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La Enfermedad de Chagas (EC) es todavía, a día de hoy, una enfermedad tropical desatendida (NTD) que afecta y pone en peligro a millones de personas en todo el mundo. Los tratamientos existentes distan de ser los ideales, ya que sus numerosos efectos adversos y sus largos intervalos de tratamiento provocan su falta de adherencia. Es por ello que existe una urgente necesidad de encontrar nuevos fármacos más seguros, eficaces, potentes y económicos que los de referencia. En las últimas décadas, diversos grupos de investigación se han centrado en la búsqueda de moléculas que sean capaces de interferir con enzimas que estén involucradas en el metabolismo de Trypanosoma cruzi (T. cruzi), y por tanto, en su supervivencia. Tras una extensa revisión bibliográfica acerca de algunas de las dianas descubiertas en el parásito, se han podido establecer los requerimientos estructurales principales para el diseño de nuevas moléculas que puedan resultar ser selectivas hacia una o varias de las enzimas contempladas. Continuando con la línea de investigación llevada a cabo por nuestro grupo, se han diseñado, sintetizado y caracterizado sesenta y nueve bases de Mannich para ser evaluadas frente a T. cruzi. Estos nuevos derivados contienen en su estructura dos heterociclos ampliamente estudiados y de reconocida actividad anti-T. cruzi: el imidazol y el benzimidazol. Todas las modificaciones estructurales definidas en el diseño de estos nuevos derivados se han realizado con el objetivo de obtener potenciales candidatos frente la EC. Estas nuevas bases de Mannich imidazólicas se han evaluado in vitro mediante un método de cribado que permite ensayar librerías de compuestos en poco tiempo, de manera eficiente y reproducible. Esta cascada de ensayos incluye experimentos sobre las distintas formas parasitarias de T. cruzi y células del huésped. De esta forma, se ha podido evaluar su actividad tripanocida y su toxicidad, y optimizar el proceso de selección de los compuestos ensayados para asegurar que, sólo aquellos más prometedores in vitro frente a T. cruzi puedan progresar a los ensayos in vivo con alta eficacia y eficiencia. De todos ellos, el compuesto 3c se presenta como el más prometedor, lo que ha permitido estudiarlo en profundidad en la fase pre-clínica: se ha evaluado su perfil toxicológico (genotoxicidad y mutagenicidad) y farmacocinético, se ha determinado su biodisponibilidad, se ha ensayado in vivo en combinación con el fármaco de referencia Benznid azol (BZ). Además, en un intento de conocer su posible mecanismo de acción, se han llevado a cabo estudios de docking para proponer su supuesto modo de unión frente a las enzimas de T. cruzi estudiadas anteriormente. Los resultados obtenidos permitirán promover estudios adicionales de interés sobre este compuesto líder y supondrán un punto de partida para futuras líneas de investigación.

Chagas disease (CD) is a centenarian neglected parasitosis caused by the protozoan Trypanosoma cruzi (T. cruzi). Despite the continuous efforts of many organizations and institutions, CD is still an important human health problem worldwide. A lack of a safe and affordable treatment has led drug discovery programmes to focus, for years, on the search for molecules enabling interference with enzymes that are essential for T. cruzi survival. In this work, the authors want to offer a brief overview of the different validated targets that are involved in diverse parasite pathways: glycolysis, sterol synthesis, the de novo biosynthesis of pyrimidine nucleotides, the degradative processing of peptides and proteins, oxidative stress damage and purine salvage and nucleotide synthesis and metabolism. Their structural aspects, function, active sites, etc. were studied and considered with the aim of defining molecular bases in the search for new effective treatments for CD. This review also compiles, as much as possible, all the inhibitors reported to date against these T. cruzi targets, serving as a reference for future research in this field.

