduced. Similar to WT, popen of E166D was increased by lowering pHint. At negative voltages, E166D presents a persistent inward current that is blocked by p-chlorophenoxy-acetic acid (CPA) and increased at low pHext. The pHext dependence of the persistent current is analogous to a similar steady inward current in WT CLC-0. Surprisingly, however, the underlying unitary conductance of the persistent current in E166D is about an order of magnitude smaller than that of the transient deactivating inward Cl- current. Collectively, our data support the possibility that the mutated CLC-0 channel E166D can assume two distinct open states. Voltage-independent protonation of D166 from the outside favors a low conductance state, whereas protonation from the inside favors the high conductance state.

Structural requisites of 2-(p-chlorophenoxy)propionic acid analogues for activity on native rat skeletal muscle chloride conductance and on heterologously expressed CLC-1.

(1) The 2-(p-chlorophenoxy)propionic acid (CPP) modulates in a stereoselective manner the macroscopic chloride conductance (gCl), the electrical parameter sustained by the CLC-1 channel, of skeletal muscle. In order to determine the structural requirements for modulating native gCl and to identify high-affinity ligands, the effects of newly synthesised CPP analogues have been evaluated on gCl of rat EDL muscle fibres by means of the two-microelectrode current-clamp technique. (2) Each type of the following independent modification of CPP structure led to a three- to 10-fold decrease or to a complete lack of gCl-blocking activity: replacement of the electron-attractive chlorine atom of the aromatic ring, substitution of the oxygen atom of the phenoxy group, modification at the chiral centre and substitution of the carboxylic function with a phosphonate one. (3) The analogues bearing a second chlorophenoxy group on the asymmetric carbon atom showed a significant gCl-blocking activity. Similar to racemate CPP, the analogue with this group, spaced by an alkyl chain formed by three methylenic groups, blocked gCl by 45% at 100 micro M. (4) These latter derivatives were tested on heterelogously expressed CLC-1 performing inside-out patch-clamp recordings to further define how interaction between drug and channel protein could take place. Depending on the exact chemical nature of modification, these derivatives strongly blocked CLC-1 with K(D) values at -140 mV ranging from about 4 to 180 micro M. (5) In conclusion, we identified four molecular determinants pivotal for the interaction with the binding site on muscle CLC-1 channels: (a) the carboxylic group that confers the optimal acidity and the negative charge; (b) the chlorophenoxy moiety that might interact with a hydrophobic pocket; (c) the chiral centre that allows the proper spatial disposition of the molecule; (d) an additional phenoxy group that remarkably stabilises the binding by interacting with a second hydrophobic pocket.

Gating competence of constitutively open CLC-0 mutants revealed by the interaction with a small organic Inhibitor.

Opening of CLC chloride channels is coupled to the translocation of the permeant anion. From the recent structure determination of bacterial CLC proteins in the closed and open configuration, a glutamate residue was hypothesized to form part of the Cl--sensitive gate. The negatively charged side-chain of the glutamate was suggested to occlude the permeation pathway in the closed state, while opening of a single protopore of the double-pore channel would reflect mainly a movement of this side-chain toward the extracellular pore vestibule, with little rearrangement of the rest of the channel. Here we show that mutating this critical residue (Glu166) in the prototype Torpedo CLC-0 to alanine, serine, or lysine leads to constitutively open channels, whereas a mutation to aspartate strongly slowed down opening. Furthermore, we investigated the interaction of the small organic channel blocker p-chlorophenoxy-acetic acid (CPA) with the mutants E166A and E166S. Both mutants were strongly inhibited by CPA at negative voltages with a >200-fold larger affinity than for wild-type CLC-0 (apparent KD at -140 mV approximately 4 micro M). A three-state linear model with an open state, a low-affinity and a high-affinity CPA-bound state can quantitatively describe steady-state and kinetic properties of the CPA block. The parameters of the model and additional mutagenesis suggest that the high-affinity CPA-bound state is similar to the closed configuration of the protopore gate of wild-type CLC-0. In the E166A mutant the glutamate side chain that occludes the permeation pathway is absent. Thus, if gating consists only in movement of this side-chain the mutant E166A should not be able to assume a closed conformation. It may thus be that fast gating in CLC-0 is more complex than anticipated from the bacterial structures.

Molecular modeling of p-chlorophenoxyacetic acid binding to the CLC-0 channel.

