Preclinical pharmacokinetics and metabolism of a novel prototype DNA-PK inhibitor NU7026.

In this study we investigated the in vitro time dependence of radiosensitisation, pharmacokinetics and metabolism of NU7026, a novel inhibitor of the DNA repair enzyme DNA-dependent protein kinase (DNA-PK). At a dose of 10 muM, which is nontoxic to cells per se, a minimum NU7026 exposure of 4 h in combination with 3 Gy radiation is required for a significant radiosensitisation effect in CH1 human ovarian cancer cells. Following intravenous administration to mice at 5 mg kg(-1), NU7026 underwent rapid plasma clearance (0.108 l h(-1)) and this was largely attributed to extensive metabolism. Bioavailability following interperitoneal (i.p.) and p.o. administration at 20 mg kg(-1) was 20 and 15%, respectively. Investigation of NU7026 metabolism profiles in plasma and urine indicated that the compound undergoes multiple hydroxylations. A glucuronide conjugate of a bis-hydroxylated metabolite represented the major excretion product in urine. Identification of the major oxidation site as C-2 of the morpholine ring was confirmed by the fact that the plasma clearance of NU7107 (an analogue of NU7026 methylated at C-2 and C-6 of the morpholine ring) was four-fold slower than that of NU7026. The pharmacokinetic simulations performed predict that NU7026 will have to be administered four times per day at 100 mg kg(-1) i.p. in order to obtain the drug exposure required for radiosensitisation.

Evaluation of the cassette dosing approach for assessing the pharmacokinetics of geldanamycin analogues in mice.

PURPOSE: There is currently much interest in developing analogues of the benzoquinone ansamycin geldanamycin that may overcome the limitations of 17-(allylamino)-17-demethoxygeldanamycin (17AAG), which is the first known inhibitor of heat shock protein 90 (Hsp90) to enter clinical trials. Studies were performed to assess whether cassette dosing, the coadministration of several compounds to a single animal, is a suitable approach to evaluate the preclinical pharmacokinetics of geldanamycin analogues in high throughput. METHODS: Five geldanamycin analogues (17AAG, NSC 255110, NSC 682300, NSC 683661, NSC 683663) were administered intravenously to mice in combination at 5 mg/kg each and as single agents at 5 mg/kg and 50 mg/kg, or 12.5 mg/kg for NSC 682300. The compounds were also incubated with mouse liver microsomes individually and in combination at 15 microM each. Quantitative analysis was performed by LC/MS/MS. Plasma and tissue pharmacokinetic parameters were evaluated by non-compartmental analysis. In vitro metabolic stability was assessed by monitoring disappearance of the parent compound. RESULTS: Of the compounds that were detectable following individual administration at 5 mg/kg, 17AAG and NSC 683661 exhibited nonlinear pharmacokinetics. In addition, the plasma area under the curve (AUC) and the half-life of these compounds was greater following cassette dosing at 5 mg/kg compared to single administration at the same dose. When pharmacokinetic parameters were calculated up to the same time point following cassette and individual administration at the higher dose, three of the compounds displayed non-linear increases in AUC and slower clearances following cassette compared to single compound dosing. When all measurable concentrations at the higher dose were included, the half-life of NSC 683663 was nine-fold longer following individual compared to cassette administration. 17AAG displayed the highest AUC following cassette dosing, whereas NSC 683663 displayed the highest AUC following single-compound dosing. Excluding NSC 683663, the rank order from the highest to the lowest AUC was the same; however, NSC 682300, which ranked fifth, was administered at a four-fold lower individual dose than the other compounds. Exposure of the liver and kidneys to the compounds was greater than that of plasma. Despite being administered at a lower dose, NSC 682300 displayed the highest kidney AUC of the five compounds. The same ranking was maintained between cassette and single compound dosing in the kidney. With the exception of NSC 682300, in vitro metabolic stability was predictive of in vivo pharmacokinetics in the plasma and liver. The extent of metabolism of four of the five compounds was lower following microsomal incubation in combination compared to incubation alone, suggestive of likely drug-drug interaction in the cassette. However, for 17AAG this may be partly due to metabolism of NSC 683661 and NSC 683663 to this compound. CONCLUSIONS: Whilst cassette dosing has advantages for use in drug discovery, it is probably unsuitable to evaluate the pharmacokinetics of geldanamycin analogues due to non-linear pharmacokinetics and drug-drug interaction. The issues identified for this compound series should also be considered in assessing the suitability of cassette dosing for other chemotypes.

Solid-phase synthesis of novel inhibitors of farnesyl transferase.

A novel diphosphate mimic, the 2,3,6-trifluoro-5-hydroxy-4-nitrophenoxy group (1), has been employed as the template in the solid-phase synthesis of novel farnesyl transferase inhibitors using the Mitsunobu reaction. The most potent inhibitor (farnesyloxy-5-hydroxy-2,3,6-trifluoro-4-nitrobenzene) displayed an IC50 of 6.3 microM versus farnesyl transferase.

Inhibition of protein prenylation by metabolites of limonene.

