Myriam Malet-Martino

NMR techniques in biomedical and pharmaceutical analysis

This article focuses on the description of some of the NMR techniques used in the field of biomedical and pharmaceutical research. Indeed, the NMR method has special characteristics which make it uniquely suitable for these kinds of studies. It is non-selective so that all the low molecular weight compounds in the sample investigated are detected simultaneously in a single run. NMR also provides rich structural information which is an important asset to characterize complex mixture components. NMR is quantitative, i.e. the area of a NMR signal is directly proportional to the number of corresponding nuclei and thus, at variance with other techniques, the response factor is not dependent on the molecular structure. It is also a non-invasive tool that permits in vivo studies in humans. Compared with other techniques, NMR is significantly insensitive, which represents the main drawback of the technique. The recent technological developments of the technique have nevertheless considerably improved its sensitivity. The first part of this article presents an overview of the advantages and limitations of NMR for in vitro quantitative analysis of complex matrices in liquid or semi-solid phases. The second part deals with the NMR-based metabolomics methodology. The third part describes the in vivo clinical magnetic resonance spectroscopy techniques. The fourth part reports some examples of NMR applications in the biomedical and pharmaceutical research fields.

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.

Analytical challenges in drug counterfeiting and falsification—The NMR approach

Counterfeiting of products is a global problem. As long as clothes, clocks, leather wear, etc. are faked there is no danger, but when it comes to drugs, counterfeiting can be life-threatening. In the last years sub-standard active pharmaceutical ingredients (APIs) were found more often even though the use of the quality-ensuring methods of international pharmacopoeias should have detected additional impurities and the low content of the API. Methods orthogonal to the separating methods used in the pharmacopoeias are necessary to find counterfeits. Beside Raman and NIR spectroscopies as well as powder X-ray analysis, NMR spectroscopy being a primary ratio method of measurement is highly suitable to identify and quantify a drug and its related substances as well as to recognize a drug of sub-standard quality. DOSY experiments are suitable to identify the ingredients of formulations and therefore to identify wrong and/or additional ingredients. This review gives an overview of the application of quantitative NMR spectroscopy and DOSY NMR in anticounterfeiting.

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

7-((4-Substituted)piperazin-1-yl) derivatives of ciprofloxacin: synthesis and in vitro biological evaluation as potential antitumor agents

Ciprofloxacin (CP), an antibiotic has been shown to have antiproliferative and apoptotic activities in several cancer cell lines. Moreover, several reports have highlighted the interest of increasing the lipophilicity to improve the antitumor efficacy. These studies have led us to synthesize new CP derivatives of various lipophilicities and to evaluate their activity in five human cancer cell lines. With an easy and cost-efficient procedure, 31 7-((4-substituted)piperazin-1-yl) derivatives of CP were prepared that displayed IC(50) values ranging from microM to mM concentrations and are non-toxic in vivo in healthy mice as shown by their maximal tolerated dose (MTD) indices >80 mg/kg. Several derivatives displayed higher in vitro antitumor activity than parent CP however this was not dependent on the lipophilicity of the substituent. Among all synthesized derivatives, the most potent were 2 and 6h whose IC(50) values were 10 microM in three (derivative 2) or four (derivative 6h) cancer cell lines.

Characterization of choline compounds with in vitro 1H magnetic resonance spectroscopy for the discrimination of primary brain tumors.

RATIONALE AND OBJECTIVES The authors sought to compare 1H magnetic resonance spectroscopy (MRS) spectra from extracts of low-grade and high-grade gliomas, especially with respect to the signals of choline-containing compounds. METHODS Perchloric acid extracts of six high-grade and six low-grade gliomas were analyzed by 1H MRS at 9.4 Tesla. RESULTS The signals of glycerophosphocholine (GPC) at 3.23 ppm, phosphocholine (PC) at 3.22 ppm, and choline (Cho) at 3.21 ppm were identified in both types of tumors. The absolute concentrations of all Cho-containing compounds (GPC + PC + Cho) in high-grade and low-grade gliomas were significantly different. The relative contributions of each of the Cho-containing compounds to the total choline signal were also statistically different. For high-grade gliomas, the choline signal is composed of GPC, PC, and Cho in a well-balanced contribution, whereas in low-grade gliomas, the signal is largely due to GPC with a small involvement of PC and Cho. CONCLUSIONS The differences in the concentration and the repartition of Cho-containing compounds seem to be a marker of high-grade gliomas. They could also help to discriminate between high- and low-grade gliomas in some difficult cases, especially if there is histologic uncertainty between anaplastic astrocytomas and low-grade oligodendrogliomas.

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.