Thiazolidinediones and type 2 diabetes: new drugs for an old disease

Thiazolidinediones are a new class of drugs for the treatment of type 2 diabetes, and act by improving insulin sensitivity in adipose tissue, liver and skeletal muscle. Rosiglitazone and pioglitazone are registered for use in monotherapy, and in combination with sulfonylureas and metformin. Pioglitazone is also licensed for use in combination with insulin. There is level II evidence that in patients with inadequate glycaemic control both drugs reduce the level of HbA(1c) and fasting plasma glucose (FPG) when used as monotherapy and in combination with sulfonylurea or metformin or insulin; and both drugs increase levels of HDL and LDL and lower free fatty acid levels, but only pioglitazone significantly lowers triglyceride levels. Both drugs lower fasting insulin and C-peptide levels. In monotherapy, they may be slightly less potent at reducing the level of HbA(1c) than sulfonylureas or metformin. The maximal effect of these agents may not be seen for 6-14 weeks after commencement. Both drugs are well tolerated but liver function must be checked at baseline every second month for the first year, and periodically thereafter. The drugs are currently contraindicated in patients with moderate to severe liver dysfunction and alanine aminotransferase levels more than 2.5 times normal, New York Heart Association III-IV cardiac status, pregnancy, lactation and in children. The main side effects include weight gain, oedema, and mild dilutional anaemia.

The measurement of actual acidity in acid sulfate soils and the determination of sulfidic acidity in suspension after peroxide oxidation

Improvements to the routine methods for the determination of actual acidity in suspension for acid sulfate soils (ASS) are introduced. The titratable sulfidic acidity (TSA) results using an improved peroxide-based method were compared with the theoretical acidity predicted by the chromium reducible sulfur method for 9 acid sulfate soils. The regression between these 2 measures of sulfidic acidity was highly significant, the slope of the regression line not significantly different from unity (P = 0.05) and the intercept not significantly different from zero. This contrasts with results of other workers using earlier peroxide oxidation methods, where TSA substantially underestimated the theoretical acidity predicted by reduced inorganic sulfur analysis. Comparison was made between the 2 principal measurements from the improved peroxide method (TSA and S-POS), with S-POS converted to theoretical sulfidic acidity to allow comparison. The relationship between these 2 measurements was highly significant. The effects of titration in suspension, as well as raising titration end points to pH 6.5, were investigated, principally with respect to the titratable actual acidity (TAA) result. TAA results obtained by KCl extraction were compared with those obtained using BaCl2, MgCl2, and water extraction. TAA in 1 M KCl suspensions titrated to pH 6.5 agreed well with titratable actual acidity measured using the 25-h extraction approach of the Lin et al. (2000a) BaCl2 method. Both BaCl2 and KCl solutions were ineffective at fully recovering acidity from synthetic jarosite without repeated extraction and titration. The application of correction factors for the estimation of total actual acidity in ASS is not supported by the results of this investigation. Acid sulfate soils that contain substantial quantities of jarosite or other acid-producing but relatively insoluble sulfate minerals continue to prove problematic to chemically analyse; however, an approach for estimating this component is discussed.

Inhibition of tubulin assembly and covalent binding to microtubular protein by valproic acid glucuronide in vitro

Acyl glucuronides are reactive metabolites of carboxylate drugs, able to undergo a number of reactions in vitro and in vivo, including isomerization via intramolecular rearrangement and covalent adduct formation with proteins. The intrinsic reactivity of a particular acyl glucuronide depends upon the chemical makeup of the drug moiety. The least reactive acyl glucuronide yet reported is valproic acid acyl glucuronide (VPA-G), which is the major metabolite of the antiepileptic agent valproic acid (VPA). In this study, we showed that both VPA-G and its rearrangement isomers (iso-VPA-G) interacted with bovine brain microtubular protein (MTP, comprised of 85% tubulin and 15% microtubule associated proteins {MAPs}). MTP was incubated with VPA, VPA-G and iso-VPA-G for 2 h at room temperature and pH 7.5 at various concentrations up to 4 mM. VPA-G and iso-VPA-G caused dose-dependent inhibition of assembly of MTP into microtubules, with 50% inhibition (IC50) values of 1.0 and 0.2 mM respectively, suggesting that iso-VPA-G has five times more inhibitory potential than VPA-G. VPA itself did not inhibit microtubule formation except at very high concentrations (greater than or equal to2 mM). Dialysis to remove unbound VPA-G and iso-VPA-G (prior to the assembly assay) diminished inhibition while not removing it. Comparison of covalent binding of VPA-G and iso-VPA-G (using [C-14]-labelled species) showed that adduct formation was much greater for iso-vTA-G. When [C-14]-iso-VPA-G was reacted with MTP in the presence of sodium cyanide (to stabilize glycation adducts), subsequent separation into tubulin and MAPs fractions by ion exchange chromatography revealed that 78 and 22% of the covalent binding occurred with the MAPs and tubulin fractions respectively. These experiments support the notion of both covalent and reversible binding playing parts in the inhibition of microtubule formation from MTP (though the acyl glucuronide of VPA is less important than its rearrangement isomers in this regard), and that both tubulin and (perhaps more importantly) MAPs form adducts with acyl glucuronides. (C) 2002 Elsevier Science Inc. All rights reserved.