Chagas disease is considered a neglected tropical disease that continues to endanger millions of people around the world. The current chemotherapy presents some disadvantages such as variable antiparasitic activity, undesired side effects and/or long treatment duration. Therefore, the development of new drugs is mandatory. Several studies suggest the Mannich base derivatives as an attractive scaffold for new antiparasitic agents. Our research group has developed new Mannich bases with promising antichagasic activity. In this context, we have designed and synthesized forty-five Mannich bases to be tested against the T. cruzi parasite. The structural modifications made for the design of new derivatives have been made in order to obtain more effective and less toxic potential candidates to combat this parasitic disease. The novel Mannich bases containing the ferrocenyl, phenethylamines, anilines or piperazines groups, among others, have been tested against three different T. cruzi strains and forms. The cytotoxicity on Vero cells was also evaluated to determine their selectivity index. According to potency and SI, the most promising compounds were selected to evaluate the genotoxicity capacity via SOS/umu-test. Once demonstrated that these compounds are non-genotoxic, two compounds were selected for in vivo study in BALB/c mice. After the in vivo test, the non-mutagenic capacity of the compound with the most promising antichagasic profile was evaluated by the Ames test. On the other hand, the possible mechanism of action of these compounds was studied through the alteration of excreted metabolites by the parasite during glucose metabolism, the detection of mitochondrial alterations and the inhibition of superoxide dismutase. Finally, computational studies were executed to propose the binding mode of the studied compounds to iron superoxide dismutase enzyme. The results obtained will allow us to promote further preclinical studies of the most promising compounds.

Chagas Disease (CD) and Leishmaniasis are two parasitic diseases transmitted by vectors and caused by protozoa from the Trypanosomatidae family. Both diseases are included within the neglected tropical diseases group and are caused by Trypanosoma cruzi and the Leishmania genus protozoa respectively. Chagas disease is endemic in 21 countries in the Americas and affects about 5 and 6 million people, causing 7,000 deaths annually. With regard to leishmaniasis, it is present in 98 countries of all continents, causing about 40,000 deaths annually. During its life cycle, T. cruzi and Leishmania spp acquire different development forms depending on the medium where they are. T. cruzi presents three stages: The trypomastigote form, highly infective for the mammalian host, epimastigote form, which is the replicative form in the insect vector and amastigote form, which is the reproductive form in mammalian cells. The genus Leishmania presents two evolutionary forms, the promastigote form, which is the infective form for humans and the amastigote form, which is the replicative form in mammalian cells. Regarding the treatment of CD and Leishmaniasis, despite its high prevalence and that they were discovered many years ago, there is still no fully an effective treatment from an activity, safety and cost point of view. In CD, only two drugs, Nifurtimox and Benznidazole, have an accepted clinical use. Both drugs have important limitations such as the multiple adverse reactions, their lower efficacy in the chronic phase of the disease, the complicated dosage, the long duration of the treatment and the resistant strains. With regard to leishmaniasis, the variety of available treatments is much wider than for CD. These treatments have a high efficacy, however, they have important limitations such as toxicity, adverse effects, high cost, the development of resistance, the long periods of treatment, the administration route and the need for hospitalization. Taking into account this situation of necessity in the acquisition and identification of new trypanocidal and leishmanicidal agents, after an extensive bibliographic review and considering the experience of the research group, a molecule repositioning strategy was applied as a starting point for the design of the presented arylaminoketone Mannich bases type derivatives. Sixty-two new derivatives were synthesized using two one-step and economically efficient synthetic methods. Fifty-eight were obtained by the Mannich reaction and four by an aliphatic nucleophilic substitution. The family of Mannich bases derivatives that has been published has shown great potential for the development of new agents against CD and Leishmaniasis. Compounds with excellent in vitro efficacy results have been identified against both kinetoplastids, with very low cytotoxicity against mammalian cells, a non-genotoxic profile and non-mutagenic. The leading compounds had a high in vivo activity against T. cruzi in the acute phase. In addition, no reactivation of parasitemia was observed in a immunosuppression murine model and they were very effectively in the presence prevention of the parasite in the target organs. In addition, the identified lead compounds had a high bioavailability. Because of this results we propose these compounds as a very promising molecules to continue their preclinical development as possible anti- kinetoplastids agentst.

We report the synthesis and in vitro activity against T. cruzi epimastigotes of 15 novel quinoxaline derivatives. Ten of the derivatives presented IC50 values lower than the reference drugs Nfx and Bzn; four of them standed out with IC50 values lower than 1.5 M. Moreover, unspecific cytotoxicity and genotoxicity studies are also reported. Compound 14 showed a SI higher than 24, whereas compound 10 was the only one that was negative in the genotoxicity screening.

