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Current Clinical Trials of Flavopiridol

Current Clinical Trials of Flavopiridol

Flavopiridol [2-(2-chlorophenyl 5 ,7-dihydroxy-8-[cis-(3-hydroxy-1-methyl-4-piperidinyl)-4H-1-benzopyran-4-one,
hydrochloride] is a semisynthetic flavone with a novel structure compared with
that of polyhydroxylated flavones, such as quercetin and genistein.[1] It is
derived from rohitukine, an alkaloid isolated from the stem bark of Dysoxylum
binectariferum, a plant indigenous to India.[2] Originally synthesized and
supplied by Hoechst India Limited, flavopiridol is provided to the Division of
Cancer Treatment and Diagnosis of the National Cancer Institute (NCI) by Aventis
Pharmaceuticals, Inc.

Mechanism of Action

Cell-cycle regulation is dependent on cyclin-dependent kinases (cdks), which
require association with cyclin proteins for activation.[3] Flavopiridol was the
first compound with the potent ability to disrupt cell-cycle progression by
inhibition of regulatory phosphorylations to be considered for clinical
development.

Flavopiridol inhibits several cellular kinases and has demonstrated
cytostatic and cytotoxic activity in vitro and in vivo in numerous human tumor
cell lines and xenograft models (including human breast, prostate, and lung
carcinoma) at clinically achievable concentrations.[1,4] Flavopiridol is capable
of disrupting progression of cells through the cell cycle at the G1/S and G2/M
transitions.[1,5,7] The direct inhibition of cdks 1, 2, and 4 via competitive
inhibition of adenosine triphosphate binding by flavopiridol has been
demonstrated.[5,7,9] Flavopiridol also inhibits cdk7/cyclin H, thus preventing
the phosphorylation and subsequent activation of several cdks[6,8] and
down-regulates cyclin D1, the cyclin associated with cdks 4 and 6.[10]

Flavopiridol-induced G1 arrest may be related to inhibition of cdk 2 and 4
activity, as well as diminution of cyclin D levels; G2/M arrest may be due in
part to inhibition of cdk1 activity. Cdk4 and 2 kinase activities, as well as
cyclins D, E, and A protein levels, are diminished following flavopiridol
exposure in a number of in vitro models. In MCF-7 cells, flavopiridol-induced
G1/S arrest is associated with the loss of cdk4 and 2 activity and reduced
cyclin D levels preceded by hypophosphorylation of Rb protein. The flavopiridol-induced
decline in cyclin D1 is an early, specific event, due in part to the
transcriptional repression of the cyclin D1 promoter.[11] Similarly, in
cdk4-deficient MCF-10A breast epithelial cells, flavopiridol-induced G1 arrest
coincided with Rb dephosphorylation and dose-dependent inhibition of cdk6-kinase
activity associated with the loss of cyclin D1 expression.[12]

The efficacy of flavopiridol is not based solely on cell cycle arrest, since
this agent induces death in noncycling A549 lung cancer cells by a process that
depends on RNA and protein synthesis.[4] Parker and co-investigators[13]
observed apoptosis in SUDHL-4 leukemia cells without evidence of cell-cycle
arrest, suggesting that the antiproliferative effects can be separated from the
proapoptotic activity of this agent.

Regulation of gene expression is another potential mechanism of action for
flavopiridol. In human monocytes, flavopiridol causes down-regulation of
vascular endothelial growth factor (VEGF) messenger (m)RNA and protein
expression induced by hypoxia. Flavopiridol does not affect hypoxia-induced
transcriptional activation of VEGF but significantly decreases the VEGF mRNA
half-life, suggesting that flavopiridol may have antiangiogenic activity.[14]
Flavopiridol also inhibits the positive transcription elongation factor, which
is a protein kinase composed of cdk9 and a cyclin subunit (cyclin T1 or cyclin
T2)[15] and controls the elongation phase of transcription by RNA polymerase
II.[16] The IC50 of flavopiridol is directly related to the concentration of the
positive transcription elongation factor.[17] (It is not known if the
antiproliferative effects of flavopiridol are due to inhibition of the positive
transcription elongation factor or other cyclin-dependent kinases). A
comprehensive review of the mechanisms of action of flavopiridol was recently
published.[18]

