The use of informatics and automation to remove bottlenecks in drug discovery.

Advances in molecular biology and genetics have furnished more targets than could be reasonably progressed, forcing the pharmaceutical industry to invest in increasing chemistry and screening throughput. Combinatorial chemistry, automation and miniaturization are described as the keys to success. Many pharmaceutical companies assisted by the vendor community have risen to the challenges, delivering more functional and reliable robotics; a number of recent developments are described. These, in turn, have highlighted other deficiencies, for example in target selection, integration and scheduling, and assay and reaction optimization. These areas provide the challenges for the future and have already sparked several new initiatives, a number of which are described.

Automated assay optimization with integrated statistics and smart robotics.

The transition from manual to robotic high throughput screening (HTS) in the last few years has made it feasible to screen hundreds of thousands of chemical entities against a biological target in less than a month. This rate of HTS has increased the visibility of bottlenecks, one of which is assay optimization. In many organizations, experimental methods are generated by therapeutic teams associated with specific targets and passed on to the HTS group. The resulting assays frequently need to be further optimized to withstand the rigors and time frames inherent in robotic handling. Issues such as protein aggregation, ligand instability, and cellular viability are common variables in the optimization process. The availability of robotics capable of performing rapid random access tasks has made it possible to design optimization experiments that would be either very difficult or impossible for a person to carry out. Our approach to reducing the assay optimization bottleneck has been to unify the highly specific fields of statistics, biochemistry, and robotics. The product of these endeavors is a process we have named automated assay optimization (AAO). This has enabled us to determine final optimized assay conditions, which are often a composite of variables that we would not have arrived at by examining each variable independently. We have applied this approach to both radioligand binding and enzymatic assays and have realized benefits in both time and performance that we would not have predicted a priori. The fully developed AAO process encompasses the ability to download information to a robot and have liquid handling methods automatically created. This evolution in smart robotics has proven to be an invaluable tool for maintaining high-quality data in the context of increasing HTS demands.

Metabolism of the "mixed" cytochrome P-450 inducer hexachlorobenzene by rat liver microsomes.

Hexachlorobenzene (HCB) was metabolised by phenobarbital-induced liver microsomes from male rats to pentachlorobenzene, pentachlorophenol, tetrachloro-1,2-benzenediol and tetrachloro-1,4-benzenediol (1:88:2:9). Metabolites were identified and quantified by electron capture g.l.c. Structures were confirmed by selective ion monitoring g.l.c.-m.s. The formation of pentachlorophenol was dependent on the presence of NADPH and O2 and inhibited by CO, SKF 525A and metyrapone. Conversion of HCB to pentachlorophenol was stimulated by pretreatment of rats with phenobarbital (PB) but not by 3-methylcholanthrene (3-MC), or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast, the conversion of pentachlorophenol to tetrachloro-1,4-benzenediol was markedly induced by 3-MC but poorly by PB. HCB, Aroclor 1254 and isosafrole stimulated both hydroxylations. The cytochrome P-450c inhibitor 9-hydroxyellipticine inhibited conversion of pentachlorophenol to tetrachlorobenzenediols by HCB and beta-naphthoflavone induced micromes. In addition to hydroxylation reactions, evidence was obtained for the conjugation of HCB with glutathione catalysed by a microsomal glutathione transferase. Radioactivity from [14C]HCB was bound to microsomal protein during aerobic incubations. Binding was inhibited by GSH and N-acetyl-cysteine. Preliminary studies suggested that the reactive species was derived from tetrachloro-1,4-benzoquinone. No correlation was found between levels of metabolites or covalent binding produced by the two sexes and the marked sex dependent hepatic porphyrogenic and carcinogenic effects of HCB.

Dentin permeability: effects of temperature on hydraulic conductance.

The rates of fluid movement across dentin discs, in vitro, were measured at 10, 20, 30, 40, and 50 degrees C in unetched and acid-etched dentin. Increasing the temperature 40 degrees (i.e., from 10 to 50 degrees C) resulted in a 1.8-fold increase in fluid flow in unetched dentin, which was of a magnitude similar to the decrease in viscosity that occurred over the same temperature range. In acid-etched dentin, the 40 degrees C temperature change produced more than a four-fold increase in fluid conductance, more than double that which could be accounted for by changes in viscosity. Analysis of the data suggests that this additional increment in hydraulic conductance is due to thermal expansion-induced increases in tubular diameter.

