Development and validation of a drug activity biomarker that shows target inhibition in cancer patients receiving enzastaurin, a novel protein kinase C-beta inhibitor.
OBJECTIVE
To evaluate the effects of the novel protein kinase C (PKC) inhibitor enzastaurin on intracellular phosphoprotein signaling using flow cytometry and to use this approach to measure enzastaurin effects on surrogate target cells taken from cancer patients that were orally dosed with this agent.


METHODS
The activity of PKC was assayed in intact cells using a modification of published techniques. The U937 cell line and peripheral blood mononuclear cells were stimulated with phorbol ester, fixed, permeabilized, and reacted with an antibody specific for the phosphorylated forms of PKC substrates. The processed samples were quantitatively analyzed using flow cytometry. The assay was validated for selectivity, sensitivity, and reproducibility. Finally, blood was obtained from volunteer cancer patients before and after receiving once daily oral doses of enzastaurin. These samples were stimulated ex vivo with phorbol ester and were assayed for PKC activity using this approach.


RESULTS
Assay of U937 cells confirmed the selectivity of the antibody reagent and enzastaurin for PKC. Multiparametric analysis of peripheral blood mononuclear cells showed monocytes to be the preferred surrogate target cell. Day-to-day PKC activity in normal donors was reproducible. Initial results showed that five of six cancer patients had decreased PKC activity following enzastaurin administration. In a following study, a group of nine patients displayed a significant decrease in PKC activity after receiving once daily oral doses of enzastaurin.


CONCLUSIONS
An inhibition of surrogate target cell PKC activity was observed both in vitro and ex vivo after exposure to the novel kinase inhibitor, enzastaurin.
Green
Lisa J
LJ
Lilly Research Laboratories, Indianapolis, Indiana 46285, USA. lisa.green@lilly.com
Marder
Philip
P
Ray
Chad
C
Cook
Carolyn A
CA
Jaken
Susan
S
Musib
Luna C
LC
Herbst
Roy S
RS
Carducci
Michael
M
Britten
Carolyn D
CD
Basche
Michele
M
Eckhardt
S Gail
SG
Thornton
Donald
D
D000971:Antineoplastic Combined Chemotherapy Protocols / Q000008:administration & dosage / Q000494:pharmacology*; D015415:Biomarkers / Q000032:analysis* / Q000378:metabolism; D000069287:Capecitabine; D045744:Cell Line, Tumor; D017321:Clinical Trials, Phase I as Topic / Q000706:statistics & numerical data; D003841:Deoxycytidine / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000494:pharmacology; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D005434:Flow Cytometry; D005472:Fluorouracil / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000494:pharmacology; D005500:Follow-Up Studies; D006801:Humans; D007211:Indoles / Q000008:administration & dosage / Q000493:pharmacokinetics / Q000494:pharmacology*; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000201:enzymology; D009000:Monocytes / Q000187:drug effects / Q000201:enzymology; D009369:Neoplasms / Q000188:drug therapy*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000276:immunology; D064546:Protein Kinase C beta; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology; D013329:Structure-Activity Relationship; D016896:Treatment Outcome
D015415:Biomarkers; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D007211:Indoles; D003841:Deoxycytidine; D000069287:Capecitabine; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D005472:Fluorouracil; C504878:enzastaurin