The application of absolute quantitative 1H NMR spectroscopy in drug discovery and development

ABSTRACT

Introduction: The identification of a drug candidate and its structural determination is the most important step in the process of the drug discovery and for this, nuclear magnetic resonance (NMR) is one of the most selective analytical techniques.

Area covered: The present review illustrates the various perspectives of absolute quantitative ¹H NMR spectroscopy in drug discovery and development. It deals with the fundamentals of quantitative NMR (qNMR), the physiochemical properties affecting qNMR, and the latest referencing techniques used for quantification. The precise application of qNMR during various stages of drug discovery and development, namely natural product research, drug quantitation in dosage forms, drug metabolism studies, impurity profiling and solubility measurements is elaborated. To achieve this, the authors explore the literature of NMR in drug discovery and development between 1963 and 2015. It also takes into account several other reviews on the subject.

Expert opinion: qNMR experiments are used for drug discovery and development processes as it is a non-destructive, versatile and robust technique with high intra and interpersonal variability. However, there are several limitations also. qNMR of complex biological samples is incorporated with peak overlap and a low limit of quantification and this can be overcome by using hyphenated chromatographic techniques in addition to NMR.     

Substitute of Animals in Drug Research: An Approach Towards Fulfillment of 4R's

The preclinical studies for drug screening involve the use of animals which is very time consuming and expensive and at times leads to suffering of the used organism. Animal right activists around the world are increasingly opposing the use of animals. This has forced the researchers to find ways to not only decrease the time involved in drug screening procedures but also decrease the number of animals used and also increase the humane care of animals. To fulfill this goal a number of new in vitro techniques have been devised which are called 'Alternatives' or 'Substitutes' for use of animals in research involving drugs. These 'Alternatives' are defined as the adjuncts which help to decrease the use as well as the number of animals in biomedical research. Russell and Burch have defined these alternatives by three R's - Reduction, Refinement and Replacement. These alternative strategies include physico-chemical methods and techniques utilizing tissue culture, microbiological system, stem cells, DNA chips, micro fluidics, computer analysis models, epidemiological surveys and plant-tissue based materials. The advantages of these alternatives include the decrease in the number of animals used, ability to obtain the results quickly, reduction in the costs and flexibility to control the variables of the experiment. However these techniques are not glittering gold and have their own shortcomings. The disadvantages include the lack of an appropriate alternative to study the whole animal's metabolic response, inability to study transplant models and idiosyncratic responses and inability to study the body's handling of drugs and its subsequent metabolites. None-the-less these aalternative methods to certain extent help to reduce the number of animals required for research. But such alternatives cannot eliminate the need for animals in research completely. Even though no animal model is a complete set of replica for a process within a human body, the intact animal does provide a better model of the complex interaction of the physiological processes.     

Review on in vivo and in vitro methods evaluation of antioxidant activity

A good number of abstracts and research articles (in total 74) published, so far, for evaluating antioxidant activity of various samples of research interest were gone through where 407 methods were come across, which were repeated from 29 different methods. These were classified as in vitro and in vivo methods. And those are described and discussed below in this review article. In the later part of this review article, frequency of in vitro as well as in vivo methods is analyzed with a bar diagram. Solvents are important for extracting antioxidants from natural sources. Frequency of solvents used for extraction is also portrayed and the results are discussed in this article. As per this review there are 19 in vitro methods and 10 in vivo methods that are being used for the evaluation of antioxidant activity of the sample of interest. DPPH method was found to be used mostly for the in vitro antioxidant activity evaluation purpose while LPO was found as mostly used in vivo antioxidant assay. Ethanol was with the highest frequency as solvent for extraction purpose.     

Molecular-level characterization of probucol nanocrystal in water by in situ solid-state NMR spectroscopy

The molecular state of colloidal probucol nanoparticles with additives was evaluated by ¹³C in situ solid-state NMR spectroscopy. The nanoparticles were obtained by dispersing a ternary co-ground mixture of probucol/polyvinylpyrrolidon (PVP)/sodium dodecyl sulfate (SDS) in water. Their mean particle size was found to be approximately 150 nm by dynamic light scattering and cryogenic-scanning electron microscopy measurements. The results of the ¹³C in situ solid-state NMR spectroscopy showed that probucol existed in the crystalline state (form I) in water. ¹³C liquid-state NMR results indicated that PVP and SDS interacted with probucol in water. Their broad signals suggested that the surface interaction of the probucol nanocrystal with PVP and SDS stabilized the suspension. In addition, a freeze-dried sample of the suspension was studied by ¹³C solid-state NMR and powder X-ray diffraction experiments, which confirmed the presence of the probucol nanocrystals. The combination of the in situ solid-state, solid-state, and liquid-state NMR measurement results provided molecular-level insights about the role of intermolecular interactions in the design of nanoformulations.     

Automated NMR structure determination and disulfide bond identification of the myotoxin crotamine from Crotalus durissus terrificus.

