Validation and development of MTH1 inhibitors for treatment of cancer.

BACKGROUND: Previously, we showed cancer cells rely on the MTH1 protein to prevent incorporation of otherwise deadly oxidised nucleotides into DNA and we developed MTH1 inhibitors which selectively kill cancer cells. Recently, several new and potent inhibitors of MTH1 were demonstrated to be non-toxic to cancer cells, challenging the utility of MTH1 inhibition as a target for cancer treatment. MATERIAL AND METHODS: Human cancer cell lines were exposed in vitro to MTH1 inhibitors or depleted of MTH1 by siRNA or shRNA. 8-oxodG was measured by immunostaining and modified comet assay. Thermal Proteome profiling, proteomics, cellular thermal shift assays, kinase and CEREP panel were used for target engagement, mode of action and selectivity investigations of MTH1 inhibitors. Effect of MTH1 inhibition on tumour growth was explored in BRAF V600E-mutated malignant melanoma patient derived xenograft and human colon cancer SW480 and HCT116 xenograft models. RESULTS: Here, we demonstrate that recently described MTH1 inhibitors, which fail to kill cancer cells, also fail to introduce the toxic oxidized nucleotides into DNA. We also describe a new MTH1 inhibitor TH1579, (Karonudib), an analogue of TH588, which is a potent, selective MTH1 inhibitor with good oral availability and demonstrates excellent pharmacokinetic and anti-cancer properties in vivo. CONCLUSION: We demonstrate that in order to kill cancer cells MTH1 inhibitors must also introduce oxidized nucleotides into DNA. Furthermore, we describe TH1579 as a best-in-class MTH1 inhibitor, which we expect to be useful in order to further validate the MTH1 inhibitor concept.

Blood parameters and corneal-reflex of finishing pigs with and without lung affections observed post mortem in two abattoirs stunning with CO2.

In two pig abattoirs of different slaughter capacities, the stunning efficacy of CO2 on finishing pigs with and without pneumonic lesions (observed post mortem) was reflected against the corneal-reflex and blood parameters (blood pH, pCO2 and pO2) from individual finishers. Stunning duration was 120 s (abattoir A) and 90 s (abattoir B), respectively. Pneumonia in finisher pigs is frequently observed during post mortem inspection, which may raise concerns about a delay of unconsciousness because of hampered gas exchange in the lungs. The aim of this study was to examine possible pneumonia consequences for stunning efficacy under commercial conditions. For that, corneal reflex, O2 and CO2 partial pressure in the blood as well as blood pH were measured in 2650 finishers from abattoir A and 2100 from abattoir B. The partial pressure of O2 after stunning accounted to about 3 kPa, the partial pressure of CO2 was found at levels of about 24 kPa in abattoir A (after 120 s CO2 exposure) and 17.5 kPa in abattoir B (after 90 s CO2 exposure). In abattoir A, the blood pH was at 6.9, and at 7.0 in abattoir B. The corneal reflex was observed in 6.2% of pigs in abattoir A and 17.1% of pigs in abattoir B. A correlation between pneumonic lesions and blood status was not observed. However, for some individual farms, a significant correlation between pneumonia and corneal reflex was observed.

Effects of selective COX-1 and -2 inhibition on formalin-evoked nociceptive behaviour and prostaglandin E(2) release in the spinal cord.

