Application of the fluorescent probe 1-anilinonaphthalene-8-sulfonate to the measurement of the nonspecific binding of drugs to human liver microsomes.

The fluorescence of 1-anilinonaphthalene-8-sulfonate (ANS) in the presence of human liver microsomes (HLMs) is altered by drugs that bind nonspecifically to the lipid bilayer. The present study characterized the relationship between the nonspecific binding (NSB) of drugs to HLMs as measured by equilibrium dialysis and the magnitude of the change in baseline ANS fluorescence. Fraction unbound in incubations of HLMs (f(u(mic))) was determined for 16 drugs (12 bases, 3 acids, and 1 neutral) with log P values in the range 0.1 to 6.7 at three concentrations (100, 200, and 500 µM). Changes in ANS fluorescence induced by each of the drugs in the presence of HLMs were measured by spectrofluorometry. Values of f(u(mic)) determined by equilibrium dialysis ranged from 0.08 to 1.0. Although NSB of the basic drugs tended to increase with increasing log P, exceptions occurred. Basic drugs generally caused an increase in ANS fluorescence, whereas the acidic and neutral drugs resulted in a decrease in ANS fluorescence. There were highly significant (p < 0.001) linear relationships between the modulus (absolute value) of the increment/decrement in ANS fluorescence and both f(u(mic)) (r = 0.90 to 0.96) and log(1 - f(u(mic))/f(u(mic))) (r = 0.85 to 0.92) at the three drug concentrations. Agreement between measured f(u(mic)) and that predicted by ANS fluorescence was very good (<10% variance) for a validation set of six compounds. The ANS fluorescence method provides an accurate measure of the NSB of drugs to HLMs. Physicochemical determinants other than log P and charge type influence the NSB of drugs to HLMs.

Interfering ribonucleic acids that suppress expression of multiple unrelated genes.

BACKGROUND: Short interfering RNAs (siRNAs) have become the research tool of choice for gene suppression, with human clinical trials ongoing. The emphasis so far in siRNA therapeutics has been the design of one siRNA with complete complementarity to the intended target. However, there is a need for multi-targeting interfering RNA in diseases in which multiple gene products are of importance. We have investigated the possibility of using a single short synthetic duplex RNA to suppress the expression of VEGF-A and ICAM-1; genes implicated in the progression of ocular neovascular diseases such as diabetic retinopathy. RESULTS: Duplex RNA were designed to have incomplete complementarity with the 3'UTR sequences of both target genes. One such duplex, CODEMIR-1, was found to suppress VEGF and ICAM-1 by 90 and 60%, respectively in ARPE-19 cells at a transfected concentration of 40 ng/mL. Use of a cyan fusion reporter with target sites constructed in its 3'UTR demonstrated that the repression of VEGF and ICAM-1 by CODEMIR-1 was indeed due to interaction with the target sequence. An exhaustive analysis of sequence variants of CODEMIR-1 demonstrated a clear positive correlation between activity against VEGF (but not ICAM-1) and the length of the contiguous complementary region (from the 5' end of the guide strand). Various strategies, including the use of inosine bases at the sites of divergence of the target sequences were investigated. CONCLUSION: Our work demonstrates the possibility of designing multitargeting dsRNA to suppress more than one disease-altering gene. This warrants further investigation as a possible therapeutic approach.

Identification of the human cytochromes P450 catalysing the rate-limiting pathways of gliclazide elimination.

AIMS: To identify the human cytochrome P450 (CYP) enzymes responsible for the formation of the 6beta-hydroxy (6beta-OHGz), 7beta-hydroxy (7beta-OHGz) and hydroxymethyl (MeOH-Gz) metabolites of gliclizide (Gz). METHODS: 6beta-OHGz, 7beta-OHGz and MeOH-Gz formation by human liver microsomes and a panel of recombinant human P450s was measured using a high-performance liquid chromatography procedure, and the kinetics of metabolite formation was determined for each pathway. Effects of prototypic CYP enzyme selective inhibitors were characterized for each of the microsomal metabolic pathways. RESULTS: Microsomes from six human livers converted Gz to its 6beta-OHGz, 7beta-OHGz, and MeOH-Gz metabolites, with respective mean (+/- SD) K(m) values of 461 +/- 139, 404 +/- 143 and 334 +/- 75 microm and mean V(max) values of 130 +/- 55, 82 +/- 31 and 268 +/- 115 pmol min(-1) mg(-1), respectively. V(max)/K(m) ratios for the microsomal reactions parallelled relative metabolite formation in vivo. Sulfaphenazole inhibited microsomal 6beta-OHGz, 7beta-OHGz and MeOH-Gz formation by 87, 83 and 64%, respectively, whereas S-mephenytoin caused significant inhibition (48%) of only MeOH-Gz formation. Recombinant CYP2C9, CYP2C18 and CYP2C19 catalysed all hydroxylation pathways, whereas CYP2C8 formed only 6beta-OHGz and 7beta-OHGz. CONCLUSION: Taken together, the results indicate that CYP2C9 is the major contributor to Gz metabolic clearance, although CYP2C19 may also be involved in MeOH-Gz formation (the major metabolic pathway). Factors known to influence CYP2C9 activity will provide the main source of variability in Gz pharmacokinetics.