More than 100 million people are at risk of contracting Chagas disease. It is estimated that in 2008, Chagas disease was responsible for the death of more than 10,000 people. Despite the fact that there are more than 100 years since the disease was discove red, safe and effective treatments still have to be found. Therefore, new drugs active against Chagas disease are urgently required. Quinoxaline derivatives show very interesting biological properties (anti- infective, cytotoxic, anticandida, antiprotozoal)and evaluation of their medicinal chemistry is still in progress. In this regard, we have spent more than a decade in the search for anti-Chagasic agents. In this review, we summarize our on-going work to identify new anti-T. cruzi agents with the quinoxaline-di-N-oxide scaffold. We present the structure-activity relationship observed among the different substitutions in C-2, C-3, C-6 and C-7 position of the quinoxaline ring. Mais de 100 milhões de pessoas estão em risco de contrair a doença de Chagas. Estima-se que em 2008, mais de 10.000 pessoas morreram devido a esta patologia. Apesar de a doença ter sido descoberta há mais de 100 anos, ainda é preciso encontrar tratamento seguro e eficaz. Portanto, novos fármacos eficazes contra a doença de Chagas necessitam urgentemente serem descobertos. Derivados de quinoxalina apresentam interessantes propriedades biológicas (anti- infecciosa, citotóxica, anti-candida, anti- protozoário) e a avaliação de suas propriedades farmacológicas ainda está em andamento. Com respeito a isso, nós estamos há mais de uma década em busca de agentes anti-chagásicos. Nesta revisão mostramos nosso trabalho em andamento para identificação de novos agentes anti-T. cruzi com o padrão estrutural quinoxalina-di-N- óxido e apresentamos a relação estrutura-atividade observada entre as diferentes substituições nas posições C-2, C-3, C-6 e C-7 do anel daquinoxalina

The experimental work presented in this memory is based on the hypothesis that quinoxalines di-N-oxide, considered to be the core of the structure, which present a carboxamide moiety on position two and aliphatic linker between this group and an aromatic system, can be proposed as potent anti-tuberculosis agents. The main purpose of this project is the synthesis of new quinoxaline di-N-oxide derivatives as anti-tuberculosis agents. The strategy consists of the design and synthesis of several derivatives of each series, and their in vitro biological evaluation against M.tb. Some compounds will be selected, based on their structural variability and biological results, for an in depth study of the physicochemical properties and electrochemical behavior of this family of derivatives. A series of specific objectives are proposed in order to achieve the main purpose of this project: 1. Bibliographic review: state of the art. 2. Design of new quinoxaline di-N-oxide derivatives. 3. Synthesis of the designed compounds. 4. Characterization of the synthesized compounds. 5. In vitro biological evaluation of the synthesized derivatives. 6. Study of the lipophilicity of quinoxaline di-N-oxide derivatives. 7. Study of the electrochemical behavior of quinoxaline di-N-oxide derivatives. 8. Study of possible structure-property and structure-activity relationships (SPR, SAR). 9. Search of new lead compounds and design of future work plans.

As a continuation of our research and with the aim of obtaining new agents against Chagas disease, an extremely neglected disease which threatens 100 million people, eighteen new quinoxaline 1,4-di-Noxide derivatives have been synthesized following the Beirut reaction. The synthesis of the new derivatives was optimized through the use of a new and more efficient microwave-assisted organic synthetic method. The new derivatives showed excellent in vitro biological activity against Trypanosoma cruzi. Compound 17, which was substituted with fluoro groups at the 6- and 7-positions of the quinoxaline ring, was the most active and selective in the cytotoxicity assay. The electrochemical study showed that the most active compounds, which were substituted by electron-withdrawing groups,possessed a greater ease of reduction of the N-oxide groups

Ever since the idea arose that melatonin might promote sleep and resynchronize circadian rhythms, many research groups have centered their efforts on obtaining new melatonin receptor ligands whose pharmacophores include an aliphatic chain of variable length united to an N-alkylamide and a methoxy group (or a bioisostere), linked to a central ring. Substitution of the indole ring found in melatonin with a naphthalene or quinoline ring leads to compounds of similar affinity. The next step in this structural approximation is to introduce a quinoxaline ring (a bioisostere of the quinoline and naphthalene rings) as the central nucleus of future melatoninergic ligands

We report the synthesis, anti-inflammatory and antioxidant activities of novel quinoxaline and quinoxaline 1,4-di-N-oxide derivatives. Microwave assisted methods have been used in order to optimize reaction times and to improve the yields. The tested compounds presented important scavenging activities and promising in vitro inhibition of soybean lipoxygenase. Two of the best lipoxygenase inhibitors (compounds 7b and 8f) were evaluated as in vivo anti-inflammatory agents using the carrageenin-induced edema model. One of them (compound 7b) showed important in vivo anti-inflammatory effect (41%) similar to that of indomethacin (47%) used as the reference drug.

