Nest Carbon Dioxide Masks GABA-Dependent Seizure Susceptibility in the Naked Mole-Rat.

African naked mole-rats were likely the first mammals to evolve eusociality, and thus required adaptations to conserve energy and tolerate the low oxygen (O2) and high carbon dioxide (CO2) of a densely populated fossorial nest. As hypercapnia is known to suppress neuronal activity, we studied whether naked mole-rats might demonstrate energy savings in GABAergic inhibition. Using whole-colony behavioral monitoring of captive naked mole-rats, we found a durable nest, characterized by high CO2 levels, where all colony members spent the majority of their time. Analysis of the naked mole-rat genome revealed, uniquely among mammals, a histidine point variation in the neuronal potassium-chloride cotransporter 2 (KCC2). A histidine missense substitution mutation at this locus in the human ortholog of KCC2, found previously in patients with febrile seizures and epilepsy, has been demonstrated to diminish neuronal Cl- extrusion capacity, and thus impairs GABAergic inhibition. Seizures were observed, without pharmacological intervention, in adult naked mole-rats exposed to a simulated hyperthermic surface environment, causing systemic hypocapnic alkalosis. Consistent with the diminished function of KCC2, adult naked mole-rats demonstrate a reduced efficacy of inhibition that manifests as triggering of seizures at room temperature by the GABAA receptor (GABAAR) positive allosteric modulator diazepam. These seizures are blocked in the presence of nest-like levels of CO2 and likely to be mediated through GABAAR activity, based on in vitro recordings. Thus, altered GABAergic inhibition adds to a growing list of adaptations in the naked mole-rat and provides a plausible proximate mechanism for nesting behavior, where a return to the colony nest restores GABA-mediated inhibition.

Effect of added alkalizer and surfactant on dissolution and absorption of the potassium salt of a weakly basic poorly water-soluble drug.

Telcagepant potassium salt (MK-0974) is an oral calcitonin gene-related peptide receptor inhibitor investigated for the treatment of acute migraine. Under gastric pH conditions, the salt rapidly gels, then converts to an insoluble neutral form that creates an impervious shell on the tablet surface, resulting in a slow and variable release dissolution rate and poor bioavailability. Early attempts to develop a solid dosage form, including solid dispersion and nanosuspension formulations, resulted in low exposures in preclinical studies. Thus, a liquid-filled soft gelatin capsule (SGC) formulation (oblong 20) was used for clinical studies. However, a solid dosage form was desirable for commercialization. The slow dissolution of the tablet formulations was overcome by using a basifying agent, arginine, and inclusion of a nonionic surfactant, poloxamer 407. The combination of arginine and poloxamer in the formulation created a local transient basic microenvironment that promoted the dissolution of the salt and prevented rapid precipitation of the neutral form on the tablet surface to form the gel layer. The tablet formulation achieved fast absorption and comparable exposure to the SGC formulation. The final optimized 280 mg tablet formulation was successfully demonstrated to be bioequivalent to the 300 mg SGC formulation.

Procedure for determination of immunosuppressive drugs in whole blood with liquid chromatography-isotope dilution mass spectrometry.

BACKGROUND: Cyclosporin A, sirolimus, tacrolimus, and everolimus are immunosuppressive drugs used for therapy after organ transplantation. There are several analytical procedures for monitoring the drug level in blood, e.g. immunological methods and high-performance liquid chromatography combined with mass spectrometry (MS). From external quality assessment schemes, it became evident that the analytical results show high dispersion and further standardization is required. METHODS: Liquid/liquid extraction of the drugs from whole blood samples was performed using ammonium acetate buffer, pH 9.5, and tert-butylmethyl ether/ethyl acetate (1:1 v/v). Separation of the immunosuppressive drugs was achieved by HPLC using a phenyl-hexyl-RP column with a ternary gradient elution profile, consisting of water, methanol, and acetonitrile containing 0.1% v/v formic acid and 0.1 mmol/L Cs+. Quantification of immunosuppressive drugs was performed by isotope-dilution mass spectrometry using [2H12]-Cyclosporin A [13C, 2H3]-Rapamycin, [13C, 2H2]-Tacrolimus, and [13C2, 2H4]-42-O-(2-Hydroxyethyl)rapamycin as internal standards. RESULTS: The recovery of the new procedure was determined by analysis of spiked blood samples. The recovery in spiked EDTA whole blood samples was 100.8 - 102.5% for cyclosporin A, 101.6 - 103.0% for sirolimus, 100.0 - 101.2% for tacrolimus, and 99.5 - 102.4% for everolimus. The imprecision of the new measurement procedure, expressed as the coefficient of variation (CV), was 1.17 - 2.60% for cyclosporin A in the concentration range between 8.1 and 979 microg/L, 0.92 - 1.72% for sirolimus in the concentration range between 2.1 and 33.2 microg/L, 0.44 - 1.06% for tacrolimus in the concentration range between 2.0 and 30.8 microg/L and 0.82 - 4.34% for everolimus in the concentration range between 2.1 and 31.4 microg/L. CONCLUSIONS: An isotope dilution LC-MS/MS procedure for determination of four immunosuppressive drugs was developed to provide a basis for further development toward a reference measurement procedure.

