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

R M Phillips, P M Loadman, B P Cronin

Research output: Contribution to journalArticle

60 Citations (Scopus)

Abstract

The 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 microm thick) with t(1/2) 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-beta-en-alpha-ol (EO9) and doxorubicin through multicell layers was significantly slower (t(1/2) = 4.62 and 13.1 h respectively). Inclusion of dicoumarol increases the rate of EO9 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 EO9 does not readily penetrate a multicell layer, in conjunction with its rapid elimination in vivo (t(1/2) < 10 min), suggests that EO9 is unlikely to penetrate more than a few microm from a blood vessel within its pharmacokinetic lifespan. These results suggest that the failure of EO9 in the clinic is due to a combination of poor drug penetration and rapid elimination in vivo.

Original languageEnglish
Pages (from-to)2112-2119
Number of pages8
JournalBritish Journal of Cancer
Volume77
Issue number12
DOIs
Publication statusPublished - Jun 1998
Externally publishedYes

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apaziquone
tirapazamine
Pharmaceutical Preparations
Neoplasms
In Vitro Techniques
Dicumarol
Drug Combinations
Doxorubicin
Plastics
Blood Vessels
Cultured Cells
Colon
Pharmacokinetics
High Pressure Liquid Chromatography
Oxygen
Carcinoma
Drug Therapy
Membranes

Cite this

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title = "Evaluation of a novel in vitro assay for assessing drug penetration into avascular regions of tumours",
abstract = "The 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 microm thick) with t(1/2) 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-beta-en-alpha-ol (EO9) and doxorubicin through multicell layers was significantly slower (t(1/2) = 4.62 and 13.1 h respectively). Inclusion of dicoumarol increases the rate of EO9 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 EO9 does not readily penetrate a multicell layer, in conjunction with its rapid elimination in vivo (t(1/2) < 10 min), suggests that EO9 is unlikely to penetrate more than a few microm from a blood vessel within its pharmacokinetic lifespan. These results suggest that the failure of EO9 in the clinic is due to a combination of poor drug penetration and rapid elimination in vivo.",
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Evaluation of a novel in vitro assay for assessing drug penetration into avascular regions of tumours. / Phillips, R M; Loadman, P M; Cronin, B P.

In: British Journal of Cancer, Vol. 77, No. 12, 06.1998, p. 2112-2119.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Evaluation of a novel in vitro assay for assessing drug penetration into avascular regions of tumours

AU - Phillips, R M

AU - Loadman, P M

AU - Cronin, B P

PY - 1998/6

Y1 - 1998/6

N2 - The 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 microm thick) with t(1/2) 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-beta-en-alpha-ol (EO9) and doxorubicin through multicell layers was significantly slower (t(1/2) = 4.62 and 13.1 h respectively). Inclusion of dicoumarol increases the rate of EO9 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 EO9 does not readily penetrate a multicell layer, in conjunction with its rapid elimination in vivo (t(1/2) < 10 min), suggests that EO9 is unlikely to penetrate more than a few microm from a blood vessel within its pharmacokinetic lifespan. These results suggest that the failure of EO9 in the clinic is due to a combination of poor drug penetration and rapid elimination in vivo.

AB - The 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 microm thick) with t(1/2) 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-beta-en-alpha-ol (EO9) and doxorubicin through multicell layers was significantly slower (t(1/2) = 4.62 and 13.1 h respectively). Inclusion of dicoumarol increases the rate of EO9 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 EO9 does not readily penetrate a multicell layer, in conjunction with its rapid elimination in vivo (t(1/2) < 10 min), suggests that EO9 is unlikely to penetrate more than a few microm from a blood vessel within its pharmacokinetic lifespan. These results suggest that the failure of EO9 in the clinic is due to a combination of poor drug penetration and rapid elimination in vivo.

KW - Antibiotics, Antineoplastic/pharmacokinetics

KW - Antineoplastic Agents/pharmacokinetics

KW - Aziridines/pharmacokinetics

KW - Carcinoma/blood supply

KW - Cell Division/drug effects

KW - Chromatography, High Pressure Liquid

KW - Colonic Neoplasms/blood supply

KW - Dicumarol/pharmacology

KW - Doxorubicin/pharmacokinetics

KW - Humans

KW - Indolequinones

KW - Indoles/pharmacokinetics

KW - Oxygen/metabolism

KW - Triazines/pharmacokinetics

KW - Tumor Cells, Cultured

U2 - 10.1038/bjc.1998.355

DO - 10.1038/bjc.1998.355

M3 - Article

VL - 77

SP - 2112

EP - 2119

JO - British Journal of Cancer

JF - British Journal of Cancer

SN - 0007-0920

IS - 12

ER -