Stéphane Balayssac

2D and 3D DOSY 1H NMR, a useful tool for analysis of complex mixtures: Application to herbal drugs or dietary supplements for erectile dysfunction

Seventeen herbal dietary supplements, marketed as natural substances for the enhancement of sexual function, were analyzed by diffusion ordered spectroscopy (DOSY) (1)H NMR. The method allowed a global analysis of the samples with detection of both active and inactive ingredients present in these complex matrixes. Eight formulations contained compounds related to the synthetic phosphodiesterase-5 inhibitors. Sildenafil, tadalafil, vardenafil, hydroxyhomosildenafil, thiosildenafil, and the newly identified adulterant thiomethisosildenafil were detected. Quantification of these active ingredients was carried out by HPLC or NMR. In addition to these actives, about 30 compounds or excipients were characterized. This study ended up with a three-dimensional DOSY-COSY (1)H NMR experiment on a herbal formulation which provided both virtual separation and structural information.

Inactivation of the HIF-1α/PDK3 Signaling Axis Drives Melanoma toward Mitochondrial Oxidative Metabolism and Potentiates the Therapeutic Activity of Pro-Oxidants

Cancer cells can undergo a metabolic reprogramming from oxidative phosphorylation to glycolysis that allows them to adapt to nutrient-poor microenvironments, thereby imposing a selection for aggressive variants. However, the mechanisms underlying this reprogramming are not fully understood. Using complementary approaches in validated cell lines and freshly obtained human specimens, we report here that mitochondrial respiration and oxidative phosphorylation are slowed in metastatic melanomas, even under normoxic conditions due to the persistence of a high nuclear expression of hypoxia-inducible factor-1α (HIF-1α). Pharmacologic or genetic blockades of the HIF-1α pathway decreased glycolysis and promoted mitochondrial respiration via specific reduction in the expression of pyruvate dehydrogenase kinase-3 (PDK3). Inhibiting PDK3 activity by dichloroacetate (DCA) or siRNA-mediated attenuation was sufficient to increase pyruvate dehydrogenase activity, oxidative phosphorylation, and mitochondrial reactive oxygen species generation. Notably, DCA potentiated the antitumor effects of elesclomol, a pro-oxidative drug currently in clinical development, both by limiting cell proliferation and promoting cell death. Interestingly, this combination was also effective against BRAF V600E-mutant melanoma cells that were resistant to the BRAF inhibitor vemurafenib. Cotreatment of melanomas with DCA and elesclomol in vivo achieved a more durable response than single agent alone. Our findings offer a preclinical validation of the HIF-1/PDK3 bioenergetic pathway as a new target for therapeutic intervention in metastatic melanoma, opening the door to innovative combinations that might eradicate this disease.

Analysis of adulterated herbal medicines and dietary supplements marketed for weight loss by DOSY 1H-NMR

Twenty herbal medicines or dietary supplements marketed as natural slimming products were analysed by diffusion ordered spectroscopy (DOSY) 1H-nuclear magnetic resonance (NMR) and DOSY-COSY 1H-NMR. The method allows analysis of the whole sample with the detection of both active and inactive ingredients in these complex matrices. Among the 20 formulations analysed, two were strictly herbal and four had a composition corresponding to declared ingredients on the packaging or the leaflet. The others were all adulterated. Eight formulations contain sibutramine alone at doses ranging from 4.4 to 30.5 mg/capsule. Five formulations contain sibutramine (from 5.0 to 19.6 mg/capsule or tablet) in combination with phenolphthalein (from 4.4 to 66.1 mg/capsule), and the last formulation was adulterated with synephrine (19.5 mg/capsule). Quantification of the actives was carried out with 1H-NMR. Several other compounds were also characterized including methylsynephrine, vitaberin, sugars, vitamins, etc. DOSY NMR is thus proposed as a useful tool for detection of unexpected adulteration.