As reported in our previous papers, a series of quinoxaline-2-carboxamide 1,4-di-N-oxide derivatives were synthesized and studied as anti-tuberculosis agents. Here, the capability of the shake-flask method was studied and the retention time (expressed as log K) of 20 compounds were determined by RP-HPLC analysis. We found that the prediction of log P by the RP-HPLC analysis can result in a high accuracy and can replace the shake-flask method avoiding the experimental problems presented by quinoxaline di-N-oxides. The studied compounds were subjected to the ALOGPS module with the aim of comparing experimental log Po/w values and predicted data. Moreover, a preliminary in silico screening of the QSAR relationship was made confirming the influence of reduction peak potential, lipophilicity, H-bond donor capacity and molecular dimension descriptors on anti-tuberculosis activity.

Continuing with the efforts to identify new active compounds against malaria and leishmaniasis, fourteen new 3-amino-1,4-di-N-oxide quinoxaline-2-carbonitrile derivatives were synthesized and evaluated for their in vitro antimalarial and antileishmanial activity against Plasmodium falciparum Colombian FCR-3 strain and Leishmania amazonensis strain MHOM/BR/76/LTB-012A. Further computational studies to analyze graphic SAR and ADME properties were undertaken. Results indicate that compounds with one halogenous group substituted in position 6 and 7 provide an efficient approach for further development of antimalarial and antileishmanial agents. In addition, interesting ADME properties were found

The increase in the prevalence of drug-resistant tuberculosis cases demonstrates the need of discovering new and promising compounds with antimycobacterial activity. As a continuation of our research and with the aim of identifying new antitubercular drugs candidates, a new series of quinoxaline 1,4-di-N-oxide derivatives containing isoniazid was synthesized and evaluated for in vitro anti-tuberculosis activity against Mycobacterium tuberculosis H37Rv strain. Moreover, various drug-like properties of new compounds were predicted. Taking into account the biological results and the promising drug-likeness profile of these compounds, make them valid leads for further experimental research.

New drugs active against drug-resistant tuberculosis are urgently needed to extend the range of TB treatment options to cover drug resistant infections. Quinoxaline derivatives show very interesting biological properties (antibacterial, antiviral, anticancer, antifungal, antihelmintic, insecticidal) and evaluation of their medicinal chemistry is still in progress. In this review we report the properties and the recent developments of quinoxaline 1,4-di-N-oxide derivatives as potential anti-tuberculosis agents. Specific agents are reviewed that have excellent antitubercular drug properties, are active on drug resistant strains and non-replicating mycobacteria. The properties of select analogs that have in vivo activity in the low dose aerosol infection model in mice will be reviewed.

Piperazine and pyrrolidine derivatives were synthesized and evaluated for their capacity to inhibit the growth of Plasmodium falciparum chloroquine-resistant (FCR-3) strain in culture. The combined presence of a hydroxyl group, a propane chain and a fluor were shown to be crucial for the antiplasmodial activity. Five compounds of the aryl-alcohol series inhibited 50% of parasite growth at doses ≤ 10 µM. The most active compound 1-(4-fluoronaphthyl)-3-[4-(4-nitro-2-trifluoromethylphenyl)piperazin-1-yl]propan-1-ol was almost 20 to 40 times more active on Plasmodium falciparum (IC50: 0.5 µM) than on tumorogenic and non tumorogenic cells. Calculated physicochemical parameters showed a good potential for intestinal absorption, but due to difficulty in being solubilised prior to oral administration, it was weakly active against Plasmodium berghei infected mice (ED50: 35%). In silico molecular docking study and molecular electrostatic potential calculation revealed that this compound bound to the active site of Plasmodium plasmepsin II enzyme.

Two series of pyrazol and propenone quinoxaline derivatives were tested for parasiticidal activity (against amastigotes of Leishmania peruviana and trypomastigotes of Trypanosoma cruzi) and for toxicity against proliferative and non-proliferative cells. The pyrazol series was almost inactive against T. cruzi but, 2,6-Dimethyl-3-[5-(3,4,5-trimethoxy-phenyl)-4,5-dihydro-1H-pyrazol-3-yl] - quinoxaline 1,4-dioxide inhibited 50% of Leishmania growth at 8.9 µM with no impact against proliferative kidney cells and low toxicity against Thp-1 and murine macrophages. The compounds of the propenone series were moderately active against T. cruzi. Among them, 2 compounds were particularly interesting: (2E)-1-(7-Fluoro-3-methyl-quinoxalin-2-yl)-3-(3,4,5-trimethoxy-phenyl)-propenone, that showed a selective activity against proliferative cells (cancer and parasites), being inactive against normal murine peritoneal macrophages and (2E)-3-(3,4,5-Trimethoxy-phenyl)-1-(3,6,7-trimethyl-quinoxalin-2-yl)-propenone that was only active against Leishmania and inactive against the other tested cells. Furthermore in silico studies were performed for ADME properties and docking studies, both series of compounds respected the Lipinski’s rules and show linear correlation between tripanosomaticidal activities and LogP. Docking studies revealed that compounds of the second series could interact with the poly (ADP-ribose) polymerase protein of Trypanosoma cruzi.