Nociception evoked prostaglandin (PG) release in the spinal cord considerably contributes to the induction of hyperalgesia and allodynia. To evaluate the relative contribution of cyclooxygenase-1 (COX-1) and COX-2 in this process we assessed the effects of the selective COX-1 inhibitor SC560 and the selective COX-2 inhibitor celecoxib on formalin-evoked nociceptive behaviour and spinal PGE(2) release. SC560 (10 and 20 mg/kg) significantly reduced the nociceptive response and completely abolished the formalin-evoked PGE(2) raise. In contrast, celecoxib (10 and 20 mg/kg) was ineffective in both regards, i.e. the flinching behaviour was largely unaltered and the formalin-induced PGE(2) raise as assessed using microdialysis was only slightly, not significantly reduced. This suggests that the formalin-evoked rapid PG release was primarily caused by COX-1 and was independent of COX-2. Mean free spinal cord concentrations of celecoxib during the formalin assay were 32.0 +/- 4.5 nM, thus considerably higher than the reported IC50 for COX-2 (3-7 nM). Therefore, the lack of efficacy of celecoxib is most likely not to be a result of poor tissue distribution. COX-2 mRNA and protein expression in the spinal cord were not affected by microdialysis alone but the mRNA rapidly increased following formalin injection and reached a maximum at 2 h. COX-2 protein was unaltered up to 4 h after formalin injection. The time course of COX-2 up-regulation suggests that the formalin-induced nociceptive response precedes COX-2 protein de novo synthesis and may therefore be unresponsive to COX-2 inhibition. Considering the results obtained with the formalin model it may be hypothesized that the efficacy of celecoxib in early injury evoked pain may be less than that of unselective NSAIDs.

Determination of celecoxib in human plasma and rat microdialysis samples by liquid chromatography tandem mass spectrometry.

Methods for the determination of celecoxib in human plasma and rat microdialysis samples using liquid chromatography tandem mass spectrometry are described. Celecoxib and an internal standard were extracted from plasma by solid-phase extraction with C18 cartridges. Thereafter compounds were separated on a short narrow bore RP C18 column (30 x 2 mm). Microdialysis samples did not require extraction and were injected directly using a narrow bore RP C18 column (70 x 2 mm). The detection was by a PE Sciex API 3000 mass spectrometer equipped with a turbo ion spray interface. The compounds were detected in the negative ion mode using the mass transitions m/z 380-->316 and m/z 366-->302 for celecoxib and internal standard, respectively. The assay was validated for human plasma over a concentration range of 0.25-250 ng/ml using 0.2 ml of sample. The assay for microdialysis samples (50 microl) was validated over a concentration range of 0.5-20 ng/ml. The method was utilised to determine pharmacokinetics of celecoxib in human plasma and in rat spinal cord perfusate.

COX-2 independent induction of cell cycle arrest and apoptosis in colon cancer cells by the selective COX-2 inhibitor celecoxib.

The regular use of various nonsteroidal anti-inflammatory drugs (NSAIDs) was shown to decrease the incidence of colorectal cancer. This effect is thought to be caused predominantly by inhibition of cyclooxygenase-2 (COX-2) and, subsequently, prostaglandin synthesis. However, recent studies have suggested that COX-independent pathways may contribute considerably to these antiproliferative effects. To evaluate the involvement of COX-dependent and COX-independent mechanisms further, we assessed the effects of celecoxib (selective COX-2 inhibitor) and SC560 (selective COX-1 inhibitor) on cell survival, cell cycle distribution, and apoptosis in three colon cancer cell lines, which differ in their expression of COX-2. Both drugs induced a G0/G1 phase block and reduced cell survival independent of whether or not the cells expressed COX-2. Celecoxib was more potent than SC560. The G0/G1 block caused by celecoxib could be attributed to a decreased expression of cyclin A, cyclin B1, and cyclin-dependent kinase-1 and an increased expression of the cell cycle inhibitory proteins p21Waf1 and p27Kip1. In addition, celecoxib, but not SC560, induced apoptosis, which was also independent of the COX-2 expression of the cells. In vivo, celecoxib as well as SC560 reduced the proliferation of HCT-15 (COX-2 deficient) colon cancer xenografts in nude mice, but both substances had no significant effect on HT-29 tumors, which express COX-2 constitutively. Thus, our in vitro and in vivo data indicate that the antitumor effects of celecoxib probably are mediated through COX-2 independent mechanisms and are not restricted to COX-2 over-expressing tumors.

Interleukin-1beta induces chronic activation and de novo synthesis of neutral ceramidase in renal mesangial cells.

