Synthesis physicochemical profile and PAMPA study of new NO-donor edaravone co-drugs.

A new class of co-drugs were synthesised by joining antioxidant edaravone with a vasodilating substructure containing NO-donor nitrooxy functions, and characterised for their stability in different media, lipophilicity and permeability profile. The products display good stability in water/co-solvent at different pH. Conversely, they are rapidly metabolised into edaravone and NO-donor moieties when incubated in human serum or rat-liver homogenates. In the latter conditions time dependent production of nitrite/nitrate (NO(x)) occurs. The compounds display wide-ranging lipophilicity. PAMPA studies predict good gastrointestinal absorption for a number of these compounds. The title products are potentially useful for treating ROS-related conditions accompanied by decreased NO availability.

Treatment with a nitric oxide-donating NSAID alleviates functional muscle ischemia in the mouse model of Duchenne muscular dystrophy.

In patients with Duchenne muscular dystrophy (DMD) and the standard mdx mouse model of DMD, dystrophin deficiency causes loss of neuronal nitric oxide synthase (nNOSµ) from the sarcolemma, producing functional ischemia when the muscles are exercised. We asked if functional muscle ischemia would be eliminated and normal blood flow regulation restored by treatment with an exogenous nitric oxide (NO)-donating drug. Beginning at 8 weeks of age, mdx mice were fed a standard diet supplemented with 1% soybean oil alone or in combination with a low (15 mg/kg) or high (45 mg/kg) dose of HCT 1026, a NO-donating nonsteroidal anti-inflammatory agent which has previously been shown to slow disease progression in the mdx model. After 1 month of treatment, vasoconstrictor responses to intra-arterial norepinephrine (NE) were compared in resting and contracting hindlimbs. In untreated mdx mice, the usual effect of muscle contraction to attenuate NE-mediated vasoconstriction was impaired, resulting in functional ischemia: NE evoked similar decreases in femoral blood flow velocity and femoral vascular conductance (FVC) in the contracting compared to resting hindlimbs (ΔFVC contraction/ΔFVC rest=0.88 ± 0.03). NE-induced functional ischemia was unaffected by low dose HCT 1026 (ΔFVC ratio=0.92 ± 0.04; P>0.05 vs untreated), but was alleviated by the high dose of the drug (ΔFVC ratio=0.22 ± 0.03; P<0.05 vs untreated or low dose). The beneficial effect of high dose HCT 1026 was maintained with treatment up to 3 months. The effect of the NO-donating drug HCT 1026 to normalize blood flow regulation in contracting mdx mouse hindlimb muscles suggests a putative novel treatment for DMD. Further translational research is warranted.

Moving from fast to ballistic gradient in liquid chromatography/tandem mass spectrometry pharmaceutical bioanalysis: Matrix effect and chromatographic evaluations.

The paper describes the steps taken by the authors to move from a fast to a ballistic gradient in routine liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of plasma samples from pharmacokinetic (PK) profiling of new chemical entities. The reduction of column dimensions from 50 x 4.6 mm to 30 x 2.1 mm followed by optimization of chromatographic separation led to a decrease in the typical runtime from 5 (fast) to 2 min (ballistic) using an API4000 tandem mass spectrometer in Turbo Ionspray mode for detection. Three analytical standards representing typical molecular structures from our sample repository were used to spike plasma from four different species (rat, dog, human and mouse). Two different approaches were used to evaluate matrix effect: post-column infusion and comparison of the peak areas of neat standards and standards spiked after extraction into different pools of plasma; the influence of PEG400 as a typical dosing vehicle was also considered. Two different protein precipitation procedures were taken into account for sample extraction prior to injection. Peak shape, width and height, selectivity and sensitivity of the method were taken into account for chromatographic evaluation. The ballistic method was successfully cross-validated with the conventional fast gradient chromatographic assay.

A quantitative high-throughput trapping assay as a measurement of potential for bioactivation.

Idiosyncratic adverse drug reactions (ADRs) are one of the most common causes of pharmaceutical withdrawals and labeling changes. Most ADRs are caused by drugs that form reactive species that can bind covalently to macromolecules such as proteins. The current methodology for the measurement of covalent binding relies on the use of radiolabeled material that requires an investment in time and resources not typically expended until later in the discovery process. Efforts are also made to identify reactive intermediates by the use of chemical trapping agents, such as reduced glutathione and cyanide, to form stable adducts that are characterized by liquid chromatography-tandem mass spectrometry and/or nuclear magnetic resonance spectroscopy. Here, we describe a high-throughput assay for the measurement of reactive intermediate formation. The method involves incubation of cold compound with liver microsomes in the presence of [14C]potassium cyanide. Hard electrophilic species would react with the trapping agent, resulting in the formation of a radiolabeled conjugate. Unreacted trapping agent is removed using solid-phase extraction, and the amount of radiolabeled conjugate present is determined by liquid scintillation counting. This newly developed screen has proved to be specific, sensitive, robust, and a powerful tool for assessing bioactivation potential.