Preclinical toxicity evaluation of erythrocyte-encapsulated thymidine phosphorylase in BALB/c mice and beagle dogs: an enzyme-replacement therapy for mitochondrial neurogastrointestinal encephalomyopathy.

Erythrocyte-encapsulated thymidine phosphorylase (EE-TP) is currently under development as an enzyme replacement therapy for mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal recessive disorder caused by a deficiency of thymidine phosphorylase. The rationale for the development of EE-TP is based on the pathologically elevated metabolites (thymidine and deoxyuridine) being able to freely diffuse across the erythrocyte membrane where the encapsulated enzyme catalyses their metabolism to the normal products. The systemic toxic potential of EE-TP was assessed when administered intermittently by iv bolus injection to BALB/c mice and Beagle dogs for 4 weeks. The studies consisted of one control group receiving sham-loaded erythrocytes twice weekly and two treated groups, one dosed once every 2 weeks and the other dosed twice per week. The administration of EE-TP to BALB/c mice resulted in thrombi/emboli in the lungs and spleen enlargement. These findings were also seen in the control group, and there was no relationship to the number of doses administered. In the dog, transient clinical signs were associated with EE-TP administration, suggestive of an immune-based reaction. Specific antithymidine phosphorylase antibodies were detected in two dogs and in a greater proportion of mice treated once every 2 weeks. Nonspecific antibodies were detected in all EE-TP-treated animals. In conclusion, these studies do not reveal serious toxicities that would preclude a clinical trial of EE-TP in patients with MNGIE, but caution should be taken for infusion-related reactions that may be related to the production of nonspecific antibodies or a cell-based immune response.

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.