Improving Metabolite Knowledge in Stable Atherosclerosis Patients by Association and Correlation of GC-MS and 1H NMR Fingerprints

The plasma of patients with stable carotid atherosclerosis (n = 9), and healthy subjects (n = 10) have been fingerprinted with both GC-MS and (1)H NMR. Principal component analysis (PCA), partial least-squares-discriminant analysis (PLS-DA) and orthogonal partial least-squares-discriminant analysis (OPLS-DA) have been applied to the profiles from each technique both separately and in combination. These techniques complement each other and enable a clearer picture of the biological samples to be interpreted not only for classification purposes, but also more importantly to define the metabolic state of patients with carotid atherosclerosis. The results showed at least 24 metabolites that were significantly modified in the group of atherosclerotic patients by this nontargeted procedure. Most of the changes can be associated to alterations of the metabolism characteristics of insulin resistance that can be strongly related to the metabolic syndrome. In addition, correlations among variables accounting for the classification show amino acids as variables whose changes showed a high degree of correlation. GC-MS and (1)H NMR fingerprints can provide complementary information in the identification of altered metabolic pathways in patients with carotid atherosclerosis. Moreover, correlations among the results with both techniques, instead of a single study, can provide a deeper insight into the patient state.

Counterfeit drugs: analytical techniques for their identification

AbstractIn recent years, the number of counterfeit drugs has increased dramatically, including not only “lifestyle” products but also vital medicines. Besides the threat to public health, the financial and reputational damage to pharmaceutical companies is substantial. The lack of robust information on the prevalence of fake drugs is an obstacle in the fight against drug counterfeiting. It is generally accepted that approximately 10% of drugs worldwide could be counterfeit, but it is also well known that this number covers very different situations depending on the country, the places where the drugs are purchased, and the definition of what constitutes a counterfeit drug. The chemical analysis of drugs suspected to be fake is a crucial step as counterfeiters are becoming increasingly sophisticated, rendering visual inspection insufficient to distinguish the genuine products from the counterfeit ones. This article critically reviews the recent analytical methods employed to control the quality of drug formulations, using as an example artemisinin derivatives, medicines particularly targeted by counterfeiters. Indeed, a broad panel of techniques have been reported for their analysis, ranging from simple and cheap in-field ones (colorimetry and thin-layer chromatography) to more advanced laboratory methods (mass spectrometry, nuclear magnetic resonance, and vibrational spectroscopies) through chromatographic methods, which remain the most widely used. The conclusion section of the article highlights the questions to be posed before selecting the most appropriate analytical approach. FigureDOSY spectrum of a counterfeit artesunate tablet containing paracetamol instead of artesunate

Detection, identification and quantification by 1H NMR of adulterants in 150 herbal dietary supplements marketed for improving sexual performance

One hundred and fifty dietary supplements (DS) marketed to increase sexual performance were analyzed. All these formulations were claimed to contain only natural compounds, plant extracts and/or vitamins. (1)H NMR spectroscopy was used for detecting the presence of adulterants and for their identification and quantification. Mass spectrometry was used as a complementary method for confirming the chemical structures. 61% of DS were adulterated with phosphodiesterase-5 inhibitors (PDE-5i) (27% with the PDE-5i medicines sildenafil, tadalafil and vardenafil, and 34% with their structurally modified analogues). Among them, 64% contained only one PDE-5i and 36% mixtures of two, three and even four. The amounts of PDE-5i medicines were higher than the maximum recommended dose in 25% of DS tainted with these drugs. Additional 5.5% DS included other drugs for the treatment of sexual dysfunction (yohimbine, flibanserin, phentolamine, dehydroepiandrosterone or testosterone). Some DS (2.5%) contained products (osthole, icariin) extracted from plants known to improve sexual performance. Only 31% of the samples could be considered as true herbal/natural products. A follow-up over time of several DS revealed that manufacturers make changes in the chemical composition of the formulations. Lack of quality or consistent manufacture (contamination possibly due to inadequate cleaning of the manufacturing chain, presence of impurities or degradation products, various compositions of a given DS with the same batch number, inadequate labelling) indicated poor manufacturing practices. In conclusion, this paper demonstrates the power of (1)H NMR spectroscopy as a first-line method for the detection of adulterated herbal/natural DS and the need for more effective quality control of purported herbal DS.

Analysis of herbal dietary supplements for sexual performance enhancement: First characterization of propoxyphenyl-thiohydroxyhomosildenafil and identification of sildenafil, thiosildenafil, phentolamine and tetrahydropalmatine as adulterants

Nine herbal dietary supplements intended to be beverages for enhancing sexual performance were analyzed before their possible launch on the market. Four of them contained a sildenafil analog reported for the first time as an adulterant. After isolation and characterization using NMR, MS, IR and UV, this analog was named propoxyphenyl-thiohydroxyhomosildenafil as the ethoxy chain on the phenyl ring of the already known analog thiohydroxyhomosildenafil was replaced by a propoxy moiety. One formulation was tainted with thiosildenafil, another unapproved PDE-5 inhibitor. Sildenafil along with the natural alkaloid tetrahydropalmatine that has no documented effect for enhancing erectile dysfunction were identified in two formulations. Another formulation was adulterated with phentolamine, a drug that is not approved for boosting male sexual performance when taken orally. The last formulation containing osthole, a bioactive natural coumarine improving sexual dysfunction, is most probably truly natural.

