The pharmacokinetics and dosing of oral 4-methylumbelliferone for inhibition of hyaluronan synthesis in mice.

Recently, there has been considerable interest in using 4-methylumbelliferone (4-MU) to inhibit hyaluronan (HA) synthesis in mouse models of cancer, autoimmunity and a variety of other inflammatory disorders where HA has been implicated in disease pathogenesis. In order to facilitate future studies in this area, we have examined the dosing, treatment route, treatment duration and metabolism of 4-MU in both C57BL/6 and BALB/c mice. Mice fed chow containing 5% 4-MU, a dose calculated to deliver 250 mg/mouse/day, initially lose substantial weight but typically resume normal weight gain after 1 week. It also takes up to a week to see a reduction in serum HA in these animals, indicating that at least a 1-week loading period on the drug is required for most protocols. At steady state, more than 90% of the drug is present in plasma as the glucuronidated metabolite 4-methylumbelliferyl glucuronide (4-MUG), with the sulphated metabolite, 4-methylumbelliferyl sulphate (4-MUS) comprising most of the remainder. Chow containing 5% but not 0·65% 4-MU was effective at preventing disease in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis, as well as in the DORmO mouse model of autoimmune diabetes. While oral 4-MU was effective at preventing EAE, daily intraperitoneal injections of 4-MU were not. Factors potentially affecting 4-MU uptake and plasma concentrations in mice include its taste, short half-life and low bioavailability. These studies provide a practical resource for implementing oral 4-MU treatment protocols in mice.

Evaluation of in vivo T cell kinetics: use of heavy isotope labelling in type 1 diabetes.

CD4(+) memory cell development is dependent upon T cell receptor (TCR) signal strength, antigen dose and the cytokine milieu, all of which are altered in type 1 diabetes (T1D). We hypothesized that CD4(+) T cell turnover would be greater in type 1 diabetes subjects compared to controls. In vitro studies of T cell function are unable to evaluate dynamic aspects of immune cell homoeostasis. Therefore, we used deuterium oxide ((2) H(2)O) to assess in vivo turnover of CD4(+) T cell subsets in T1D (n = 10) and control subjects (n = 10). Serial samples of naive, memory and regulatory (T(reg)) CD4(+) T cell subsets were collected and enrichment of deoxyribose was determined by gas chromatography-mass spectrometry (GC-MS). Quantification of T cell turnover was performed using mathematical models to estimate fractional enrichment (f, n = 20), turnover rate (k, n = 20), proliferation (p, n = 10) and disappearance (d*, n = 10). Although turnover of T(regs) was greater than memory and naive cells in both controls and T1D subjects, no differences were seen between T1D and controls in T(reg) or naive kinetics. However, turnover of CD4(+) memory T cells was faster in those with T1D compared to control subjects. Measurement and modelling of incorporated deuterium is useful for evaluating the in vivo kinetics of immune cells in T1D and could be incorporated into studies of the natural history of disease or clinical trials designed to alter the disease course. The enhanced CD4(+) memory T cell turnover in T1D may be important in understanding the pathophysiology and potential treatments of autoimmune diabetes.