Preclinical Activity

Flavopiridol should exert cytostatic activity because of the pivotal role of
the cdks in the cell division cycle. Evidence demonstrating its cytostatic
activity includes the finding that it inhibits the growth of a broad spectrum of
human tumor cell lines in vitro. In the NCI tumor cell line panel, flavopiridol
had significant inhibitory activity against all of the more than 60 human tumor
cell lines with no clear selectivity for tumor type. IC50 values ranged from
approximately 50 to 200 nM,[1] similar to concentrations required to inhibit
cdks. Flavopiridol-induced growth inhibition seems to be independent of tumor Rb,
cyclin D1, p16, and p53 status.[19,21]

Administration of flavopiridol after or concomitant with antineoplastic
agents, including mitomycin C (Mutamycin), paclitaxel, gemcitabine (Gemzar),
SN-38 (the active metabolite of CPT-11), imatinib mesylate (Gleevec), and
doxorubicin can promote chemotherapy-induced apoptosis.[22-29] Recent reports
suggest a marked increase in apoptosis when differentiating agents such as
phorbol 12-myristate 13-acetate (PMA), suberoylanilide hydroxamic acid (SAHA),
and depsipeptide are combined with flavopiridol.[30-32] Cytotoxic synergy was
more pronounced when non-small-cell lung cancer (NSCLC) A549 cells were
exposed to flavopiridol after rather than before or concomitant with paclitaxel,
cytarabine, topotecan (Hycamtin), doxorubicin, and etoposide.[33]

Clinical Data

NCI-sponsored clinical trials of flavopiridol were initiated in 1994.
Preclinical data suggested that prolonged exposure was necessary to achieve
maximal antitumor effect.[1] Two phase I trials used a 72-hour infusion
every-2-weeks schedule. In a trial at the NCI, diarrhea was dose-limiting, and
the maximum tolerated dose was 50 mg/m²/24h ´ 3.[34] Aggressive prophylaxis of
diarrhea allowed for further dose escalation to a maximum tolerated dose of
78 mg/m²/24h ´ 3, with dose-limiting hypotension seen at higher doses.
Anorexia and asthenia were additional major toxicities. Mean steady state plasma
flavopiridol concentrations achieved at the maximum tolerated doses were 271 nM
(range: 174-2,943 nM) and 344 nM (range: 130-1557 nM), respectively, with
postinfusion peaks suggestive of enterohepatic recirculation.

Diarrhea was also dose limiting in a trial using the same schedule conducted
at the University of Wisconsin.[35,36] The maximum tolerated dose was 40
mg/m²/24h ´ 3; nausea, vomiting, and orthostatic hypotension occurred at the
maximum tolerated dose. In this trial, a steady state concentration of 415 nM
was achieved at the maximum tolerated does. Antitumor activity against renal
cell carcinoma, colon carcinoma, non-Hodgkin lymphoma, and gastric carcinoma (a
prolonged complete response) was seen in these studies.

Results of four single-agent flavopiridol studies incorporating a continuous
infusion 50 mg/m²/24h ´ 3 every 14 days in patients with renal cell, gastric,
colon, and non-small- cell lung carcinoma confirmed an adverse event profile
dominated by diarrhea, nausea, vomiting, and asthenia.[37-40] In addition, 19 of
89 patients (21%) experienced venous thromboses, including 12 at the central
venous catheter site. Two patients experienced transient ischemic attacks, and
one, a myocardial infarction. Two complete responses in patients with renal cell
cancer were the only objective responses reported in these trials.

Additional schedules of administration are being pursued because of the
disappointing degree of antitumor activity achieved with the 72-hour schedule
and because of additional preclinical data that suggested higher plasma
concentrations of flavopiridol may be necessary to obtain tumoricidal
activity.[41] NCI investigators followed their original phase I infusional study
with an exploration of daily 1-hour infusions for 1 to 5 days every 21 days.
They defined maximum tolerated doses of 37.5, 50, and 62.5 mg/m²/d for 5-, 3-,
and 1-day administrations, respectively, and documented median peak plasma
concentrations of 1.7, 3.2, and 3.8 µM with these schedules.[42,43] Neutropenia
was the primary dose-limiting toxicity, but diarrhea and a proinflammatory
syndrome of anorexia, tumor pain, fever, and asthenia were also prominent. Five
patients (9%) experienced thrombotic events (three lower-extremity deep-vein
thromboses and two catheter-related thromboses).

Investigators at the National Cancer Center East in Japan determined that 80
mg/m² was tolerable on a weekly 24-hour infusion schedule and achieved a mean
Cmax of 718 nM.[44]

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