Blood-brain barrier models: in vitro to in vivo translation in preclinical development of CNS-targeting biotherapeutics.

INTRODUCTION: The majority of therapeutics, small molecule or biologics, developed for the CNS do not penetrate the blood-brain barrier (BBB) sufficiently to induce pharmacologically meaningful effects on CNS targets. To improve the efficiency of CNS drug discovery, several in vitro models of the BBB have been used to aid early selection of molecules with CNS exposure potential. However, correlative studies suggest relatively poor predictability of in vitro BBB models underscoring the need to combine in vitro and in vivo BBB penetration assessment into an integrated preclinical workflow. AREAS COVERED: This review gives a brief general overview of in vitro and in vivo BBB models used in the pre-clinical evaluation of CNS-targeting drugs, with particular focus on the recent progress in developing humanized models. The authors discuss the advantages, limitations, in vitro-in vivo correlation, and integration of these models into CNS drug discovery and development with the aim of improving translation. EXPERT OPINION: Often, a simplistic rationalization of the CNS drug discovery and development process overlooks or even ignores the need for an early and predictive assessment of the BBB permeability. Indeed, past failures of CNS candidates in clinical trials argue strongly that the early deployment of in vitro and in vivo models for assessing BBB permeability, mechanisms of transport and brain exposure of leads, and the co-development of BBB delivery strategies will improve translation and increase the clinical success of CNS pipelines.

Dose-dependent effects of dietary alpha- and gamma-tocopherols on genetic instability in mouse Mutatect tumors.

Vitamin E in foodstuffs is a mixture of tocopherols. In mouse Mutatect tumors, a model designed to detect DNA mutations, the hypoxanthine phosphoribosyltransferase (Hprt) gene mutation frequency is associated with the number of tumor-infiltrating neutrophils and both are markedly decreased in mice fed high levels of alpha-tocopherol. Dietary alpha-tocopherol is also associated with a decrease in neutrophil-associated loss of an interleukin 8 (IL-8)-expressing transgene in this tumor model. We examined Hprt gene mutation frequency (expressed as the number of 6-thioguanine-resistant colonies per 10(5) clonable tumor cells), IL-8 transgene loss, and myeloperoxidase activity (an indirect measure of neutrophil number) in tumors from Mutatect mice fed diets supplemented with various concentrations of D-alpha-tocopherol acetate and/or D-gamma-tocopherol acetate or neither tocopherol for 4 weeks. Hprt gene mutation frequency and myeloperoxidase activity were statistically significantly lower in tumor cells from mice fed alpha-tocopherol at 50 or 100 mg/kg body weight per day than in tumor cells from mice fed 0 mg/kg body weight per day alpha-tocopherol (P<.001 for each comparison). IL-8 transgene loss occurred in 28 of 28 tumors (100%; 95% confidence interval [CI] = 86% to 100%) from mice fed alpha-tocopherol at 50 mg or less/kg body weight per day and seven of 18 tumors (39%; 95% CI = 24% to 54%) from mice fed 100 mg/kg body weight per day (P<.001, Fisher's exact test, referent groups [pooled] 0, 25, and 50 mg/kg). gamma-Tocopherol had no detectable effect on any of the three endpoints. Thus, dietary alpha-tocopherol decreases two forms of genetic instability in a dose-dependent manner in this experimental tumor model.

Dietary vitamin E affects neutrophil distribution and genetic instability in murine Mutatect tumors.

Vitamin E is best known for its ability to scavenge reactive oxygen and nitrogen species. Solid tumors are frequently infiltrated with leukocytes, a potential source of these reactive species. The Mutatect tumor model is a fibrosarcoma that can be grown subcutaneously in syngeneic C57BL/6 mice. We previously showed that these tumors are infiltrated with neutrophils and that the number of neutrophils correlates with the number of hypoxanthine phosphoribosyl transferase (hprt) mutations and loss of an interleukin-8 (IL-8) transgene. Neutrophils are a source of nitric oxide, and tumors contain nitrotyrosine, a marker of damage by nitric oxide-related species. We also showed previously that dietary vitamin E supplements markedly lower the frequency of hprt mutants and the level of myeloperoxidase (a neutrophil marker) in a tumor fraction containing "loosely bound" cells. In the present report, we examine the effect of dietary vitamin E in greater detail. No effect on inducible nitric oxide synthase expression or nitrotyrosine levels was observed. However, dietary vitamin E induced a major redistribution of neutrophils from the loosely bound cellular fraction to the "stromal" fraction, while the total number of neutrophils in tumors was essentially unchanged. The loss of the IL-8 transgene seen earlier in Mutatect tumors was largely prevented. Vitamin E also prevented the large increase in hprt mutants (in the cellular and stromal fractions). Thus vitamin E appears to be protective against genotoxicity by scavenging reactive species, but also its ability to affect the distribution of neutrophils within tumors may be important.