The conditioned place preference test for assessing welfare consequences and potential refinements in a mouse bladder cancer model.

Most pre-clinical analgesic efficacy assays still involve nociceptive testing in rodents. This is despite concerns as to the relevance of these tests for evaluating the pain-preventative properties of drugs. More appropriate methods would target pain rather than nociception, but these are currently not available, so it remains unknown whether animal pain equates to the negatively affective and subjective/emotional state it causes in humans. Mouse cancer models are common despite the likelihood of substantial pain. We used Conditioned Place Preference (CPP) testing, assessments of thermal hyperalgesia and behaviour to determine the likelihood that MBT-2 bladder cancer impacts negatively on mouse welfare, such as by causing pain. There was no CPP to saline, but morphine preference in tumour bearing mice exceeded that seen in tumour-free controls. This occurred up to 10 days before the study end-point alongside reduced body weight, development of hyperalgesia and behaviour changes. These effects indicated mice experienced a negative welfare state caused by malaise (if not pain) before euthanasia. Due to the complexity of the assessments needed to demonstrate this, it is unlikely that this approach could be used for routine welfare assessment on a study-by-study basis. However, our results show mice in sufficiently similar studies are likely to benefit from more intensive severity assessment and re-evaluation of end-points with a view to implementing appropriate refinements. In this particular case, a refinement would have been to have euthanased mice at least 7 days earlier or possibly by provision of end-stage pain relief. CPP testing was found to be a helpful method to investigate the responses of mice to analgesics, possibly on a subjective level. These findings and those of other recent studies show it could be a valuable method of screening candidate analgesics for efficacy against cancer pain and possibly other pain or disease models.

Preclinical selection of a novel poly(ADP-ribose) polymerase inhibitor for clinical trial.

Poly(ADP-ribose) polymerase (PARP)-1 (EC 2.4.2.30) is a nuclear enzyme that promotes the base excision repair of DNA breaks. Inhibition of PARP-1 enhances the efficacy of DNA alkylating agents, topoisomerase I poisons, and ionizing radiation. Our aim was to identify a PARP inhibitor for clinical trial from a panel of 42 potent PARP inhibitors (K(i), 1.4-15.1 nmol/L) based on the quinazolinone, benzimidazole, tricyclic benzimidazole, tricyclic indole, and tricyclic indole-1-one core structures. We evaluated chemosensitization of temozolomide and topotecan using LoVo and SW620 human colorectal cells; in vitro radiosensitization was measured using LoVo cells, and the enhancement of antitumor activity of temozolomide was evaluated in mice bearing SW620 xenografts. Excellent chemopotentiation and radiopotentiation were observed in vitro, with 17 of the compounds causing a greater temozolomide and topotecan sensitization than the benchmark inhibitor AG14361 and 10 compounds were more potent radiosensitizers than AG14361. In tumor-bearing mice, none of the compounds were toxic when given alone, and the antitumor activity of the PARP inhibitor-temozolomide combinations was unrelated to toxicity. Compounds that were more potent chemosensitizers in vivo than AG14361 were also more potent in vitro, validating in vitro assays as a prescreen. These studies have identified a compound, AG14447, as a PARP inhibitor with outstanding in vivo chemosensitization potency at tolerable doses, which is at least 10 times more potent than the initial lead, AG14361. The phosphate salt of AG14447 (AG014699), which has improved aqueous solubility, has been selected for clinical trial.

An evaluation of thymidine phosphorylase as a means of preventing thymidine rescue from the thymidylate synthase inhibitor raltitrexed.

The antitumour effect of thymidylate synthase inhibitors such as raltitrexed (RTX) may be reversed by salvage of thymidine (Thd). Since thymidine phosphorylase (TP) depletes Thd, the potential for tumour-selective depletion of Thd using antibody-mediated delivery of TP to tumours was investigated. In vitro studies demonstrated that 25 x 10(-3) units/ml TP depleted extracellular Thd (3 microM) and restored sensitivity to the growth inhibitory effects of RTX in Lovo and HT29 cell lines. Thymidine concentrations in xenograft tumours were inversely proportional to the activity of TP in the tumour, and the presence of a subcutaneous Lovo xenograft reduced plasma Thd concentrations from 0.92 +/- 0.07 to 0.37 +/- 0.04 microM. Intravenous administration of native TP enzyme depleted plasma Thd to 5 nM, but following rapid elimination of TP, plasma Thd returned to pretreatment values. There was no effect on tumour TP or Thd. Conjugation of TP to the A5B7 F(ab)2 antibody fragment, which targets carcinoembryonic antigen (CEA) expressed on colorectal cell-lines such as Lovo, did result in selective accumulation of TP in the tumour. However, there was no tumour-selective depletion of Thd and there did not appear to be any potential benefit of combining antibody-targeted TP with RTX.