Development of a selective photoactivatable antagonist for corticotropin-releasing factor receptor, type 2 (CRF2)

A novel photoactivatable analog of antisauvagine-30 (aSvg-30), a specific antagonist for corticotropin-releasing factor (CRF) receptor, type 2 (CRF2), has been synthesized and characterized. The N-terminal amino-acid D-Phe in aSvg-30 [D-Phe11,His12] Svg((11-40)) was replaced by a phenyldiazirine, the 4-(1-azi-2,2,2-trifluoroethyl) benzoyl (ATB) residue. The photoactivatable aSvg-30 analog ATB-[ His12] Svg was tested for its ability to displace [I-125-Tyr0] oCRF or [I-125-Tyr0]Svg from membrane homogenates of human embryonic kidney (HEK) 293 cells stably transfected with cDNA coding for rat CRF receptor, type 1 ( rCRF(1)) or mouse CRF receptor, type 2beta (mCRF(2beta)). Furthermore, the ability of ATB- [His12] Svg((12-40)) to inhibit oCRF- or Svg-stimulated cAMP production of transfected HEK 293 cells expressing either rCRF(1) (HEK-rCRF(1) cells) or mCRF(2beta) (HEK-mCRF(2beta) cells) was determined. Unlike astressin and photo astressin, ATB- [His12]Svg((12-40)) showed high selective binding to mCRF(2beta) (K-i = 3.1 +/- 0.2 nM) but not the rCRF(1) receptor (K-i = 142. 5 +/- 22.3 nM) and decreased Svg-stimulated cAMP activity in mCRF(2beta)-expressing cells in a similar fashion as aSvg-30. A66-kDa protein was identified by SDS/PAGE, when the radioactively iodinated analog of ATB- [His12]Svg((12-40)) was covalently linked to mCRF(2beta) receptor. The specificity of the photoactivatable I-125-labeled CRF2beta antagonist was demonstrated with SDS/PAGE by the finding that this analog could be displaced from the receptor by antisauvagine-30, but not other unrelated peptides such as vasoactive intestinal peptide (VIP).

The synthesis and structure of an n-terminal dodecanoic acid conjugate of a-conotoxin MII

The alpha-conotoxin MII is a 16 amino acid long peptide toxin isolated from the marine snail, Conus magus. This toxin has been found to be a highly selective and potent inhibitor of neuronal nicotinic acetylcholine receptors of the subtype alpha3beta2. To improve the bioavailability of this peptide, we have coupled to the N-terminus of conotoxin MII, 2-amino-D,L-dodecanoic acid (Laa) creating a lipidic linear peptide which was then successfully oxidised to produce the correctly folded conotoxin MII construct.

Activation of ion secretion via proteinase-activated receptor-2 in human colon

Proteinase-activated receptor (PAR) type 2 (PAR-2) has been shown to mediate ion secretion in cultured epithelial cells and rat jejunum. With the use of a microUssing chamber, we demonstrate the role of PAR-2 for ion transport in native human colonic mucosa obtained from 30 normal individuals and 11 cystic fibrosis (CF) patients. Trypsin induced Cl- secretion when added to the basolateral but not luminal side of normal epithelia. Activation of Cl- secretion by trypsin was inhibited by indomethacin and was further increased by cAMP in normal tissues but was not present in CF colon, indicating the requirement of luminal CF transmembrane conductance regulator. Effects of trypsin were largely reduced by low Cl-,by basolateral bumetanide, and in the presence of barium or clotrimazole, but not by tetrodotoxin. Furthermore, trypsin-induced secretion was inhibited by the Ca2+-ATPase inhibitor cyclopiazonic acid and in low-Ca2+ buffer. The effects of trypsin were almost abolished by trypsin inhibitor. Thrombin, an activator of PAR types 1, 3, and 4, had no effects on equivalent short-circuit currents. The presence of PAR-2 in human colon epithelium was confirmed by RT-PCR and additional experiments with PAR-2-activating peptide. PAR-2-mediated intestinal electrolyte secretion by release of mast cell tryptase and potentiation of PAR-2 expression by tumor necrosis factor-alpha may contribute to the hypersecretion observed in inflammatory processes such as chronic inflammatory bowel disease.

