Annales Pharmaceutiques Franaises (2012) 70, 155162

Disponible en ligne sur

www.sciencedirect.com
ORIGINAL ARTICLE
Stability of ready-to-use temsirolimus infusion
solution (100 mg/L) in polypropylene containers
under different storage conditions
Stabilit des solutions de temsirolimus dilues (100 mg/L dans des poches de
polypropylne) dans diffrentes conditions de conservation
S. Poujol
a
, F. Bressolle
a,b,
, I. Solassol
a
, F. Pinguet
a
a
Oncopharmacology Laboratory, Val dAurelle Cancer Centre, 34298 Montpellier, France
b
Clinical Pharmacokinetic laboratory, Faculty of Pharmacy, Montpellier 1 University, 15,
avenue Charles-Flahault, 34093 Montpellier, France
Received 11 January 2012; accepted 21 March 2012
Available online 21 April 2012
KEYWORDS
Temsirolimus;
Infusion solutions;
100 mg/L;
Polypropylene bags;
Stability;
Different storage
conditions
Summary The aim of this study was to determine the stability of ready-to-use temsirolimus
infusion solutions under different storage conditions. Solutions were prepared in polypropylene
containers by adding temsirolimus injection to 0.9% sodium chloride infusion to reach a nal
concentration of 100 mg/L. The following storage conditions were tested: (i) 4
o
C in the refrig-
erator; (ii) 20
o
C under room light exposure and light protection; and (iii) outdoor temperature
with sunlight exposure. Moreover, stress testing was performed on drug substance at 20
o
C under
ultraviolet (UV) radiation (365 nm). A stability-indicating high-performance liquid chromatog-
raphy (HPLC) method with UV detection was developed for this analysis. Precision was below
4% and accuracy ranged from 97 to 102%. The lower limit of quantitation was 0.1 mg/L. The
degradation products produced after UV light exposure were detected upon further analysis by
mass spectrometry detection. The stability of temsirolimus is light and temperature dependent.
After storage at 20
o
C with room light exposure, the rate of degradation was around 0.25%/h;
after 1 day, 92.5% of the initial temsirolimus concentration was recovered. When protected
from light, at 4 and 20
o
C, losses were decelerated; the decrease in drug concentration was
1.0 and 1.56% per day, respectively. Under daylight exposure, a substantial decrease in drug
concentration was observed; after 1 h, losses were higher than 10%. Exposed to UV light, half of
the drug was lost after 45 min. In conclusion, temsirolimus 100 mg/L in infusion polypropylene
bags containing 0.9% sodium chloride was chemically stable when protected from light for 4
and 3 days at 4 and 20
o
C, respectively.
2012 Elsevier Masson SAS. All rights reserved.

