Evaluation of a novel in vitro assay for assessing drug penetration into avascular regions of tumours

Abstract

SummaryThe poor blood supply to solid tumours introduces many factors that affect the outcome of chemotherapy, one of which is the problem of drug delivery to poorly vascularized regions of tumours. Whereas poor drug penetration has been recognized as a contributing factor to the poor response of many solid tumours, the question of drug penetration through multicell layers has not been thoroughly addressed, largely because of restrictions imposed upon these studies by the requirement for either radiolabelled or naturally fluorescent compounds. The aim of this study is to describe modifications made to a recently published assay that broadens the scope for assessing drug penetration during the early stages of drug development and to characterize the ability of various drugs to penetrate multicell layers. DLD-1 human colon carcinoma cells were cultured on Transwell-COL plastic inserts placed into 24-well culture plates so that a top and bottom chamber were established, the two chambers being separated by a microporous membrane. Drugs were added to the top chamber at doses equivalent to peak plasma concentrations in vivo and the rate of appearance of drugs in the bottom chamber determined by high-performance liquid chromatography (HPLC). Both 3-amino-1,2,4-benzotriazine 1,4-dioxide (tirapazamine) and 7-[4'-(2-nitroimidazol-1-yl)-butyl]-theophylline (NITP) rapidly penetrated DLD-1 multicell layers (50.9 ± 12.1 gm thick) with t1,2 values of 1.36 and 2.38 h respectively, whereas the rate of penetration of 5-aziridino-3-hydroxymethyl-1-methyl-2-[1H-indole-4,7-dione] prop-,-en-a-ol (EO9) and doxorubicin through multicell layers was significantly slower (t1,2 = 4.62 and 13.1 h respectively). Inclusion of dicoumarol increases the rate of E09 penetration, whereas reducing the oxygen tension to 5% causes a reduction in tirapazamine penetration through multicell layers, suggesting that the extent of drug metabolism is one factor that determines the rate at which drugs penetrate multicell layers. The fact that E09 does not readily penetrate a multicell layer, in conjunction with its rapid elimination in vivo (tl,2 < 10 min), suggests that E09 is unlikely to penetrate more than a few ,um from a blood vessel within its pharmacokinetic lifespan. These results suggest that the failure of E09 in the clinic is due to a combination of poor drug penetration and rapid elimination in vivo.

Inhibition of endosomal sequestration of basic anticancer drugs: influence on cytotoxicity and tissue penetration

“…The compound of interest is added to one side of the MCL and its appearance on the other side is measured by appropriate analytical methods. Using this method, we and others have demonstrated limited penetration of several anticancer drugs through solid tissue (Phillips et al, 1998;Tunggal et al, 1999;Tannock et al, 2002).…”

“…While these mechanisms are important, solid tumours have a poorly formed vasculature and an additional explanation for the lack of effectiveness of many anticancer drugs may be their limited ability to penetrate through multiple cell layers of the extravascular space to reach all of the tumour cells (Durand, 1989;Jain, 1990;Hicks et al, 1997;Phillips et al, 1998;Lankelma et al, 1999;Tunggal et al, 1999;Tannock et al, 2002). The multicellular layer (MCL) model allows tumour cells to be grown in culture with many properties of tumours in vivo, including desmosomes between cells and an extracellular matrix (Hicks et al, 1997;Minchinton et al, 1997;Phillips et al, 1998;Tunggal et al, 1999Tunggal et al, , 2000Cowan and Tannock, 2001;Tannock et al, 2002). Multicellular layers provide a simple, direct and quantitative means for measuring the penetration of drugs through solid tissue.…”

Chemoprevention and Novel Treatments of Non-Muscle Invasive Bladder Cancer

“…In vivo, it has demonstrated activity against colon, non-small cell lung, renal, melanoma and central nervous system tumor models (Hendricks et al, 1993). With early promising results, it has failed to show favorable phase II outcomes with Phillips et al citing its rapid pharmacokinetic elimination and poor penetration in avascular tissues (Phillips et al, 1998). In humans, Apaziquone's half-life is less than 10 minutes, via extra-hepatic metabolism by red blood cells, with its metabolites, EO5a, having decreased cytotoxicity (Schelens et al, 1994;Vainchtein et al, 2007).…”

Induction of cachexia in mice

“…A recent study by Phillips et al (1998) describes a simple tissue culture assay for investigating the extravascular penetration properties of anticancer drugs. The reported assay is a modification of the method developed by ourselves (Cowan et al, 1996;Hicks et al, 1997) and Minchinton et al (1997) in which tumour cells are grown as multicellular layers (MCL) on commercially-available Teflon microporous membranes.…”

“…We assume that the microporous Teflon membrane supporting the MCL is only 10 µm thick, with 100% porosity, and that there are no unstirred boundary layers. Even with these unrealistically favourable assumptions, the half-time for purely diffusive transfer between an unstirred donor compartment of 100 µl and a stirred receiving compartment of 600 µl (in the absence of an MCL) would be 30 min, whereas Figure 4 of Phillips et al (1998) shows a half-time of approximately 5 min. Using these same parameters but assuming efficient mixing in the donor compartment gives slightly faster transfer (half-time 3.5 min) than observed.…”