Intravesical pH: a potentially important variable affecting efficacy and the further development of anthracycline chemotherapy for superficial bladder cancer.
OBJECTIVE
To assess, using epirubicin-sensitive and multidrug resistant (MDR) derivatives of human bladder cancer cell lines in vitro, the probable effect of intravesical pH changes, with and without the MDR antagonist verapamil, on the uptake, intracellular distribution and cytotoxicity of epirubicin during intravesical chemotherapy.


METHODS
Incubations for cytotoxicity testing were carried out in buffered medium containing epirubicin, at pH values of 6.0-8.5, with verapamil where appropriate. The cytotoxicity of epirubicin, with and without verapamil, was determined using the tetrazolium cytotoxicity assay. Intracellular epirubicin fluorescence was assessed using flow cytometry and confocal microscopy. Flow cytometric total intracellular epirubicin fluorescence was measured at pH 6.0, 6.4, 6.8, 7.2, and 7.6, and confocal microscopy was carried out at pH 6.0 and 8.0. The MDR-reversing agent verapamil was added at 100 micro g/mL to some incubations.


RESULTS
Epirubicin cytotoxicity in resistant cell lines appears considerably enhanced by adding verapamil and further improved, especially in MDR cells, by alkalinization of the drug solution to pH 8.0. Flow cytometry results showed striking and consistent differences in epirubicin handling with pH. Sensitive cells can be induced to absorb considerably more drug at alkaline pH, whilst resistant cells show no such behaviour. Nuclear drug fluorescence was greater in sensitive cells at alkaline pH, but cytoplasmic drug fluorescence in the resistant cells was little changed by pH. Adding verapamil to resistant cells restored the sensitive phenotype of drug handling.


CONCLUSIONS
Buffering epirubicin to an alkaline pH before intravesical application should increase its intrinsic cytotoxicity. The potential for synergy at certain drug combinations will be enhanced by applying these findings. MDR reversal and fatty acid augmentation of drug uptake are discussed as examples.
Harris
N M
NM
Solent Department of Urology, St. Mary's Hospital, Portsmouth, UK. neilandjane@supanet.com
Duffy
P M
PM
Crook
T J
TJ
Anderson
W R
WR
Sharpe
P
P
Hayes
M C
MC
Cooper
A J
AJ
Solomon
L Z
LZ
D000283:Administration, Intravesical; D000903:Antibiotics, Antineoplastic / Q000008:administration & dosage*; D002295:Carcinoma, Transitional Cell / Q000737:chemistry / Q000188:drug therapy*; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D004357:Drug Synergism; D015251:Epirubicin / Q000008:administration & dosage*; D005434:Flow Cytometry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors; D014407:Tumor Cells, Cultured; D001743:Urinary Bladder / Q000737:chemistry*; D001749:Urinary Bladder Neoplasms / Q000737:chemistry / Q000188:drug therapy*; D014700:Verapamil / Q000627:therapeutic use*
D000903:Antibiotics, Antineoplastic; D015251:Epirubicin; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D014700:Verapamil