As a continuation of our research and with the aim of obtaining new anti-tuberculosis agents which can improve the current chemotherapeutic anti-tuberculosis treatments, forty-three new quinoxaline-2-carboxamide 1,4-di-N-oxide derivatives were synthesized and evaluated for in vitro anti-tuberculosis activity against Mycobacterium tuberculosis strain H37Rv. Active compounds were also screened to assess toxicity to a VERO cell line. Results indicate that compounds with a methyl moiety substituted in position 3 and unsubstituted benzyl substituted on the carboxamide group provide an efficient approach for further development of anti-tuberculosis agents.

Mycobacterium tuberculosis (M.Tb) is a bacillus capable of causing a chronic and fatal condition in humans known as tuberculosis (TB). It is estimated that there are 8 million new cases of TB per year and 3.1 million infected people die annually. Thirty-six new amide quinoxaline 1,4-di-N-oxide derivatives have been synthesized and evaluated as potential anti-tubercular agents,obtaining biological values similar to the reference compound, Rifampin (RIF).

La malaria es una enfermedad infecciosa causada por el parásito Plasmodium. Según la OMS, causa 266 mil muertes anuales. La mayoría de los casos se registran en los países más pobres, lo que unido a la situación económica actual y a la falta de tratamientos eficaces, hace imprescindible la búsqueda de nuevas estructuras que generen tratamientos de corta duración y bajo coste. La Unidad de I+D de Medicamentos lleva años trabajando en este área y esta tesis doctoral recoge el desarrollo de una de sus líneas de trabajo que se centra en el diseño y síntesis de nuevos derivados de arilaminoalcoholes como agente antimaláricos. El desarrollo de las nuevas estructuras tomó como punto de partida un trabajo previo del grupo. Así en esta tesis doctoral queda descrito el desarrollo químico completo de todas las nuevas moléculas obtenidas mediante procesos sintéticos sencillos y su posterior caracterización por diferentes técnicas de elucidación estructural como la resonancia magnética nuclear, análisis elemental, espectroscopia infrarroja o punto de fusión. Treinta nuevos derivados fueron evaluados in vitro por el Grupo Malaria de Colombia, frente a cepas NF54 y FCR-3 de P. falciparum, tomando como referencia la cloroquina. A su vez se estudió su citotoxicidad in vitro en células Vero en el Instituto de Investigación para el Desarrollo (IRD) de Francia. Los resultados obtenidos en derivados hidroxílicos muestran actividad antiplasmódica muy interesante frente a cepas de Plasmodium resistentes a la cloroquina. Cuatro de los compuestos que mostraron mejores datos in vitro fueron seleccionados para el estudio de actividad in vivo en el IRD de Perú en roedores infectados con la cepa Plasmodium berghei NK65 aunque los datos obtenidos no superaron los ofrecidos por la cloroquina. Durante los ensayos biológicos se realizaron estudios computacionales que mostraron que los derivados desarrollados mostraban buenas características farmacocinéticas a priori. Los estudios de acoplamiento (docking) propusieron como diana terapéutica la enzima plasmepsina II, esencial en los procesos de degradación de la hemoglobina, vitales para el desarrollo del parásito. Los buenos resultados de docking presentan una unión de los derivados con el sitio activo de la enzima y apoyan los datos de actividad de los ensayos in vitro. Se sugiere de esta forma una estructura farmacofórica que permitirá continuar con el proyecto con el refinamiento estructural, diseño y síntesis de estructuras ya mejoradas.