An improved method for the bromination of metalated haloarenes via lithium, zinc transmetalation: a convenient synthesis of 1,2-dibromoarenes.

A facile protocol for the synthesis of 1,2-dibromoarenes is described. A standard ortho-lithiation/bromination procedure, when applied to bromoarenes, resulted in poor yields of the corresponding 1,2-dibromoarenes (13-62% yield). However, transmetalation of the transient aryllithium intermediate to an arylzinc species with ZnCl2, followed by bromination, resulted in dramatically improved yields of the synthetically useful 1,2-dibromoarenes (68-95% yield).

An efficient synthesis of a dual PPAR alpha/gamma agonist and the formation of a sterically congested alpha-aryloxyisobutyric acid via a Bargellini reaction.

A practical synthesis of benzisoxazole 1 and its conversion to alpha-aryloxyisobutyric acid 2 using 1,1,1-trichloro-2-methyl-2-propanol (chloretone) was developed. Benzisoxazole 1 was formed in high yields by the action of either methanesulfonyl chloride/base upon intermediate oxime 8 or with thionyl chloride/base, which initially forms cyclic sulfite 10. A highly reactive, short-lived intermediate derived from chloretone was detected by ReacIR and its half-life determined to be approximately 5 min. Reaction conditions for the Bargellini reaction were developed that resulted in a 95% yield of 2 from the reaction of highly hindered phenol 1 with chloretone hemihydrate and powdered NaOH in acetone. Thus highly hindered alpha-aryloxyisobutyric acids can be made in a single step in high yield.

A concise synthesis of a novel antiangiogenic tyrosine kinase inhibitor.

An efficient synthesis of the potent KDR inhibitor 3-[5-[[4-(methylsulfonyl)-1-piperazinyl]methyl]-1H-indole-2-yl]quinolin-2(1H)-one (1) is described. The process features a noncryogenic indole boronation and a dicyclohexylamine-mediated Suzuki coupling.

Stereoselective synthesis of a potent thrombin inhibitor by a novel P2-P3 lactone ring opening.

The concise synthesis of a potent thrombin inhibitor was accomplished by a mild lactone aminolysis between an orthogonally protected bis-benzylic amine and a diastereomerically pure lactone. The lactone was synthesized by the condensation of l-proline methyl ester with an enantiomerically pure hydroxy acid, which in turn was synthesized by a highly stereoselective (>500:1 er) and productive (100,000:1, S/C) enzymatic reduction of an alpha-ketoester. In addition, a second route to the enantiomerically pure lactone was accomplished by a diastereoselective ketoamide reduction.

Determination of the cardiac glycosides digoxin and digitoxin by liquid chromatography combined with isotope-dilution mass spectrometry (LC-IDMS)--a candidate reference measurement procedure.

This article describes a method of high analytical sensitivity, reproducibility and trueness for the determination of digoxin and digitoxin in serum or plasma at therapeutic levels using a combination of high-pressure liquid chromatography (HPLC), isotope-dilution mass spectrometry (IDMS) and caesium-adduct formation. A method for threefold deuterium substitution in the glycosides was developed, which could be performed within 24 hours without distillation giving yields > 98% of the theoretical value. Extraction from a serum or plasma matrix was performed using a liquid-phase extraction with ammonium acetate buffer/tertiary butylmethyl ether/ethyl acetate at pH 9.5. The HPLC-separation used a 10 x 2 mm LiChrospher RP-18 5 microm guard column in combination with a 125 x 2 mm main column of the same material and a gradient containing methanol, caesium ions and formic acid. Quantification of digoxin and digitoxin was made with IDMS using deuterated internal standards and the system run in single ion monitoring (SIM) mode. The methods had a lower limit of determination of 0.25 microg/l for digoxin and digitoxin, a trueness between 97.5 and 104% for digoxin and between 98 and 101% for digitoxin, respectively and had a coefficient of variation of less than 3% in the therapeutic range for both glycosides. Maximally 1 ml serum or plasma was needed for the procedure. The method is used to set target values for materials used in external quality assessment surveys (EQAS) run by INSTAND as part of a national EQAS-programme.)

Studies on the racemization of a stereolabile 5-aryl-thiazolidinedione.

