Robert A. Watt

Uptake and cytotoxicity of novel nitroimidazole-polyamine conjugates in ehrlich ascites tumour cells

A number of tumour cells, including Ehrlich ascites tumour cells (EATC), possess a polyamine uptake system which selectively accumulates endogenous polyamines and structurally related compounds by an active energy dependent system(s). We suggest that it may be possible to utilize this uptake system to target certain cytotoxic agents to those tumour cells possessing this system. In an initial attempt to determine the feasibility of this suggestion, we have synthesized a series of 2- and 5-nitroimidazoles linked to polyamines and determined their ability to utilize the polyamine uptake system. Within the limited series of compounds synthesized, 2-nitroimidazole-polyamine conjugates were more potent inhibitors of spermidine uptake into EATC than the 5-nitroimidazole conjugates. It has been assumed partly based on the competitive nature of this inhibition, that the ability of these compounds to inhibit spermidine uptake is also a measure of their ability to be accumulated by EATC. A greater than 700-fold variation was observed in the ability of different analogues to inhibit spermidine uptake. The most potent inhibitors retained certain structural characteristics similar to those of spermidine. Those compounds linked to polyamines were much more potent inhibitors of polyamine uptake than the parent nitroimidazoles i.e. metronidazole and misonidazole. The toxicity of the parent compounds and their polyamine conjugates in control and polyamine-depleted EATC was assessed by measuring inhibition of tritiated thymidine incorporation. Polyamine depletion, by prior exposure to difluoromethylornithine, results in a compensatory increase in the uptake of polyamines and related structures which may result in an increase in toxicity. Whilst many of the novel conjugates showed only little or moderate toxicity to control cells, the toxicity of several of the conjugates but not the parent nitroimidazoles increased in the polyamine-depleted cells. A clear distinction was also observed between the ability to inhibit spermidine uptake (and hence affinity for the uptake system) and toxicity, e.g. compound 430, a dinitroimidazole-polyamine conjugate, was the best inhibitor of spermidine uptake studied but showed no toxicity. These results support the hypothesis that linking polyamines to nitroimidazoles facilitates the entry of the latter into cells, such as EATC, which possess the polyamine uptake system and may therefore have therapeutic application in the delivery of polyamine-linked cytotoxics to certain tumours.

An NMR study on the effect of glucose availability on carbohydrate metabolism in Dipetalonema viteae and Brugia pahangi.

Adult Brugia pahangi and Dipetalonema viteae utilise a percentage of absorbed glucose (ca. 15%) in the formation of the disaccharide trehalose [8]. This paper reports an investigation, employing 13C-NMR techniques, of the utilisation of trehalose by these nematodes and also the effect of glucose availability on metabolic product composition. The metabolism of [1-13C]trehalose in D. viteae differed dramatically from that of [1-13C]glucose under normal experimental conditions. A succinate/lactate ratio of 0.73 was obtained from the metabolism of [1-13C]trehalose compared with 0.05 from [1-13C]glucose at an initial concentration of ca. 5 mM. Similar, but less consistent, results were obtained from B. pahangi adults. Macrofilariae of D. viteae were fed variable, low levels of glucose at hourly intervals for 8 h, and a significant relationship (P less than 0.001) between the glucose addition rate and the ratio of succinate to lactate production was obtained. The lower the amount of glucose added each hour, the higher was the observed succinate to lactate ratio. The percentage yield of succinate increased greatly as the amount of added glucose was diminished. Parallel experiments performed on B. pahangi macrofilariae indicated that B. pahangi did not increase their succinate output so greatly with reduced glucose availability. It is clear that in the absence of available external glucose, B. pahangi and D. viteae draw on their internal trehalose reserves as a source of carbohydrate for energy generation.(ABSTRACT TRUNCATED AT 250 WORDS)

An investigation of the glucose metabolism of Brugia pahangi and Dipetalonema viteae by nuclear magnetic resonance spectroscopy.

