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Overview of Phase I/II Pemetrexed Studies

Overview of Phase I/II Pemetrexed Studies

ABSTRACT: Pemetrexed (Alimta) is an antifolate that is effective in the inhibition of multiple enzyme targets including thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyl transferase. The compound has been evaluated in several phase I trials, both as single agent and in combination with other cytotoxic agents. The initial schedule selected for further investigation in phase II trials was pemetrexed 600 mg/m2 as a 10-minute infusion on day 1 every 21 days. During the subsequent phase II development, the dose of pemetrexed was adjusted to 500 mg/m2 due to bone marrow and gastrointestinal toxicities. The adjusted dose of pemetrexed was well tolerated throughout the late-phase drug development program. Preclinical evidence suggests that pemetrexed has additive or synergistic activity when combined with many other clinically important anticancer agents, including gemcitabine (Gemzar), fluorouracil, carboplatin (Paraplatin), oxaliplatin (Eloxatin), paclitaxel, and vinorelbine (Navelbine). Doselimiting toxicities in these studies were primarily hematologic, and there was no evidence of cumulative hematologic toxicity. During the drug development program it was discovered that supplementation with folic acid and vitamin B12 profoundly increased the tolerability of pemetrexed. The studies discussed in this review demonstrate that pemetrexed is well tolerated as a single agent and will be an important contribution to combination chemotherapy regimens.

Pemetrexed (Alimta) is a novel
multitargeted antifolate antimetabolite
that inhibits, among
other enzymes, thymidylate synthase
(TS), dihydrofolate reductase
(DHFR), and glycinamide ribonucleotide
formyl transferase (GARFT).
The primary targets of pemetrexed
control pivotal steps in the de novo
synthesis of pyrimidines and purines.
The multitargeted nature of pemetrexed
is confirmed by in vitro experiments
demonstrating that both thymidine
and hypoxanthine are required
to prevent pemetrexed-induced cytotoxicity.[

Three initial single-agent phase I
studies were conducted to explore different
treatment schedules of pemetrexed.
These schedules comprised
administration of the compound weekly
* 4 every 6 weeks, daily for 5 days
every 21 days, and once every 21
days. Pemetrexed was administered
as a 10-mg/m2 intravenous (IV) infusion
over 10 minutes.

Phase I Trials of
Single-Agent Pemetrexed

Rinaldi et al reported a single-agent
phase I study with pemetrexed administered
as a 10-minute infusion
every week for 4 weeks. The treatment
was repeated after 6 weeks in patients
with advanced solid tumors.[2] A total
of 25 patients received doses ranging
from 10 to 40 mg/m2/wk. The doselimiting
toxicity consisted of neutropenia
that was completely reversible.
Nonhematologic toxicities were mild,
and no grade 3 or 4 nonhematologic
toxicities were reported. The maximum
tolerated dose was determined
to be 40 mg/m2/wk and the recommended
phase II dose utilizing this
administration schedule was 30 mg/
m2/wk. Two patients with advanced
colorectal cancer experienced minor responses.

Based on the results of this study,
Rinaldi and colleagues conducted a
second phase I trial.[3] In this study,
pemetrexed was administered as IV
infusion over 10 minutes every 21
days. A total of 37 patients with advanced
solid tumors received doses
ranging from 50 to 700 mg/m2. The
maximum tolerated dose was found
to be 600 mg/m2 and the recommended
dose for subsequent phase II trials
was determined to be 600 mg/m2. Neutropenia,
thrombocytopenia, and cumulative
fatigue were dose-limiting
toxicities. Partial responses were noted
in patients with advanced pancreatic
cancer (n = 2) and advanced
colorectal cancer (n = 2), with minor
responses in patients with advanced
colorectal cancer (n = 6).

The third single-agent phase I trial
with pemetrexed was conducted by
McDonald and colleagues.[4] In this
study, pemetrexed was administered
as an IV infusion over 10 minutes
daily for 5 days. The treatment was
repeated every 21 days. Thirty-eight
patients with advanced malignancies
received pemetrexed with doses ranging
from 0.2 to 5.2 mg/m2. The maximum
tolerated dose was found to be 4
mg/m2, with neutropenia being the leading
dose-limiting toxicity. One patient
with metastatic non-small-cell lung
cancer (NSCLC), one with metastatic
colon cancer, and one with pancreatic
cancer experienced minor responses.

