The synthesis of [26,27-11C]dihydroxyvitamin D(3), a tracer for positron emission tomography (PET).

1Alpha,25-dihydroxyvitamin D(3), an endogenous ligand with the highest affinity for the vitamin D receptor (VDR), was labeled with 11C for use in biological experiments. The radionuclide was incorporated via the reaction of [11C]methyllithium on a methyl ketone precursor in tetrahydrofuran at -10 degrees C. Deprotection of the labeled intermediate yielded 2.5-3 GBq [26,27-11C]1alpha,25-dihydroxyvitamin D(3) [11C-1,25(OH)(2) D(3)] with specific radioactivity averaging 100 GBq/micromol at the end of synthesis and HPLC purification. The entire process took 48 min from the end of radionuclide production. In vitro binding experiments in rachitic chick purified VDR demonstrated the high affinity binding of this novel tracer. Thus; 11C-1,25(OH)(2) D(3) is available for in vivo distribution studies and may be suitable for the positron emission tomography (PET) determination of VDR levels and occupancy in animals and humans.

Metabolism of glycerol-1,2,3-trimethylsuccinate in rat hepatocytes.

The metabolism of glycerol-1,2,3-trimethylsuccinate ester was investigated in rat hepatocytes. The ester displayed a greater nutritional value than D-glucose, as a precursor of either CO2 or glycogen. In terms of 14CO2 production, the value calculated from experiments conducted in the presence of 1.9 mM [U-14C] glycerol-1,2,3-trimethylsuccinate, glycerol-1,2,3-trimethyl[1,4-14C] succinate and glycerol- 1,2,3-trimethyl[2,3-14C] succinate represented about 50 times that found in cells incubated with 1.0 mM D-[U-14C] glucose. For glycogen synthesis, the results found with the ester were approximately 7-8 times higher than those found with the hexose. A further advantage of the ester over D-glucose consisted in the fact that, at increasing concentrations of these nutrients, a maximal metabolic response may be reached at lower levels of glycerol- 1,2,3-trimethylsuccinate than D-glucose. By comparison with previous data obtained in the same experimental model, glycerol-1,2,3-trimethylsuccinate was also found to display a higher nutritional value than the dimethyl ester of succinic acid. It is proposed, therefore, that glycerol-1,2,3-trimethylsuccinate could be used to support ATP generation in cells endangered by an imbalance between the rate of synthesis and hydrolysis of this adenine nucleotide.

Metabolism of [1,3-(13)C]glycerol-1,2,3-tris(methylsuccinate) and glycerol-1,2,3-tris(methyl[2,3-(13)C]succinate) in hepatocytes from Goto-Kakizaki rats.

The metabolism of [1,3-(13)C]glycerol-1,2,3-tris(methylsuccinate) and glycerol-1,2,3-tris(methyl[2,3-(13)C] succinate) was examined in hepatocytes prepared from hereditarily diabetic Goto-Kakizaki rats. Over 120 min incubation in the presence of one of the two (13)C-labelled esters (2.5 mM), the output of (13)C-enriched glucose averaged 57.1 +/- 18.5 and 54.1 +/- 22.7 nmol per 10(6) cells, when expressed as [1,3-(13)C]glycerol and [2,3-(13)C] succinate equivalent, respectively. In the case of [1,3-(13)C]glycerol-1,2,3-tris(methyl-succinate), the molecules of glucose were symmetrically labelled. In the case of glycerol-1,2,3-tris(methyl[2,3-(13)C] succinate), however, both the single-labelled and double-labelled isotopomers of glucose contained more (13)C atoms in their C(6)-C(5)-C(4) than C(1)-C(2)-C(3) moiety. These findings indicate that glycerol-1,2,3-tris(methylsuccinate), recently proposed as a novel insulinotropic tool for the treatment of non-insulin-dependent diabetes mellitus, is efficiently metabolized in hepatocytes from diabetic rats, the high rate of gluconeogenesis coinciding with channelling of D-glyceraldehyde-3-phosphate between glyceraldehyde-3-phosphate dehydrogenase and phosphofructoaldolase.

Studies on the interaction between the vitamin D receptor and the radiolabelled 20-epi vitamin D analogue GS 1500.

Previous studies have shown that the binding affinity of a vitamin D analogue for the vitamin D receptor (VDR) does not correlate with the biological potency of the compound. In the present investigation the vitamin D analogue GS 1500, which is characterised by an altered stereochemistry at carbon C-20 (20-epi) and an aromatic ring in the side chain, was studied with respect to its interaction with the VDR. Using [3H]-GS 1500 as tracer, the receptor binding properties of GS 1500 were investigated and compared to those of 1,25(OH)2D3. The binding studies did not reveal a different binding site for GS 1500 than the one already established, and the binding affinity was in accordance with previously found values. At the level of VDR interaction with the vitamin D responsive element, GS 1500 did induce a binding complex at a lower concentration than 1,25(OH)2D3, which may help explain the difference in potency.

New 1 alpha,25-dihydroxy vitamin D3 analogues with side chains attached to C-18: synthesis and biological activity.

A new class of analogues of 1 alpha,25-dihydroxy vitamin D3 has been synthesised, in which the side chain (C-23 to C-27) has been removed and where new hydroxylated side chains have been attached to the C-18 methyl group. These analogues show antiproliferative activity in U937 and HaCaT cells comparable to that of 1 alpha,25-dihydroxy vitamin D3. Lack of calcemic activity makes these analogues potentially useful in the treatment of proliferative diseases.

Metabolism of glycerol-1,2,3-trimethylsuccinate in rat pancreatic islets.

The metabolism of 14C-labelled glycerol-1,2,3-trimethylsuccinate (2.0 mM) was examined in rat pancreatic islets. The oxidation of the glycerol moiety of the ester was negligible relative to that of its succinate residues. The oxidation of glycerol-1,2,3-trimethyl[1,4-(14)C]succinate was two times higher than that of glycerol-1,2,3-trimethyl[2,3-(14)C]succinate, this difference being matched by a higher generation of 14C-labelled acidic metabolites and amino acids from the latter than from the former tracer. The total generation of 14CO2 from the ester, uniformly labelled except in its methyl groups, was close to that found for the oxidation of 1.0 mM D-[U-(14)C]glucose. These findings thus reveal that glycerol-1,2,3-trimethylsuccinate is efficiently metabolized in islet cells and support the idea that this ester could be used as a nutrient to bypass defects of D-glucose transport and metabolism in the islet B-cell and, hence, improve proinsulin biosynthesis and insulin release in non-insulin-dependent diabetes mellitus.

Derivatives of 2-methylenepropane-1,3-diol as new antagonists of platelet activating factor.

Two new achiral platelet activating factor (PAF) antagonists, N-[5-[[2-methylene-3- [[(octadecylamino)carbonyl]oxy]propoxy]carbonyl]pentyl]pyridinium bromide and 3-[6-[[2-methylene-3- [[(octadecylamino)carbonyl]oxy]propoxy]carbonyl]hexyl]thiazolium bromide were synthesized from 2-methylenepropane-1,3-diol. Platelet aggregation in platelet-rich plasma from rabbits, induced by racemic C16-PAF, was competitively antagonized by 9 or 10. At concentrations less than or equal to 10(-4) M, neither compound 9 nor compound 10 caused platelet aggregation, nor did they inhibit platelet aggregation induced by collagen or adenosine diphosphate. Bronchoconstriction in the guinea pig and hypotension in the rat, induced by racemic C16-PAF, were also effectively antagonized by 9 and 10. Both appear to be more potent as PAF antagonists than Takeda's CV-3988.