Effects of rosiglitazone and linoleic acid on human preadipocyte differentiation

Background Peroxisome proliferator activated receptor gamma (PPARgamma) is a ligand-activated transcription factor known to be central to both adipose tissue development and insulin action. Growth of adipose tissue requires differentiation of preadipocytes with acquisition of specific cellular functions including insulin sensitivity, leptin secretion and the capacity to store triglyceride. Dietary fatty acids and members of the thiazolidinedione class of compounds have been reported to influence adipogenesis at the transcriptional level. Here, we compare the effects of a dietary fatty acid, linoleic acid, and a thiazolidinedione, rosiglitazone, on biochemical and functional aspects of human preadipocyte differentiation in vitro . Materials and methods Human omental and subcutaneous preadipocytes were subcultured 2-3 times and subsequently differentiated for 21 days in the presence of either linoleic acid or rosiglitazone. Differentiation was assessed using a number of biochemical and functional parameters. Results Omental and subcutaneous preadipocytes differentiated in the presence of linoleic acid showed marked cytoplasmic triacylglycerol accumulation however, no biochemical markers of differentiation (LPL expression, G3PDH gene expression and enzyme activity and leptin expression or secretion) were detected. In contrast, treatment of these cells with rosiglitazone induced full biochemical differentiation as judged by all markers assessed, despite comparatively little lipid accumulation. The rosiglitazone effects were subcutaneous depot-specific. Cells treated with linoleic acid showed decreased glucose uptake cf rosiglitazone-treated cells. A luciferase reporter assay demonstrated that rosiglitazone potently activates h-peroxisome proliferator activated receptor gamma while linoleic acid had no effect. Conclusions These studies demonstrate that (a) human preadipocytes have the potential to accumulate triacylglycerol irrespective of their stage of biochemical differentiation; (b) while omental preadipocytes are refractory to biochemical differentiation in vitro , they are able to accumulate triacylglycerol; and (c) rosiglitazone and linoleic acid may exert their effects via different biochemical pathways.

Fatty acid binding protein is a major determinant of hepatic pharmacokinetics of palmitate and its metabolites

Disposition kinetics of [H-3] palmitate and its low-molecular-weight metabolites in perfused rat livers were studied using the multiple-indicator dilution technique, a selective assay for [H-3] palmitate and its low-molecular-weight metabolites, and several physiologically based pharmacokinetic models. The level of liver fatty acid binding protein (L-FABP), other intrahepatic binding proteins (microsomal protein, albumin, and glutathione S-transferase) and the outflow profiles of [H-3] palmitate and metabolites were measured in four experimentalgroups of rats: 1) males; 2) clofibrate-treated males; 3) females; and 4) pregnant females. A slow-diffusion/bound model was found to better describe the hepatic disposition of unchanged [H-3] palmitate than other pharmacokinetic models. The L-FABP levels followed the order: pregnant female > clofibrate-treated male > female > male. Levels of other intrahepatic proteins did not differ significantly. The hepatic extraction ratio and mean transit time for unchanged palmitate, as well as the production of low-molecular-weight metabolites of palmitate and their retention in the liver, increased with increasing L-FABP levels. Palmitate metabolic clearance, permeability-surface area product, retention of palmitate by the liver, and cytoplasmic diffusion constant for unchanged [H-3] palmitate also increased with increasing L-FABP levels. It is concluded that the variability in hepatic pharmacokinetics of unchanged [H-3] palmitate and its low-molecular-weight metabolites in perfused rat livers is related to levels of L-FABP and not those of other intrahepatic proteins.

Flavonoids, phenolic acids and abscisic acid in Australian and New Zealand Leptospermum honeys

Flavonoids, phenolic acids and abscisic acid of Australian and New Zealand Leptospermum honeys were analyzed by HPLC. Fifteen flavonoids were isolated in Australian jelly bush honey (Leptospermum polygalifolium), with an average content of 2.22 mg/100 g honey. Myricetin (3,5,7,3',4',5'-hexahydroxyflavone), luteolin (5,7,3',4'-tetrahydroxyflavone) and tricetin (5,7,3',4',5'-pentahydroxyflavone) were the main flavonoids identified. The mean content of total phenolic acids in jelly bush honey was 5.14 mg/100 g honey, with gallic and coumaric acids as the potential phenolic acids. Abscisic acid was quantified as twice the amount (11.6 mg/100 g honey) of the phenolic acids in this honey. The flavonoid profile mainly consisted of quercetin (3,5,7,3',4'-pentahydroxyflavone), isorhamnetin (3,5,7,4'-tetrahydroxyflavone 3'-methyl ethyl), chrysin (5,7-dihydroxyflavone), luteolin and an unknown flavanone in New Zealand manuka (Leptospermum scoparium) honey with an average content of total flavonoids of 3.06 mg/100 g honey. The content of total phenolic acids was up to 14.0 mg/100 g honey, with gallic acid as the main component. A substantial quantity (32.8 mg/100 g honey) of abscisic acid was present in manuka honey. These results showed that flavonoids and phenolic acids could be used for authenticating honey floral origins, and abscisic acid may aid in this authentication. (C) 2002 Published by Elsevier Science Ltd.