Molecular simulation techniques were applied to predict the interaction of the CLC-0 Cl(-) channel and the channel-blocking molecule p-chlorophenoxyacetic acid (CPA). A three-dimensional model of the CLC-0 channel was constructed on the basis of the homology with the bacterial Cl(-) channel StCLC, the structure of which has been solved by X-ray crystallography. Docking of the CPA molecule was obtained by using a geometric recognition algorithm, yielding 5000 possible conformations. By restraining the simulation to those conformations in which CPA is near the intracellular mouth of the channel, the CPA-protein complex models were reduced to three sets of conformations, which are interconvertible within 2 ns when molecular dynamics is applied to the system. Point mutations of CLC-0 at three different positions predicted to interact with CPA in these configurations did, however, not greatly alter CPA inhibition, suggesting a deeper final binding location. In the model, binding of CPA to a more internal position in the ionic pathway was obtained by applying a constant force vector to CPA, pushing it toward the center of the channel. This technique allowed us to outline the possible intrachannel pathway of CPA and to describe qualitatively the binding sites and energy barriers of this pathway. The consistency of the obtained models and the experimental data indicates that the CLC-0-CPA complex model is reasonable and can be used in further biological studies, such as rational design of blocking agents of and mutagenesis of CLC Cl(-) channels.

Molecular requisites for drug binding to muscle CLC-1 and renal CLC-K channel revealed by the use of phenoxy-alkyl derivatives of 2-(p-chlorophenoxy)propionic acid.

CLC channels are a gene family of Cl(-) channels that serve a variety of functions, several of which are involved in genetic diseases. Few specific ligands of CLC channels are known that could be useful as pharmacological tools or potential drugs. We synthesized various derivatives of 2-(p-chlorophenoxy)propionic acid, the S(-)-enantiomer of which is a specific blocker of the muscle channel CLC-1. In particular, compounds with different alkyl or phenoxy-alkyl groups on the chiral center, isosteres of the oxygen in the aryloxy moiety, or bioisosteres of the carboxy function were prepared. We found that compounds containing a phenoxy and a phenoxy-alkyl group on the chiral center (bis-phenoxy derivatives) specifically inhibited renal CLC-K channels from the extracellular side with an affinity in the 150-microM range and with almost no effect on other CLC channels when applied from the outside. Surprisingly, the same substances inhibited CLC-1 from the intracellular side in a voltage-dependent manner with an apparent K(D) of <5 microM at -140 mV, thus being the most potent blockers of a CLC channel known so far. Although the chlorine atom in para- position of the second phenoxy group was essential for inhibition of CLC-K channels from the outside, it could be substituted by a methoxy group without changing the potency of block for CLC-1 from the inside. These newly identified substances provide powerful tools for studying the structure-function relationship and the physiological role of CLC channels and may represent a starting point for the development of useful drugs targeting CLC-K channels.

Mechanisms of block of muscle type CLC chloride channels (Review).

CLC proteins are a nine-member gene family of Cl- channels that have diverse roles in the plasma membrane and in intracellular organelles. The recent structure determination of bacterial CLC homologues by Dutzler et al. was a breakthrough for the structure-function analysis of CLC channels. This review describes the mechanisms of inhibition of muscle type CLC channels by two classes of small organic substances: 9-anthracene carboxylic acid (9AC) and p-chlorophenoxy propionic acid (CPP). Both substances block muscle type CLC channels (CLC-0 and CLC-1) from the intracellular side. For CPP, one could show that it inhibits the individual protopores of the double-barrelled channel. A major difference between the two types of blockers is the extremely slow binding- and unbinding-kinetics of 9AC (time scale of min), compared to that of CPP block (time scale of s), while the general mechanism of block seems to be quite similar. In the case of the chiral CPP only the S(-) enantiomer is effective. Both substances exhibit a strongly voltage-dependent block with strong inhibition at negative voltages and relief of block at depolarizing potentials at which the channels tend to open maximally. A quantitative kinetic model was developed for the CPP block of CLC-0 in which the closed state has a much larger affinity for CPP than the open state and opening of drug-bound channels is greatly slowed compared to drug-free channels. First experiments with mutated CLC-0 channels and with derivatives of CPP strongly support the pore localization of the CPP binding site. This work provides the basis for the use of these small organic substances as tools to investigate the pharmacological properties of mammalian CLC channels guided by the crystallographic structure of bacterial CLC homologues. They might also turn out to be useful to obtain information about the intricate coupling of gating and permeation that characterizes CLC channels.