The enantiomers of the stereolabile peroxisome proliferator-activated receptor (PPAR) agonist, 1, were isolated by preparative chiral chromatography and their absolute configuration established using a combination of chromatographic and NMR methods. Enantiomer interconversion was investigated under a variety of conditions, with rapid racemization being observed in most solvents, including all aqueous systems studied, irrespective of pH. Rapid racemization in both dog and human plasma was confirmed by chiral HPLC with MS detection.

How to make things work again--troubleshooting using the GC-IDMS determination of triacylglycerols as an example.

This article describes the process of "repairing" a method which has gone out of control, using the gas-chromatographic isotope-dilution mass spectrometric (GC-IDMS) determination of total glycerides in serum, measured as glycerol. The original method used 13C2-glycerol as aqueous internal standard and 12C-tripalmitin dissolved in toluene as external standard. The modified method used tripalmitin as internal and external standard, the former being labelled uniformly on the glycerol moiety of tripalmitin (13C3-tripalmitin). In addition, glycerol-free human serum albumin was added to the external standards to "trap" the glycerol physically during evaporation of the extraction solvent after alkaline hydrolysis. The modified method was more stable than the original one and the intensity of the MS-signal in the modified method was at least 100 times stronger in the external standards than in the original method. The precision of the modified method in measuring total glycerides in serum samples (as total glycerol) was better than in the original method, the coefficients of variation being under 1.5% at concentrations between 0.8 and 3.5 mmol/l.

Practical synthesis of aryl triflates under aqueous conditions.

[reaction: see text] A practical and efficient synthesis of aryl triflates under biphasic basic aqueous conditions is described. The current methodology provides entry into these valuable substrates that omits the use of amine bases and allows facile isolation by simple solvent evaporation after phase separation. Good yields can also be obtained without the use of organic solvent.

The measurement of theophylline in human serum or plasma using gas chromatography and isotope dilution-mass spectrometry (GC-IDMS) taking other substituted xanthines into consideration.

A method is described which uses a combination of gas chromatography and isotope dilution-mass spectrometry (GC-IDMS) to determine the concentration of theophylline (1,3-dimethyl xanthine) in human plasma or serum samples. The effects of similar substituted xanthines - namely theobromine (3,7-dimethyl xanthine), paraxanthine (1,7-dimethyl xanthine) 1,3-dimethyl-7-(2-hydroxyethyl) xanthine (internal standard HPLC) and caffeine (1,3,7-trimethyl xanthine) were tested to confirm the specificity of the method. The derivatisation of all xanthines was performed with N-methyl-N-trimethylsilyl trifluroacetamide (MSTFA). The internal standard used was 2-(13)C ,1,3-(15)N2-theophylline. The extraction and derivatisation procedures were examined in detail and optimised stepwise during the development of the method. High-performance liquid chromatography (HPLC) was used for comparison.

Determination of theophylline by HPLC and GC-IDMS, the effect of chemically similar xanthine derivatives on the specificity of the method and the possibility of paracetamol as interfering substance.

The aim of this study was to develop and compare high-performance liquid chromatography (HPLC) and gas chromatography coupled with isotope dilution-mass spectrometry (GC-IDMS) methods with a common extraction procedure for the determination of substituted xanthines in biological matrices such as serum and urine. For HPLC both isocratic and gradient methods were evaluated. Difficulties occurred in separation of all 6 xanthines of interest - uric acid, theobromine, theophylline, paraxanthine, caffeine and 1,3-dimethyl-7-(2-hydroxyethyl) xanthine as internal standard. In addition, paracetamol was seen to interfere at higher concentrations, which meant that a system had to be developed to separate all 7 components of interest. The final solution chosen consisted of precipitation of serum samples with 6 mol/l trichloroacetic acid followed by neutralisation with 3 mol/l KOH and chromatography on a 150 x 4.6 mm Nautilus C-18 column (Macherey & Nagel) using an isocratic elution consisting of 0.02 mol/l acetate-phosphate buffer, pH 3.0 containing 9.6% v/v acetonitrile and monitoring at 273 +/- 7 nm. Comparisons with GC-IDMS and FPIA were acceptable. Run times of 10 minutes were possible. An additional "safe time" of 5 minutes was allowed to elute any substances with similar absorption maxima which were sometimes present in commercial control sera. Precision of the method was 1.64% (intra-assay) and 2.87% (inter-assay) at 4.1 mg/l and 1.51% respectively 2.15% at 25 mg/l including extraction and measurement steps. Recovery was between 86 and 101% between 1.25 and 100 mg/l and peak time deviations for all 7 components between 0.07% and 0.34% (coefficient of variation) in 7 consecutive measurements.