Micrograms Of Compound Research Articles

Fast imaging-based single particle analysis method for solubility determination

The solubility and dissolution rates of chemical compounds are crucial properties in several fields of industry and research. However, accurate, rapid and green methods for their measurement, which only consume micrograms of compound, are lacking. Here, the unique approach of non-specific, image-based single particle analysis (SPA) for solubility testing is directly compared to and thus validated on the mid-solubility range with the current gold standard shake-flask method with UV–Vis spectroscopy employed for determining sample concentrations. Five biologically active compounds representing a range of physicochemical properties including pKa and logP were analyzed with both methods. The comparison of SPA and the shake-flask (SF) analysis shows excellent linear correlation (R2 = 0.99). Higher variability of the SPA method is attributed to variability between the properties of individual particles, which cannot be detected with traditional methods. Due to the similar average solubility values compared to those produced with SF, it is concluded that the SPA method has great potential as an analytical tool for small-scale solubility studies. It also has several practical advantages over the current gold standard shake-flask method, such as speed, low consumables consumption, and no requirement for prior knowledge of compound chemistry.

A Novel Method for High Throughput Lipophilicity Determination by Microscale Shake Flask and Liquid Chromatography Tandem Mass Spectrometry

Modern small molecule drug design requires the optimization of not only the binding characteristics of the molecule but also its physicochemical properties for ADMET performance. A key physical property is lipophilicity and medicinal chemists need rapid access to high quality data in order to drive their decision making. Traditionally lipophilicity (log D) measurements are performed with a shake flask method and UV determination. This method suffers from low sensitivity and is not easily converted to a high throughput format. Over the past decade, several groups have taken different approaches to improve this assay, including replacing the shake flask method with one that utilizes reverse phase HPLC. Here we describe a new microscale shake flask method that utilizes UPLC-MS/MS to achieve increased throughput, sensitivity and accuracy. Approaches for assessing data quality are also described. This platform technology only requires micrograms of compound and is routinely used by most small molecule drug discovery project teams at Genentech.

Advanced Methods for Natural Product Drug Discovery in the Field of Nutraceuticals

Advances in analytical methods and bioassay development have helped to push forward the research in natural products. In plant extracts and nutraceuticals, bioactive compounds are part of a complex mixture. The development of high-resolution methods related to HPLC for both chemical and biological profiling has significantly increased the efficiency of classical bioactivity-guided fractionation procedures. Furthermore, the level of sensitivity obtained by these methods give the possibility to work with few micrograms of compound. This represents a key advantage for rapid localisation of the biological activity and subsequent identification of the compounds of interest. The same methods are also used to study the extracts from a metabolomic view point. The possibility to study them as a whole can highlight synergetic effects, which are likely to occur in plant extracts and nutraceuticals. In this paper, the main trends are summarised and the developments made in our laboratory on profiling crude extracts with UHPLC-TOF-MS, natural product identification at the microgram level using microflow NMR and integration of these methods with biological evaluation are highlighted.

Quantifying in Situ Rates of Phlorotannin Synthesis and Polymerization in Marine Brown Algae

Using three species of marine brown algae, we describe a stable isotope labeling technique to quantify: (1) in situ rates of phlorotannin synthesis, (2) phlorotannin polymerization or aging, and (3) the related allocation of carbon resources to secondary metabolism. In our field and laboratory assays, Lobophora variegata (Bahamas), Sargassum pteropleuron (Bahamas), and Fucus distichus (California, USA) assimilated various quantities of 13C, but all allocated ≤ 1% of the assimilated carbon to the production of phlorotannins. We quantified rates of phlorotannin synthesis both as micrograms of compound produced per gram of tissue per unit of time and as micrograms of compound produced per gram of C assimilated per unit of time. Rates of synthesis, normalized to account for differences in potential photosynthetic rates, are comparable to previously reported rates of phlorotannin accumulation. The aging of phlorotannins from low- ( 30 kDa) molecular-size polymers was observed in S. pteropleuron within a 28-hr period. Our results indicate that, using this labeling technique, it is possible to make precise measurements of allelochemical metabolism and resource allocation, which are useful both in critically evaluating the assumptions made by ecological models of plant chemistry and in estimating the partial metabolic cost of specific secondary metabolites.