Exploiting Mitochondrial Dysfunction for Effective Elimination of Imatinib-Resistant Leukemic Cells

Challenges today concern chronic myeloid leukemia (CML) patients resistant to imatinib. There is growing evidence that imatinib-resistant leukemic cells present abnormal glucose metabolism but the impact on mitochondria has been neglected. Our work aimed to better understand and exploit the metabolic alterations of imatinib-resistant leukemic cells. Imatinib-resistant cells presented high glycolysis as compared to sensitive cells. Consistently, expression of key glycolytic enzymes, at least partly mediated by HIF-1α, was modified in imatinib-resistant cells suggesting that imatinib-resistant cells uncouple glycolytic flux from pyruvate oxidation. Interestingly, mitochondria of imatinib-resistant cells exhibited accumulation of TCA cycle intermediates, increased NADH and low oxygen consumption. These mitochondrial alterations due to the partial failure of ETC were further confirmed in leukemic cells isolated from some imatinib-resistant CML patients. As a consequence, mitochondria generated more ROS than those of imatinib-sensitive cells. This, in turn, resulted in increased death of imatinib-resistant leukemic cells following in vitro or in vivo treatment with the pro-oxidants, PEITC and Trisenox, in a syngeneic mouse tumor model. Conversely, inhibition of glycolysis caused derepression of respiration leading to lower cellular ROS. In conclusion, these findings indicate that imatinib-resistant leukemic cells have an unexpected mitochondrial dysfunction that could be exploited for selective therapeutic intervention.

Two-dimensional DOSY experiment with Excitation Sculpting water suppression for the analysis of natural and biological media

The Bipolar Pulse Pair Stimulated Echo NMR pulse sequence was modified to blend the original Excitation Sculpting water signal suppression. The sequence is a powerful tool to generate rapidly, with a good spectrum quality, bidimensional DOSY experiments without solvent signal, thus allowing the analysis of complex mixtures such as plant extracts or biofluids. The sequence has also been successfully implemented for a protein at very-low concentration in interaction with a small ligand, namely the salivary IB5 protein binding the polyphenol epigallocatechine gallate. The artifacts created by this sequence can be observed, checked and removed thanks to NPK and NMRnotebook softwares to give a perfect bidimensional DOSY spectrum.

1H NMR Metabolomic Signatures in Five Brain Regions of the AβPPswe Tg2576 Mouse Model of Alzheimer's Disease at Four Ages

In the quest for biomarkers of onset and progression of Alzheimers disease, a 1H NMR-based metabolomic study was performed on the simple single-transgenic Tg2576 mouse model. These mice develop a slow cognitive decline starting by 6 months and express amyloid plaques from 10 months of age. The metabolic profiles of extracts from five brain regions (frontal cortex, rhinal cortex, hippocampus, midbrain, and cerebellum) of Tg2576 male mice were compared to those of controls, at 1, 3, 6 and 11 months of age. The most obvious differences were due to brain regions. Age was also a discriminating parameter. Metabolic perturbations were already detected in the hippocampus and the rhinal cortex of transgenic mice as early as 1 month of age with decreased concentrations of glutamate (Glu) and N-acetylaspartate (NAA) compared to those in wild-type animals. Metabolic changes were more numerous in the hippocampus and the rhinal cortex of 3 month-old transgenic mice and involved Glu, NAA, myo-inositol, creatine, phosphocholine, and γ-aminobutyric acid (only in the hippocampus) whose concentrations decreased. A metabolic disruption characterized by an increase in the hippocampal concentrations of Glu, creatine, and taurine was detected in 6 month-old transgenic mice. At this time point, the chemical profile of the cerebellum was slightly affected. At 11 months, all the brain regions analyzed (except the frontal cortex) were metabolically altered, with mainly a marked increase in the formation of the neuroprotective metabolites creatine and taurine. Our findings demonstrate that metabolic modifications occur long before the onset of behavioral impairment.