The lipid signaling molecule ceramide is formed by the action of acid and neutral sphingomyelinases and degraded by acid and neutral ceramidases. Short-term stimulation of mesangial cells with the pro-inflammatory cytokine interleukin-1beta (IL-1beta) leads to a rapid and transient increase in neutral sphingomyelinase activity (Kaszkin, M., Huwiler, A., Scholz, K., van den Bosch, H., and Pfeilschifter, J. (1998) FEBS Lett. 440, 163-166). In this study, we report on a second delayed peak of activation occurring after hours of IL-1beta treatment. This second phase of activation was first detectable after 2 h of treatment and steadily increased over the next 2 h, reaching maximal values after 4 h. In parallel, a pronounced increase in neutral ceramidase activity was observed, accounting for a constant or even decreased level of ceramide after long-term IL-1beta treatment, despite continuous sphingomyelinase activation. The increase in neutral ceramidase activity was due to expressional up-regulation, as detected by an increase in mRNA levels and enhanced de novo protein synthesis. The increase in neutral ceramidase protein levels and activity could be blocked dose- dependently by the p38 MAPK inhibitor SB 202190, whereas the classical MAPK pathway inhibitor U0126 and the protein kinase C inhibitor Ro 318220 were ineffective. Moreover, cotreatment of cells for 24 h with IL-1beta and SB 202190 led to an increase in ceramide formation. Interestingly, IL-1beta-stimulated neutral ceramidase activation was not reduced in mesangial cells isolated from mice deficient in MAPK-activated protein kinase-2, which is a downstream substrate of p38 MAPK, thus suggesting that the p38 MAPK-mediated induction of neutral ceramidase occurs independently of the MAPK-activated protein kinase-2 pathway. In summary, our results suggest a biphasic regulation of sphingomyelin hydrolysis in cytokine-treated mesangial cells with delayed de novo synthesis of neutral ceramidase counteracting sphingomyelinase activity and apoptosis. Neutral ceramidase may thus represent a novel cytoprotective enzyme for mesangial cells exposed to inflammatory stress conditions.

A convenient method to discriminate between cytochrome P450 enzymes and flavin-containing monooxygenases in human liver microsomes.

Liver microsomes are a frequently used probe to investigate the phase I metabolism of xenobiotics in vitro. Structures containing nucleophilic hetero-atoms are possible substrates for cytochrome P450 enzymes (P450) and flavin-containing monooxygenases (FMO). Both enzymes are located in the endoplasmatic reticulum of hepatocytes and both need oxygen and NADPH as cofactors. The common method to distinguish between the two enzyme systems is to use the thermal inactivation of FMO and to inhibit P450 completely with carbon monoxide, N-octylamine or N-benzylimidazole. In the literature no indication could be found that the heat inactivation of FMO does not affect any of the human P450 enzymes or that the overall P450 inhibitors inhibit the different human P450 enzymes sufficiently and do not affect the FMO. The effect of N-benzylimidazole and heat inactivation was tested on specific activities of seven P450 enzymes in human liver microsomes, 1A2, 2A6, 2C9, 2C19, 2D6, 3A4/5, and 2E1, using methoxyresorufin O-demethylation, coumarin 7-hydroxylation, (S)-warfarin 4-hydroxylation, (S)-(+)-mephenytoin 4-hydroxylation, dextrometorphan O-demethylation, oxidation of denitronifedipine, and chlorzoxazone 6-hydroxylation respectively. The sulfoxidation of methimazole (MMI) was used as a specific probe for the determination of FMO activity. Methimazole sulfoxidation was compared with the well known assay for FMO metabolism, the formation of N,N-dimethylaniline (DMA) N-oxide, to be confirmed as an exclusively FMO mediated reaction. The participation of P450 and FMO in the sulfoxidation of four sulfur containing peptides, ametryne; terbutryne, prometryne and methiocarb was investigated using human liver microsomes. All four reactions were demonstrated to be catalysed predominantly by cytochrome P450.