The DNAzymes Rs6, Dz13, and DzF have potent biologic effects independent of catalytic activity.

DNAzymes are catalytic DNA molecules capable of cleaving RNA substrates and therefore constitute a possible gene-suppression technology. We examined whether the previously reported potency of a DNAzyme targeting c-jun (Dz13) could be improved with judicious use of sequence and chemical modifications. Catalytic activity was measured to establish correlations between catalytic activity and biological potency. Surprisingly, Dz13 had significant cytotoxic activity against cells of rodent origin (IC(50) = 20-50 nM) despite having greatly reduced catalytic activity against a rodent target substrate (<25%), the latter being the result of a mismatch to the rodent c-jun sequence. In contrast, a modified Dz13 matching the rodent c-jun sequence (DT1501b) had no activity at similar concentrations against human or rodent cells despite being able to efficiently cleave the rodent c-jun sequence. Overall, catalytic activity against synthetic substrates did not correlate with cytotoxic activity and catalytically inactive mutants had in some cases equal or superior potency in cell cytotoxicity assays. Further examination of other previously published DNAzymes (Rs6 and DzF) revealed other occurrences of this anomalous behaviour. The active sequences all have G-rich 5 termini, suggesting that G-quadruplex formation might be involved. Consistent with this, deaza-guanosine substitutions abrogated cytotoxicity of Dz13. However, Dz13 did not show evidence of quadruplex formation as determined by circular dichroism studies and native electrophoresis. These data reveal that the biologic activity of several published DNAzymes is not mediated through the catalytic degradation of target mRNA.

Access to medicines and high-quality therapeutics: global responsibilities for clinical pharmacology.

A major theme of the 2004 World Congress of Clinical Pharmacology and Therapeutics was worldwide equity of access to medicines.

Differential contribution of active site residues in substrate recognition sites 1 and 5 to cytochrome P450 2C8 substrate selectivity and regioselectivity.

Selected active site residues in substrate recognition sites (SRS) 1 and 5 of cytochrome P450 2C8 (CYP2C8) were mutated to the corresponding amino acids present in CYP2C9 to investigate the contribution of these positions to the unique substrate selectivity and regioselectivity of CYP2C8. The effects of mutations, singly and in combination, were assessed from changes in the kinetics of paclitaxel 6alpha-hydroxylation, a CYP2C8-specific pathway, and the tolylmethyl and ring hydroxylations of torsemide, a mixed CYP2C9/CYP2C8 substrate. Within SRS1, the single mutation S114F abolished paclitaxel 6alpha-hydroxylation, while the I113V substitution resulted in modest parallel reductions in K(m) and V(max). Mutations in SRS5 (viz., V362L, G365S, and V366L) reduced paclitaxel intrinsic clearance (V(max)/K(m)) by 88-100%. Torsemide is preferentially metabolized by CYP2C9, and it was anticipated that the mutations in CYP2C8 might increase activity. However, methyl and ring hydroxylation intrinsic clearances were either unchanged or decreased by the mutations, although hydroxylation regioselectivity was often altered relative to wild-type CYP2C8. The mutations significantly increased (28-968%) K(m) values for both torsemide methyl and ring hydroxylation but had variable effects on V(max). The effects of the combined mutations in SRS1, SRS5, and SRS1 plus SRS5 were generally consistent with the changes produced by the separate mutations. Mutation of CYP2C8 at position 359 (S359I), a site of genetic polymorphism in CYP2C9, resulted in relatively minor changes in paclitaxel- and torsemide-hydroxylase activities. The results are consistent with multiple substrate binding orientations within the CYP2C8 active site and a differential contribution of active site residues to paclitaxel and torsemide binding and turnover.