Crotamine is one of four major components of the venom of the South American rattlesnake Crotalus durissus terrificus. Similar to its counterparts in the family of the myotoxins, it induces myonecrosis of skeletal muscle cells. This paper describes a new NMR structure determination of crotamine in aqueous solution at pH 5.8 and 20 degrees C, using standard homonuclear 1H NMR spectroscopy at 900MHz and the automated structure calculation software ATNOS/CANDID/DYANA. The automatic NOESY spectral analysis included the identification of a most likely combination of the six cysteines into three disulfide bonds, i.e. Cys4-Cys36, Cys11-Cys30 and Cys18-Cys37; thereby a generally applicable new computational protocol is introduced to determine unknown disulfide bond connectivities in globular proteins. A previous NMR structure determination was thus confirmed and the structure refined. Crotamine contains an alpha-helix with residues 1-7 and a two-stranded anti-parallel beta-sheet with residues 9-13 and 34-38 as the only regular secondary structures. These are connected with each other and the remainder of the polypeptide chain by the three disulfide bonds, which also form part of a central hydrophobic core. A single conformation was observed, with Pro13 and Pro21 in the trans and Pro20 in the cis-form. The global fold and the cysteine-pairing pattern of crotamine are similar to the beta-defensin fold, although the two proteins have low sequence homology, and display different biological activities.     

The modulation by xanthines of the DNA-damaging effect of polycyclic aromatic agents. Part II. The stacking complexes of caffeine with doxorubicin and mitoxantrone

Recently accumulated statistical data indicate the protective effect of caffeine consumption against several types of cancer diseases. There are also reports about protective effect of caffeine and other xanthines against tumors induced by polycyclic aromatic hydrocarbons. One of the explanations of this phenomenon is based on biological activation of such carcinogens by cytochromes that are also known for metabolism of caffeine. In the accompanying paper [Kapuscinski et al., this issue] we provide evidence (flow cytometry and the cell cycle analysis) that the cytostatic effects of caffeine (CAF) on two DNA alkylating agents, which do not require the biological activation, depend on their ability to form stacking (pi-pi) complexes. In this study, we use physicochemical techniques (computer aided light absorption and microcalorimetry), and molecular modeling to examine previously published qualitative data. This is published both by our and other group's data, indicates that CAF is able to modify the cytotoxic and/or cytostatic action of the two well known antitumor drugs doxorubicin (DOX) and mitoxantrone (MIT). To obtain the quantitative results from the experimental data we used the statistical-thermodynamical model of mixed aggregation, to find the association constants K(AC) of the CAF-drug interaction (128+/-10 and 356+/-21M(-1) for DOX-CAF and MIT-CAF complex formation, respectively). In addition, the favorable enthalpy change of CAF-MIT (DeltaH=-11.3kcal/mol) was measured by microcalorimetry titration. The molecular modeling (semi-empirical and force field method) allowed us to obtain the geometry of these complexes, which indicated the favorable energy (DeltaE) of complex formation of the protonated drug's molecules in aqueous environment (-7.4 and -8.7kcal/mol for DOX-CAF.5H(2)O and MIT-CAF.8H(2)O complex, respectively). The molecular modeling calculation indicates the existence of CAF-drug complexes in which the MIT molecules are intercalated between two CAF molecules (DeltaE=-29.9kcal/mol). These results indicate that the attenuating effect of caffeine on cytotoxic or mutagenic effects of some polycyclic aromatic mutagens cannot be the result of metabolic activation in the cells, but simply is the physicochemical process of the sequestering of aromatic molecules (e.g. carcinogens or mutagens) by formation of the stacking complexes. The caffeine may then act as the "interceptor" of potential carcinogens (especially in the upper part of digesting track) where its concentration can reach the mM level). There is, however, no indication, both, in the literature or from our experiments, that the xanthines can reverse the damage to nucleic acids at the point when the damage to DNA has already occurred.     

NMR spectroscopy: analytical applications from chemistry to the clinic

The impact of new technology in NMR instrumentation is described with reference to a range of problem areas in the pharmaceutical and biomedical fields. In particular, the contribution of very high field instruments based on superconducting magnets is considered, together with Fourier transform and related software developments. Specific application areas discussed include quantitative analysis, structure elucidation, NMR detection in high-performance liquid chromatography, analysis of body fluids, metabolic studies in single cells, analysis of intact tissue in vitro and in vivo, and NMR imaging.     

Blood sampling without blood draws for in vivo pharmacokinetic studies in rats

PURPOSE: Pharmacokinetic (PK) studies in rats typically involve removal of serial blood samples following dosing. This research illustrates the development of a fast in vivo microextraction technique that has the potential to partly replace current sampling techniques based on blood draws, especially in the case of small animals. METHODS: The proposed sampling procedure is based on solid phase microextraction (SPME), an approach that has continued to revolutionize sampling and sample preparation ever since its discovery a decade ago. In vivo microextraction is faster than conventional methods, interferes minimally with the investigated system, minimizes errors associated with sample preparation and limits exposure of lab personnel to hazardous biological samples. RESULTS: Here we show the successful application of fast microextraction during a full PK study with diazepam in rats. The results were found to correlate very well with a standard analytical method. Three calibration strategies--external, standard on the fiber, and double extraction--were employed to correlate the amount extracted with the in vivo concentration. CONCLUSIONS: Our results demonstrate the unique advantages of this technique and highlight its utility as a valuable new tool for fast bioanalysis in support of in vivo sampling and PK studies, particularly during the early drug discovery process. This approach can be used not only for drugs, but also for other exogenous or endogenous compounds.