Objectives: To evaluate a novel series of quinoxaline 1,4-di-N-oxides for in vitro activity against Mycobacterium tuberculosis and for efficacy in a mouse model of tuberculosis (TB). Methods: Ketone and amide derivatives of quinoxaline 1,4-di-N-oxide were evaluated in in vitro and in vivo tests including: (i) activity against M. tuberculosis resistant to currently used antitubercular drugs including multidrug-resistant strains (MDR-TB resistant to isoniazid and rifampicin); (ii) activity against non-replicating persistent (NRP) bacteria; (iii) MBC; (iv) maximum tolerated dose, oral bioavailability and in vivo efficacy in mice; and (v) potential for cross-resistance with another bioreduced drug, PA-824. Results: Ten compounds were tested on single drug-resistant M. tuberculosis. In general, all compounds were active with ratios of MICs against resistant and non-resistant strains of

This study extends earlier reports regarding the in vitro efficacies of the 1,4-di-N-oxide quinoxaline derivatives against Mycobacterium tuberculosis and has led to the discovery of a derivative with in vivo efficacy in the mouse model of tuberculosis. Quinoxaline-2-carboxylate 1,4-di-N-oxide derivatives were tested in vitro against a broad panel of single-drug-resistant M. tuberculosis strains. The susceptibilities of these strains to some compounds were comparable to those of strain H(37)Rv, as indicated by the ratios of MICs for resistant and nonresistant strains, supporting the premise that 1,4-di-N-oxide quinoxaline derivatives have a novel mode of action unrelated to those of the currently used antitubercular drugs. Specific derivatives were further evaluated in a series of in vivo assays, including evaluations of the maximum tolerated doses, the levels of oral bioavailability, and the efficacies in a low-dose aerosol model of tuberculosis in mice. One compound, ethyl 7-chloro-3-methylquinoxaline-2-carboxylate 1,4-dioxide, was found to be (i) active in reducing CFU counts in both the lungs and spleens of infected mice following oral administration, (ii) active against PA-824-resistant Mycobacterium bovis, indicating that the pathway of bioreduction/activation is different from that of PA-824 (a bioreduced nitroimidazole that is in clinical trials), and (iii) very active against nonreplicating bacteria adapted to low-oxygen conditions. These data indicate that 1,4-di-N-oxide quinoxalines hold promise for the treatment of tuberculosis.

The aim of this study was to identify new compounds active against Plasmodium falciparum based on our previous research carried out on 3-phenylquinoxaline- 2-carbonitrile 1,4-di-N-oxide derivatives. Twelve compounds were synthesized and evaluated for antimalarial activity. Eight of them showed an IC50 < 1 μM against the 3D7 strain. Derivative 1 demonstrated high potency (IC50= 0.63 μM) and good selectivity (SI=10.35), thereby becoming a new lead-compound.

La restricción calórica es la terapia nutricional más frecuente en el tratamiento de la obesidad, cuya eficacia depende de la respuesta oxidativa del organismo para evitar la modificación del peso corporal. En este contexto, el objetivo del presente trabajo fue examinarin vivo la oxidación mitocondrial de voluntarios obesos, antes y después de adelgazar, utilizando el test en aliento con 2-ceto[1-13C]isocaproato. El estudio se realizó en 32 voluntarios de ambos sexos: 16 controles (índice de masa corporal: 19,0-27,0 kg/m²), y 16 obesos (índice de masa corporal: 30,0-41,6 kg/m²) que siguieron un período de restricción calórica (-500 kcal) durante 10 semanas. El test con 2-ceto[1-13C]isocaproato se realizó antes y después del tratamiento, a partir de la ingestión de 1 mg/kg de trazador y 20 mg/kg de L-leucina, disueltos en 200 mL de zumo de naranja. Antes y después de la ingestión (cada 10 minutos durante 2 horas), se tomaron muestras de aliento en las que se midió el enriquecimiento en13C mediante espectrometría de masas de relación isotópica. A partir de estas determinaciones se calculó el porcentaje de trazador oxidado en las mitocondrias (%13C). Los obesos tendieron a oxidar un porcentaje menor de trazador que los controles (25,1 ± 5,5% vs 27,5 ± 4,0% p = 0,175). Tras el período de intervención, la pérdida de peso medio fue -7,8 ± 3% (p < 0,001), y se acompañó de un aumento significativo en la oxidación del trazador (25,1 ± 5,5% vs 34,3 ± 5,2% p < 0,001). De hecho, el peso corporal y el porcentaje de 2-ceto[1-13C]isocaproato oxidado fueron inversamente proporcionales (r = -0,34, p = 0,018). Por tanto, el test en aliento con 2-ceto[1(13)-C]isocaproato detectó in vivo la adaptación de la oxidación mitocondrial en obesos tratados mediante restricción calórica, ofreciendo una información complementaria sobre la pérdida de peso.