Dentin permeability. Effects of desensitizing dentifrices in vitro.

Patients using placebo dentifrices in clinical trials usually show a significant decrease in dentin sensitivity over a 2- to 4-week period. If their sensitivity were due to hydrodynamic fluid movement, then the results suggest that there was a decrease in their dentin permeability. This hypothesis was tested in vitro by measuring the ease with which fluid could flow (i.e., hydraulic conductance) across dentin discs before and after brushing the discs with a variety of dentifrices, including most of the marketed densensitizing dentifrices. All dentifrices decreased the hydraulic conductance of dentin. An experimental dentifrice containing oxalate as the active ingredient was far more effective than any of the marketed dentifrices. The results tend to support the hypothesis that, at least part of the reduction in clinical sensitivity in patients with hypersensitive dentin is due to the abrasive action of the dentifrice.

Effects of air-drying in vitro on human dentine permeability.

The effects of evaporation produced by air blasts of 0, 0.5, 2 or 5 min to dentine in vitro were evaluated by measuring dentine hydraulic conductance before and after each trial. When the tubules were filled with water, even prolonged evaporation had no effect on dentine permeability. Tubules filled with physiological salt solution produced a time-dependent decrease in dentine permeability. Tubules filled with 1.5 per cent albumin in water gave the largest reductions in dentine permeability. These effects were more marked in unetched as opposed to acid-etched dentine. The results suggest that part of the reduction in dentine sensitivity produced clinically by prolonged air blasts may be due to precipitation of organic and inorganic constituents of dentinal fluid at the surface.

Metabolism of hexachlorobenzene by rat-liver microsomes.

Hexachlorobenzene (HCB) was metabolized to dechlorinated and hydroxylated products by the mixed-function oxidase system of rat-liver microsomes (phenobarbital- or 3-methylcholanthrene-induced). Metabolites were identified by gas-liquid chromatography-mass spectrometry. 14C-HCB was bound to microsomal protein. No explanation was found for the sex-dependent hepatotoxicity of HCB in rats.

Genetic, iron status and sex factors of porphyria induced by hexachlorobenzene.

This study explored the influence of genetic strain, sex and iron on the development of hexachlorobenzene (HCB)-induced porphyria in mice and rats. Results are discussed in terms of the mechanism of action and comparison with human exposure to the chemical.

Hexachlorobenzene as a promoter of diethylnitrosamine-initiated hepatocarcinogenesis in rats and comparison with induction of porphyria.

In rats, hexachlorobenzene (HCB) causes uroporphyria and liver tumours predominantly in females. To investigate the promotional properties of HCB, male and female rats received diethylnitrosamine (DEN) in the drinking water (0.015%) for 3 weeks. After a 2-week recovery period rats were fed control diet or one containing HCB (0.02%) for 30 weeks. HCB was an efficient promoter of DEN-initiated hepatocarcinogenesis in both sexes as judged by the size and numbers of visible tumours and by the percentage of liver sections that stained strongly positive for gamma-glutamyltranspeptidase (GGT) activity. Tumours were larger in males whereas regions of GGT-positive tumour and non-tumour tissue were greater in females. Inhibition of the haem biosynthesis enzyme uroporphyrinogen decarboxylase (UD) only occurred in the liver of females treated with HCB or DEN/HCB. In the latter group, UD was inhibited both in and outside tumours whereas uroporphyrin only accumulated in non-cancerous tissue. No significant inhibition of UD was observed in the liver of males. In another study, rats received one i.p. dose of DEN (20 mg/kg) and after 3 weeks were fed HCB for 30 weeks. Numbers of GGT-positive foci were greatly increased by HCB in both sexes, but especially in males (1.4-fold greater than females). Thus HCB was shown to be a promoter of hepatocarcinogenesis. However, the lack of a consistent marked sex difference suggests that this is only a partial explanation for the induction of tumours which mainly occur in females when this chemical is administered alone for prolonged periods.

In vivo efficacies of levofloxacin and ciprofloxacin in acute murine hematogenous pyelonephritis induced by methicillin-susceptible and-resistant Staphylococcus aureus strains.