A comparison of the results from
these three phase I studies indicated a
relationship between the maximum tolerated
dose treatment schedules; however,
myelosuppression and epithelial
toxicities as manifested by mucositis
and/or diarrhea remained as predominant
toxicities (Table 1). Additional
nonhematologic toxicities included fatigue
and rash. Because of the convenient
administration schedule, the
achievable dose intensity, and the extent
of anecdotal antitumor activity observed,
the every-21-day schedule was
selected for further development of
pemetrexed. The recommended phase
II dose of 600 mg/m2 was subsequently
reduced to 500 mg/m2 in order to further
optimize the tolerability of pemetrexed
and to prepare for early clinical
trials with combination regimens.

None of the single-agent phase I
studies used supplementation of patients
with folic acid and vitamin B12.
The added value of vitamin supplementation
to the safety of pemetrexed
was only discovered after the agent
had already entered its first registration
phase III registration trial.

Pharmacokinetic parameters of
pemetrexed were evaluated in these
phase I studies and yielded the following
conclusions: at clinically used
dose ranges, pemetrexed is eliminated
from serum, with a mean terminal
elimination half-life of 2 to 3 hours.[5]
A linear relationship between area
under the concentration-time curve
(AUC) and dose was noted. Within
24 hours after administration of the
compound, 70% to 90% of the administered
dose is recovered in the
urine. Hepatic metabolism of parent
compound is minimal leading to negligible
amounts of biologically inactive
metabolites. There was no
evidence for renal toxicities of pemetrexed
in patients with normal creatinine
clearance despite the fact that
pemetrexed is renally eliminated.

Nevertheless, in order to determine
whether comedication with potentially
nephrotoxic agents may lead to renal
toxicities, a phase I trial was
recently completed which evaluated
the effects of combining ibuprofen
with pemetrexed in patients with advanced
cancer. Preliminary pharmacokinetic
data indicated that
coadministration of these two agents
did not affect creatinine clearance or
pharmacokinetic variables of pemetrexed.[

Combination Regimens
With Pemetrexed

Pemetrexed underwent extensive
evaluation in preclinical studies to
determine if the agent could be combined
with other clinically used cytotoxic
agents, including platinums,
gemcitabine (Gemzar), cyclophosphamide
(Cytoxan), the taxanes, doxorubicin,
vinorelbine (Navelbine), and
radiation.[7] Findings of synergistic
or additive antitumor effects in human
tumor xenografts and cell lines
suggested that these combinations
warranted further evaluation in clinical

Table 2 summarizes clinical studies
that have been conducted with pemetrexed
combined with a variety of other
clinically relevant compounds, and details
will be presented below. These
trials have demonstrated that pemetrexed
is a versatile and well-tolerated
drug that can be combined at full doses
with all compounds studied, laying the
basis for a broad subsequent drug development

Pemetrexed Combination
With Gemcitabine

The cytotoxicity and potential underlying
mechanisms of the combination
of pemetrexed and gemcitabine
have been evaluated in several preclinical
studies involving a variety of
tumors and using simultaneous and
sequential administration.[8-14] Data
have shown that the combination results
in synergistic cytotoxicity when
administered sequentially but antagonism
with concurrent administration.

Optimal synergy was observed with
the pemetrexed → gemcitabine sequence
in studies of HT29 colon carcinoma
xenografts[8] and MIA
PaCA-2, PANC-1, and Capan-1 pancreatic
cancer cell lines.[9] In contrast,
the highest level of synergy in a
study of colon adenocarcinoma cell
lines LoVo, WiDR, and LRWZ occurred
when gemcitabine administration
preceded that of pemetrexed,
while the reverse sequence resulted in
additive and synergistic effects.[10]
In the latter trial, an increase in TS
expression, which is associated with
resistance to conventional antifolates,
was noted in all cell lines.

Experiments in HT29 colon cancer
cells evaluated the cell-cycle-modulating
effects of pemetrexed by flow
cytometry as a potential mechanism
to increase gemcitabine potency.[8]
A decrease in HT29 proliferation rate
correlated with an accumulation of
cells in S phase after 12 to 24 hours of
pemetrexed exposure. The authors
concluded that synchronization of
HT29 cells by pemetrexed was effecting
a change in the nucleotide
pools by inhibition of target enzymes-
that in turn potentiated cytotoxicity
of exposure to gemcitabine. Other studies have also shown S-phase cell
synchronization after pemetrexed