This study followed the metabolism of [13C]glucose under anaerobic and aerobic conditions in the adult filarial nematodes Brugia pahangi and Dipetalonema viteae using non-invasive 13C nuclear magnetic resonance techniques. Adult B. pahangi and D. viteae showed a rapid uptake of labelled glucose which remained linear over at least 4 h. Both species of worm removed significantly more glucose from the medium under aerobic conditions than under anaerobic conditions. The principal product of metabolism, under both anaerobic and aerobic conditions, was lactate, which accounted for 62-71% of the original [13C]glucose. Examination of the maintenance medium following worm incubation revealed a further excretory product which was identified as succinate. This product accounted for 1-2% of labelled glucose in adult B. pahangi and 2-5% in adult D. viteae. The presence of succinate as an excretory product suggests that a partial reversed tricarboxylic acid cycle is active in these filarial nematodes. A further peak was identified in the worm homogenate and identified as trehalose. The disaccharide was not an excretory product and occurred only within the worm. The peak accounted for 13-14% of the 13C-labelled glucose in B. pahangi and 15-16% in D. viteae. Trehalose has not been previously recorded in either of these nematodes and is likely to have a storage function.

Identifying counterfeit medicines using near infrared spectroscopy

Counterfeit medicines are a growing threat to public health across the world and screening methods are needed to allow their rapid identification. A counterfeiter must duplicate both the physical characteristics and the chemical content of a proprietary product to avoid it being detected as a counterfeit product and this is almost impossible to get right. Counterfeit proprietary medicines are, therefore, relatively easy to identify by near infrared (NIR) spectroscopy which can detect physical as well as chemical differences between products by simple spectral comparison. Identifying generic products is more difficult as they use different excipients in the tablet or capsule matrix. Nevertheless, using appropriate models and a large library, NIR spectroscopy can detect counterfeit generic versions. Detecting sub-standard proprietary medicines can be carried out with NIR spectroscopy models and the most widely used is partial least squares regression (PLSR). General rules for generating accurate quantitative models are easy to describe. Quantifying the active pharmaceutical ingredient (API) in generic products can also be carried out using PLSR models with calibration samples generated by manufacturing laboratory samples or by collecting many generic versions of a medicine so as to obtain a good range of the API content in tablets and capsules. Using hand-held instruments or mobile laboratories allows NIR spectrometers to be taken to places where analyses may be made quickly, rather than taking the samples to a laboratory. This has the enormous advantage that the screening of large numbers of samples may be made in pharmacies and wholesalers. Imaging can bring a whole new dimension to NIR spectroscopy to allow the identification of the API and individual excipients as well as measuring the particle sizes of components and giving a measure of the homogeneity of the matrix. The effect of water on potential misidentifications may be obviated by only using blister-packed samples, having large spectral libraries subjected to different humidities or omitting the spectral region where water absorbs.

Implementation of enhanced correlation maps in near infrared chemical images: Application in pharmaceutical research

Recent developments in Hyperspectral Imaging equipment have made possible the use of this analytical technique for fast scanning of sample surfaces. This technique has turned out to be especially useful in Pharmacy, where information about the distribution of the components in the surface of a tablet can be obtained. One particular application of Hyperspectral Chemical Imaging is the search for singularities inside pharmaceutical tablets, e.g. coating defects. Nevertheless, one problem has to be faced: how to analyze a sample without any previous knowledge about it, or having only the minimum information about the tablet. In this work a new methodology, based on correlation coefficients, is introduced to obtain valuable information about one Hyperspectral Image (detection of defects, punctual contaminants, etc.) without any previous knowledge. The methodology combines Principal Component Analysis (PCA), correlation coefficient between one specific pixel included in the image and the rest of the image; and a new enhanced contrast function to obtain more selective chemical and spatial information about the image. To illustrate the applicability of the proposed methodology, real tablets of ibuprofen have been studied. The proposed methodology is presented as a control technique to detect batch variability, defects in final tablets and punctual contaminants, being a potential supplementary tool for quality controls. In addition, the usefulness of the proposed methodology is not exclusive to NIR-CI devices, but to any hyperspectral and multivariate image system.