The monoterpenes limonene and perillyl alcohol are undergoing clinical evaluation in cancer patients. In this paper, we report the chemical synthesis, characterisation, and quantitation in patients' plasma of a novel human metabolite of limonene, which is identified as an isomer of perillic acid. The synthesis of R-perillic acid is also described, because previous reports on the activity of perillic acid against isoprenylation enzymes refer to the S-enantiomer, although it is the R-enantiomer which is the metabolite of R-limonene. The above monoterpenes, with several related compounds, were assayed for inhibitory activity towards the isoprenylation enzymes in rat brain cytosol. Although R- and S-limonene are only weak inhibitors of the isoprenylation enzymes, their major metabolites, perillic acid and perillyl alcohol, are more potent inhibitors, with IC50 values in the low mM range. The metabolites possess greater activity towards the geranylgeranyltransferase type I enzyme than farnesyltransferase, while the novel metabolite displays IC50 values similar to those of perillic acid suggesting that it may contribute to the in vivo activity of limonene.

Occupational exposure to permethrin during its use as a public hygiene insecticide.

Permethrin is an active ingredient found in many public hygiene insecticide products and exposure to it was assessed in a survey of 45 professional users. The exposures measured were over a wide range, with more than a 100-fold difference between average levels and the highest levels. Dermal contamination was evident on 93% of the operators, the highest contamination resulting from the use of leaking application equipment, demonstrating that proper maintenance of equipment is vital. Where the insecticide was applied at ground level most contamination was on the legs, indicating the importance of appropriate footwear. Contamination of the hands occurred despite the use of protective gloves, higher levels of contamination occurring when liquids were used. Dermal contamination was not always the principle route of exposure, and high airborne concentrations were linked with use in confined areas. Airborne concentrations were also associated with the physical form of the product used and the treatment method. To help in assessing the effectiveness of protective clothing and control measures, biological monitoring was carried out. Monitoring of metabolites in urine showed that systemic uptake occurred but evidence from toxicological studies indicates that the levels found were well below those considered to cause harm.

Methylene bis (2-chloroaniline) (MbOCA): towards a biological monitoring guidance value.

Abstract We report the development and validation of a high performance liquid chromatography method for the determination of methylene bis (2-chloroaniline) (MbOCA) and its labile conjugates in urine. The method has been in regular use for the biological monitoring of workers exposed to MbOCA for the past 11 years. Following the development of a biological monitoring strategy, and the introduction of a biological action level by the Health and Safety Commission in 1984, there has been a steady fall in the proportion of workers whose urinary results are above the action level. We conclude that, in the absence of reliable health-based data, a guidance value based on the use of the 90th percentile derived from monitoring a cross-section of the industry, can be used to interpret biological monitoring results. The measurement of urinary MbOCA is a practical non-invasive way of monitoring workers which can be useful in helping to control exposure.

Metabolism of trans-cinnamic acid in the rat and the mouse and its variation with dose.

The metabolism of [3-14C/phenyl-2H5] cinnamic acid was investigated in rats and mice at a dose level of 2.5 mmol/kg body weight. Recoveries of the 14C dose were between 92 and 98% with most (82-90%) present in the 0-24 hr urine samples. Urinary metabolites were identified by their chromatographic properties and mass spectra. In both species the major metabolite was hippuric acid, which is also an endogenous urinary component. Several minor metabolites, 3-hydroxy-3-phenylpropionic acid, benzoic acid and benzoyl glucuronide, were found in both species. Two, acetophenone and cinnamoylglycine, the glycine conjugate of cinnamic acid, could be positively identified only in mouse urine. The effect of dose size on the urinary excretion of 14C-cinnamic acid metabolites was studied over the dose range 0.0005 to 2.5 mmol/kg. In the rat the pattern of metabolite excretion was very similar over the whole dose range with slight increases in the proportion of the dose excreted as minor metabolites as dose size increased. In the mouse the excretion of cinnamoylglycine was more important at the lowest dose level and decreased in relative importance as dose size increased. Changes in the other metabolites were similar to those seen in the rat.

A biological monitoring assessment of exposure to methylene dianiline in manufacturers and users.

This paper describes a cross sectional study in which biological monitoring was used to assess exposure to methylene dianiline (MDA) in a selection of United Kingdom industries that manufacture or use MDA. Samples of urine were collected from 411 workers, representing 45 factories engaged in various activities. All urine samples were analysed for MDA and its acetyl metabolites and results are reported as total MDA. In this study, 91% of postshift urine samples and 88% of preshift samples had less than 50 nmol MDA/mmol creatinine. Some evidence was obtained which showed that when exposure to MDA was through inhalation (as solid material or contaminated dust), postshift urine samples had higher MDA concentrations than samples taken preshift the next day. When exposure was most likely to be through the dermal route, urine samples taken preshift next day tended to have higher MDA concentrations than urine samples collected immediately postshift on the day of exposure. Therefore a biological monitoring sampling strategy for MDA must take account of the route of entry into the body. If exposure is likely to be via inhalation, postshift samples should be collected and if exposure is likely via the skin, preshift samples next day are more appropriate. The results show that in most factories, regardless of the route of exposure, it is possible to keep urinary MDA concentrations below 50 nmol/mmol creatinine. In the absence of a health based or hygiene based standard, the use of a "yardstick" as a target to aim for, which has been derived from good working practice across the industry, may be a useful way of helping to control exposure.