In MIA PaCA-2, PANC-1, and
Capan-1 pancreatic cell lines, Giovannetti
et al demonstrated that pemetrexed
treatment significantly
enhanced gene expression and activity
of deoxycytidine kinase (dCK), a
key enzyme involved in pyrimidine
salvage pathways and in the rate-limiting
step in gemcitabine activation.[9]
Rauchwerger et al studied the role of
the equilibrative-sensitive nucleoside
transporter (es-NT) in gemcitabine
sensitivity.[11] Cellular uptake of
gemcitabine requires transport across
the plasma membrane by sodiumindependent
(equilibrative) mechanisms
(es-NT), the activity of which
is a prerequisite for tumor growth inhibition
by gemcitabine.[12] Thus,
combining a nucleoside analog with
agents that increase NT expression,
such as TS inhibitors, would theoretically
increase the potential for cell
kill through depleting the nucleotide

In experiments carried out using
TS inhibitors (5-FU and raltitrexed
[Tomudex]) with gemcitabine administered
concurrently and sequentially
in three human pancreatic and one
human bladder cancer cell lines, TS
inhibitor pretreatment significantly
augmented cell kill relative to singleagent
gemcitabine and significantly
increased cell surface es-NT content
over basal levels in two of the pancreatic
cancer cell lines.

Results were maximal when TS
inhibitor treatment preceded gemcitabine
administration.[11] Thus, potential
mechanisms of synergy with
the pemetrexed → gemcitabine sequence
include TS inhibition, depletion
of nucleotide pools, S-phase
synchronization of cells, and activation
of es-NT and dCK. Mechanisms
for additive or synergistic effects observed
with the reverse sequence are
less clear as yet.

Based on the demonstration of preclinical
cytotoxic synergy, a phase I
trial of pemetrexed in combination
with gemcitabine was conducted in
56 patients with advanced solid tumors
who had received at least one
previous chemotherapy regimen.[15]
Adjei et al used sequential administration
of gemcitabine followed by
pemetrexed, based on their in vitro
clonogenic assays demonstrating cytotoxic
synergy in cultured human
colon carcinoma cells with this sequence
but not the reverse sequence.[
15] Patients in group I (n =
35) received gemcitabine at 1,000 or
1,250 mg/m2 IV over 30 minutes on
days 1 and 8, and pemetrexed on day
1 only, 90 minutes after gemcitabine,
at escalating doses ranging from 200
to 600 mg/m2 given IV over 10 minutes.
Courses were repeated every 3
weeks. Because 57% of courses were
associated with neutropenia that required
reduction/omission of the day
8 gemcitabine dose, group II patients
(n = 21) received the pemetrexed on
day 8 instead of day 1.

Neutropenia was the principal
dose-limiting hematologic toxicity in
both groups I and II and seemed to be
dose related; no infections were noted
in patients with severe neutropenia.
The median neutrophil count nadir
was on day 7 in group I and day 14 in
group II patients, indicating a relationship
between the nadir and pemetrexed
administration. The maximum
tolerated dose for group I was determined
to be gemcitabine at 1,000 mg/
m2 and pemetrexed at 500 mg/m2 due
to prolonged (> 5 days) grade 4 neutropenia
in four of six patients receiving
the 1,250-mg/m2 gemcitabine
dose. For group II, the maximum tolerated
dose was gemcitabine 1,250
mg/m2 and pemetrexed 500 mg/m2 due
to life-threatening and prolonged neutropenia
seen at the higher pemetrexed
dose of 600 mg/m2.

The primary nonhematologic toxicity
was elevated hepatic transaminase
level in 71% of treatment courses,
most cases of which were mild to
moderate and rapidly reversible. Other
toxicities included nausea, fatigue,
and rash. Patients receiving pemetrexed
on day 8 (group II) had fewer
and less severe toxicities and fewer
dosage interruptions than those receiving
pemetrexed on day 1 (group I).

Among 55 assessable patients, objective
responses were confirmed in 7
of 34 group I and 6 of 21 group II
patients with tumors, including colorectal
cancer (n = 3), non-small-cell
lung cancer (n = 3), cholangiocarcinoma
(n = 2), ovarian cancer (n = 2),
mesothelioma (n = 1), breast cancer
(n = 1), and adenocarcinoma of unknown
primary site (n = 1). Twelve
of these patients had partial responses
and one was considered a mixed response,
with response durations of at
least 3 months. An additional 27 patients
had stable disease, with durations
of stable disease ranging from 1
to 11 cycles after the initial evaluation
at cycle 2.