The quantification of citral in lemongrass and lemon oils by near‐infrared spectroscopy

Previous work has demonstrated the capability of near‐infrared (NIR) spectroscopy to determine the cineole content (not less than 70% w/w) of eucalyptus oil with an accuracy comparable with that of the British Pharmacopoeia (BP) assay method. The aim of the present study was to determine if the same method was capable of quantifying other chemical constituents at similar levels in essential oils and also to ascertain if NIR spectroscopy can accurately quantify compounds present at much lower levels in essential oils. Lemongrass oil contains citral at concentrations of approximately 65–85% w/w, and lemon oil contains citral at a concentration of approximately 2–5% w/w. A total of 26 samples of pure lemongrass oil and 35 samples of pure lemon oil (both including samples that were “spiked” with citral to increase the calibration range) were scanned on the FOSS NIRSystems 6500 Rapid Content Sampler using a reflectance vessel as sample presentation method. The reference method for both types of oil was the BP monograph titration assay for the citral content of lemon oil and calibrations were constructed using these reference data. For the lemongrass oils, the mean accuracy was found to be 1.00% or less and the mean bias was 0.09% or less. For the lemon oils, the mean accuracy was found to be 4.28% or less and the mean bias was −0.71% or less. The NIR method developed was rapid, simple and non‐destructive and may prove beneficial for the accurate determination of the citral content of lemongrass oils and for the approximate citral content of lemon oils.

A near-infrared method for the assay of cineole in eucalyptus oil as an alternative to the official BP method

Eucalyptus oil of British Pharmacopoeia (BP) and European Pharmacopoeia standard must contain not less than 70.0% w/w 1,8‐cineole (eucalyptol). The official assay is a freezing‐point method which involves the addition of o‐cresol to the eucalyptus oil, whereupon the o‐cresol and the 1,8‐cineole form a solid complex. The assay has several disadvantages and we aim to show that near‐infrared (NIR) spectroscopy is an attractive alternative to this method, in that it is simple to use, requires no sample preparation and is potentially as accurate as the traditional method.

A nuclear magnetic resonance study of the role of phosphoenol pyruvate carboxykinase (PEPCK) in the glucose metabolism of Dipetalonema viteae

13C-NMR has been applied to the study of the metabolism of [1-13C]glucose by macrofilariae of Dipetalonema viteae under conditions of restricted glucose supply. In a medium buffered with 13C-labelled bicarbonate, succinate labelled in the carboxyl position is formed in good yield. Quinolinic acid, a known inhibitor of phosphoenol pyruvate carboxykinase (PEPCK) has been shown to suppress the formation of labelled succinate from [1-13C]glucose. Both sets of experiments support the formation of succinate through the PEPCK-mediated carboxylation of phosphoenol pyruvate, followed by the operation of a partial tricarboxylic acid cycle.

Identification of counterfeit medicines from the Internet and the World market using near-infrared spectroscopy

Pharmaceutical counterfeiting is a life threatening problem affecting all countries. Counterfeit medicines may be encountered anywhere in conventional markets or from the Internet. This paper proposes a rapid and non-destructive near-infrared spectroscopic method for the identification of counterfeit medicines using the minimum number of authentic samples. As little as twenty spectra from ten tablets from a batch are required to compare a test sample to its authentic counterpart. In this respect, tablets are measured as received and the correlation coefficient of the SNV-D2 spectra between the authentic sample and the test sample is determined. A correlation coefficient of lower than 0.95 indicates that the batch fails identification. In this case, if enough authentic samples are available, principal component analysis (PCA) could be applied. The PCA scores plot of the authentic and counterfeit samples with the 95% equal frequency ellipses drawn around the authentic sample set is effective in identifying counterfeits. The method could identify 82 known counterfeit medicines out of 201 medicines supplied from the Internet and the World market. However, it is still a comparative method to identify potential counterfeits and cannot identify products without authentic samples.

Rapid identification of Digitalis purpurea using near-infrared reflectance spectroscopy

Glycosides from Digitalis are widely used for the treatment of various cardiac conditions. The potential for near‐infrared (NIR) spectroscopy as a technique for the rapid identification of Digitalis purpurea was studied. If successful, this method would be advantageous over traditional methods which are destructive and time‐consuming.