Ocular anaphylaxis induced in the rat by topical application of compound 48/80

In the present study we sought to develop a model of ocular anaphylaxis based on the topical application of compound 48/80 to the surface of the rat eye. Doses ranging from 50 to 1000 micrograms were found to produce graded edema of the conjunctiva and swelling of the lid. On histologic examination, 50 microns compound 48/80 produced no changes distinguishable from those in PBS-treated controls, 150 microns produced mild alterations, and 250, 500, and 1000 micrograms compound 48/80 produced a marked increase in degranulated mast cells and a mild influx of neutrophils. The time course of the response to 250 micrograms and 1000 micrograms of compound 48/80 was evaluated over a 72-h period. Both doses elicited epithelial damage. A mild reduction in the number of mast cell was seen at 6 h in rats receiving 250 or 1000 micrograms. The reduction persisted to 72 h in rats receiving 1000 micrograms. The number of neutrophils was increased at 1 and 6 h in eyes treated with 250 micrograms and at 1, 6, and 24 h in eyes treated with 1000 micrograms compound 48/80. The clinical and histologic changes induced by application of 250 micrograms compound 48/80 resemble those seen in patients with allergic conjunctivitis suggesting that a model of ocular anaphylaxis based on the topical application compound 48/80 will be clinically relevant and experimentally practical.

Neurogenic inflammation, vascular permeability, and mast cells.

Electrical stimulation (ES) of sensory nerves causes increased vascular permeability and vasodilatation, a process known as neurogenic inflammation. The purpose of this study was to assess the role of mast cells in neurogenic inflammation induced by ES of sensory nerves. ES of the rat saphenous nerve for 1, 3, 5, 15, or 30 min induced a 166 to 436% increase in the amount of 125I-albumin deposited in the skin. Through the initial 15 min of ES, the histamine content of the skin remained unchanged. However, 30 min of ES caused a 22.1% decrease in skin histamine (p less than 0.05). ES for 5 min followed by measurement of vascular permeability from 0 to 30 min thereafter resulted in maximal increases in 125I-albumin in the skin immediately after cessation of the pulse of ES. When skin histamine was measured at various intervals after a 5-min pulse of ES, no change in the histamine content was observed through the subsequent 30 min. When mast cell degranulation was assessed histologically, 5 min of ES failed to stimulate mast cell degranulation. However, 30 min of ES caused a significant increase in the proportion of degranulating mast cells. When draining venous plasma histamine was monitored before, during and after ES, no change in plasma histamine was observed. In contrast, the intradermal injection of 5 micrograms of compound 48/80 produced a significant increase in plasma histamine. In order to examine the possibility that histamine might be released but remain in the skin after ES, skin "blisters" were developed by intradermal injections of saline. There was a significant increase in the amount of 125I-albumin extravasated into blister fluid measured after 3, 5, and 10 min of ES and a significant increase in histamine after 5 or 10 min. Therefore, prolonged ES of sensory nerves can cause mast cell degranulation. However, ES causes increased vascular permeability at times when no mast cell activation can be observed. These data suggest that the initial phases of neurogenic inflammation are independent of mast cell activation.

Mutagenic activities of fecapentaene derivatives in the Ames/ Salmonella test system

The direct mutagenic activities of a pair of naturally-occurring and several synthetic fecapentaenes were measured in the Ames/ Salmonella test system. We found that strain TA100 with preincubation was the most sensitive procedure for the naturally-occurring fecapentaene-12 (FP-12). Its natural analog, FP-14, and the synthetic isomer, cis-FP-12, yielded similar mutagenic activities to FP-12 in the range of 1000–2000 TA100 revertants per microgram of compound. The synthetic analogs of FP-12 and FP-14, MFP-12 and MFP-14, wherein the glycerol moiety was replaced by methoxy, exhibited consistently higher mutagenic activities than their parent fecapentaenes (MFP-12 was about 20 times more potent than FP-12; MFP-14 was about twice the potency of FP-14). The standard rat liver metabolizing system (S9) reduced the activities of all the fecapentaenes in a dose-related manner.

Hypertonic KCI, NaCl and capsaicin intracamerally causes release of substance P-like immunoreactive material into the aqueous humor in rabbits.

We have investigated release of substance P-like immunoreactivity (SPLI) into the anterior chamber of the rabbit eye evoked by stimuli which cause non-cholinergic miosis. In a recent study such miosis was reported to be blocked by the substance P analogue (D-Arg1, D-Pro2, D-Trp7,9, Leu11)-SP. Mechanical intracranial antidromic trigeminal nerve stimulation caused marked SPLI release presumably from primary sensory nerve endings in the anterior part of the eye. Intermittent stimulation for 20 min was not more effective than stimulation for 10 min. Intracameral injection of either 20 microliters 4.65 M KCI, 20 microliters 4.65 M NaCl or 100 micrograms capsaicin also caused SPLI release. Intracameral injection of 70 microliters 150 mM KCI, 28 micrograms prostaglandin E1 or 200 micrograms of compound 48/80 did not cause detectable SPLI release.