Pharmacologic evaluations conducted
in four group I patients at the
maximum tolerated dose showed no
alteration of pemetrexed pharmacokinetics
based on gemcitabine pretreatment,
although the sample size
was small. Recommended dose and
schedule of this regimen for phase II
study was gemcitabine 1,250 mg/m2
on days 1 and 8 with pemetrexed 500
mg/m2 given 90 minutes after gemcitabine
on day 8, every 21 days.[15]
Phase II studies of the pemetrexed/
gemcitabine combination are being
carried out in advanced-stage non-
small-cell lung, breast, and pancreatic
cancer, as described elsewhere in
this supplement.

Pemetrexed and Cisplatin
In a phase I study of pemetrexed
and cisplatin, two administration
schedules were investigated based on
the hypothesis that because pemetrexed
is primarily eliminated by renal
excretion, hydration required for cisplatin
administration may potentially
modulate the clearance of pemetrexed
and thus impact on antitumor activity
or toxicity.[16] In order to investigate
this hypothesis in a clinical setting,
one patient cohort (n = 40) received
pemetrexed followed by hydration and
cisplatin on day 1 of a 21-day cycle.
Another cohort (n = 11) was treated
with pemetrexed on day 1 without
any hydration, followed by hydration
and cisplatin on day 2 of a 21-day
cycle. In both cohorts, pemetrexed was
administered as an IV infusion over
10 minutes.

The maximum tolerated dose for
both schedules was pemetrexed
600 mg/m2 and cisplatin 100 mg/m2,
demonstrating that both compounds can
be combined at fully active clinical doses. Dose-limiting toxicities consisted
mainly of myelosuppression. Ten patients
in cohort 1 experienced partial
responses, and one patient with head
and neck cancer had a complete response.
In the second cohort, two patients
experienced partial responses.
Most notably, five of 11 patients with
pleural mesothelioma developed confirmed
and independently validated
partial responses, indicating a profound
antitumor effect of this combination in
malignant pleural mesothelioma-a disease
for which at that time no established
treatment was available.
Antitumor responses were also noted
in other tumor types including NSCLC,
colorectal cancer, melanoma, and cancer
of unknown primary. The recommended
doses for subsequent clinical
studies were determined to be pemetrexed
500 mg/m2 and cisplatin 75 mg/
m2 with administration of both agents
on day 1.

Based on the provocative results of
this study, pleural mesothelioma was
chosen as the primary target tumor entity
for approval, and additional studies,
including a single-agent phase II
trial and a phase I trial with pemetrexed
and carboplatin, were initiated. A courageous
step was taken by initiating the
ultimately successful pivotal phase III
registration trial based on the results of
the phase I combination trial of pemetrexed
and cisplatin.

Pemetrexed in Combination
With Carboplatin

The combination of pemetrexed
and carboplatin was evaluated in a
phase I trial conducted by Hughes
and colleagues.[17] Twenty-seven patients
with MPM received escalating
doses of pemetrexed (400 mg/m2 to
500 mg/m2) and carboplatin (AUC 4
to 6). Pemetrexed was administered
as a 10-minute infusion and carboplatin
was administered as a 30-minute
infusion, both on day 1 every 21 days.
Pemetrexed at 500 mg/m2 and carboplatin
at AUC 6 was the maximum
tolerated dose; three of five patients
at this dose level experienced grade 4
neutropenia as the dose-limiting toxicity.
Nonhematologic toxicities at the
maximum tolerated dose included
nausea, vomiting, and stomatitis.
There were no grade 4 nonhematologic
toxicities reported at this dose
level. Two courses at all dose levels
were complicated by grade 3 elevation
of transaminase levels. Response
to therapy was a secondary outcome
and was measured in all patients.

Of the 25 patients evaluable for
response, there were eight confirmed
partial responses, for an overall response
rate of 32%. Five of the eight
patients who experienced partial responses
had stage IV disease, and five
patients had mesothelioma of epithelial
histology. All of the patients who
received treatment with pemetrexed
and carboplatin experienced cancerrelated
symptoms at the start of chemotherapy.
Nineteen (70%) of the
original 27 patients accrued experienced
relief in cancer-related symptoms
while on study. Median overall
survival was 451 days and median
time to disease progression was 405
days. Figure 1 shows the response of
a patient on this study. The recommended
phase II dose for this combination
was determined to be pemetrexed
500 mg/m2 and carboplatin AUC 5,
which allowed for administration of
full doses of both agents.


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