Levofloxacin, the active L-isomer of ofloxacin, has demonstrated strong activity against Staphylococcus aureus both in vitro and in vivo. In a murine model of hematogenous pyelonephritis, the in vivo efficacies of levofloxacin and ciprofloxacin were evaluated against two methicillin-susceptible and two methicillin-resistant S. aureus strains. All four isolates had virtually identical susceptibilities to levofloxacin and ciprofloxacin. Pyelonephritis was induced in carrageenan-primed mice by an intravenous injection of 0.5 ml of 10(7) CFU of methicillin-susceptible S. aureus isolates per ml or 10(8) CFU of methicillin-resistant S. aureus isolates per ml. At 1 h postinfection, the mice were treated orally with levofloxacin or ciprofloxacin once a day or twice a day (total daily dose of 20 to 160 mg/kg of body weight) for 4 days. Mice were euthanized 24 h after the final treatment, and the kidneys were excised and weighed. The kidneys were prepared for histological examination or were homogenized to determine the numbers of CFU per gram of tissue quantitatively. The reduction in the mean log10 number of CFU per gram as a function of total daily dose was recorded. A dose-response analysis showed that levofloxacin was superior to ciprofloxacin for all four isolates at any dose or regimen tested, independent of the methicillin susceptibility of the isolates. By using an inverse prediction technique, the equivalent effective doses of levofloxacin (once a day) were less than those of ciprofloxacin (twice a day) by 5.2 and 3.2 times, respectively, for methicillin-susceptible S. aureus 9039 and 3087. For methicillin-resistant S. aureus 667 and 2878, the equivalent effective doses of levofloxacin (once a day) were less than those of ciprofloxacin (twice a day) by 4.1 and 6.4 times, respectively. In a separate study, histological examination of all infected, untreated mice showed moderate to marked hematogenous pyelonephritis. Levofloxacin-treated mice (40 mg/kg once a day) showed no evidence of pyelonephritis in the kidneys, whereas the kidneys of mice treated with the same dose of ciprofloxacin showed only a reduction in the severity of the lesions. Treatment with ciprofloxacin (80 mg/kg twice a day) demonstrated a histology comparable to that of treatment with levofloxacin (40 mg/kg once a day). Levofloxacin (40 mg/kg once a day) reduced the log10 numbers of CFU per gram by 5 log10; however, ciprofloxacin (80 mg/kg twice a day) reduced the numbers of CFU per gram by only 3 log10. In the present murine model of pyelonephritis, levofloxacin was superior to ciprofloxacin in preventing pyelonephritis and eradicating S. aureus.

Mechanistic studies of the inhibition of hepatic uroporphyrinogen decarboxylase in C57BL/10 mice by iron-hexachlorobenzene synergism.

Porphyria was induced in C57BL/10 mice with iron overload by a single oral dose (100 mg/kg) of hexachlorobenzene (HCB). Within 2 weeks hepatic uroporphyrinogen decarboxylase (EC 4.1.1.37) was inhibited, reaching a maximum (greater than 95%) at 6-8 weeks. There was no recovery by 14 weeks, despite a fall in liver HCB concentrations to only 6% of the day-3 value. The major rise in hepatic porphyrin levels occurred after 4 weeks and secondary inhibition of uroporphyrinogen synthase (EC 4.2.1.75) was inferred from the progressively greater proportion of uroporphyrin I present relative to the III isomer. Plasma alanine aminotransferase (EC 2.6.1.2) activity was also elevated. Although, in further studies, total microsomal cytochrome P-450 content and ethoxyphenoxazone de-ethylase activity reached a peak a few days after dosing and had declined significantly at the time of maximum inhibition of the decarboxylase, additional treatment of HCB-dosed mice with a cytochrome P1-450 inducer, beta-naphthoflavone, enhanced the inhibition, whereas piperonyl butoxide, an inhibitor of cytochrome P-450, partially protected. Uroporphyrinogen decarboxylase was not radiolabelled in vivo by [14C]HCB. There was no major difference in the ability to hydroxylate HCB between hepatic microsomes from induced C57BL/10 mice and those from the insensitive DBA/2 strain. By contrast, lipid peroxidation, in the presence of NADPH, was 8-fold greater in control C57BL/10 microsomes than in DBA/2 microsomes and was stimulated by iron treatment (although not by HCB). The results suggest that the inhibition of hepatic uroporphyrinogen decarboxylase is unlikely to be due to a direct effect of a metabolite of HCB but to another process requiring a specific cytochrome P-450 isoenzyme and an unknown iron species.