Corresponding author.
E-mail address: fbressolle@yahoo.fr (F. Bressolle).
0003-4509/$ see front matter 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.pharma.2012.03.005
156 S. Poujol et al.
MOTS CLS
Temsirolimus ;
Solutions
reconstitues ;
100 mg/L ;
Poches en
polypropylne ;
Stabilit ;
Diffrentes
conditions de
stockage
Rsum Lobjectif de cette tude a t de dterminer la stabilit des solutions de tem-
sirolimus aprs reconstitution dans diffrentes conditions de conservation. Les solutions ont
t prpares dans des acons en polypropylne en ajoutant le concentr de temsirolimus pour
injection une solution de chlorure de sodium 0,9 % an dobtenir une concentration nale de
100 mg/L. Les conditions suivantes de stockage ont t testes : (i) 4
o
C dans le rfrigrateur ;
(ii) 20
o
C avec et sans exposition la lumire articielle ; et (iii) temprature extrieure avec
exposition la lumire solaire. La stabilit de la solution reconstitue a t galement teste
20
o
C sous une lampe lumire ultraviolette 365 nm. La stabilit a t mesure par chro-
matographie liquide haute performance et dtection dans lultraviolet (CLHP-UV). La dlit
de la mthode est infrieure 4 % et lexactitude varie de 97 102 %. La limite de quantication
est de 0,1 mg/L. Les produits de dgradation forms aprs exposition la lumire ultraviolette
ont t analyss par CLHP et dtection par spectromtrie de masse. La stabilit du temsirolimus
est lumire et temprature dpendent. Aprs stockage 20
o
C la lumire articielle, la vitesse
de dgradation est de lordre de 0,25 %/h ; 92,5 % de la concentration initiale de temsirolimus
sont retrouvs aprs un jour de stockage. Protg de la lumire 4 et 20
o
C, les pertes sont
beaucoup moins importantes ; la concentration diminue respectivement de 1,0 et 1,56 %/jour.
Expos la lumire solaire, une rduction substantielle de la concentration est observe ; aprs
une heure, les pertes sont suprieures 10 %. Expos la lumire ultraviolette, la moiti de la
concentration de temsirolimus a disparu aprs 45 minutes. En conclusion, la solution de tem-
sirolimus 100 mg/L est stable trois jours 20
o
C protg de la lumire et quatre jours 4
o
C
dans des acons en polypropylne contenant du chlorure de sodium 0,9 %.
2012 Elsevier Masson SAS. Tous droits rservs.
Introduction
Temsirolimus (sirolimus-42-[2,2-bis-(hydroxymethyl)]-
propionate) is an ester analog of rapamycin, a macrolide
antibiotic with antifungal, antitumor, and immunosup-
pressive activities [1]. This compound exists as different
diastereoisomers (Fig. 1); regarding stereochemistry, three
isomers A, B and C can exist and they interconvert in
solution. Isomer B is the predominant isomer ( 97%) in
both solution and solid states [2,3]. Temsirolimus inhibits
the mammalian target of rapamycin (mTOR) kinase, a
component of intracellular signaling pathway involved in
cell growth and proliferation [4,5], and in the response
of such cells to hypoxic stress [6]. Temsirolimus binds
to FK506-binding protein 12 (FKBP12), and the resultant
proteindrug complex inhibits the kinase activity of mTOR
[7,8]. mTOR is a serine/threonine kinase which plays a
role in the phosphatidylinositol 3-kinase/AKT pathway that
is upregulated in some tumors [4,5,9]. Blockade of mTOR
signaling by temsirolimus inhibits the production of proteins
that regulate progression to the cell cycle [9,10] and angio-
genesis [11,12]. Temsirolimus received approval by the
US Food and Drug Administration in May 2007, and by the
European Medicines Agency (EMA) in November 2007 for the
treatment of advanced renal cell carcinoma. This drug has
United Kingdom marketing authorization for the rst-line
treatment of patients with advanced renal cell carcinoma
who have at least three of the six prognostic risk factors
[13]. Recently, temsirolimus received approval by EMA in
September 2011 for the treatment of adult patients with
relapsed or refractory mantle cell lymphoma. The safety,
tolerability and efcacy of temsirolimus have been well
established in clinical trials [14,15]. Drug related toxicity
included rash, mucositis, asthenia, nausea, hyperglycemia,
hypophosphatemia, anemia, and hypertriglyceridemia.
Clinical activity in other tumor types, such as relapsed or
refractory non-Hodgkin lymphoma [16,17], endometrial
cancer [18], neuroendocrine carcinomas [19], sarcoma
[20], and metastatic breast cancer [21] has been observed.
Temsirolimus is therefore an important new agent for
cancer treatment.
Temsirolimus is administered as a solution to be given by
intravenous infusion over 30 to 60 min. The nished prod-
uct, Torisel

, is a two-vial system consisting of a concentrate

solution containing 25 mg/mL temsirolimus (in one vial) and
a specically formulated diluent (in another vial) composed
of polysorbate 80, polyethylene glycol 400, dehydrated alco-
hol and nitrogen. Before use, the temsirolimus concentrate
has to be diluted with the diluent, followed by a dilution with
0.9% sodium chloride for intravenous injection. According
to the manufacturers guidelines for quality assurance, the
solution is stable at least 6 h at 25
o
C when protected from
sunlight and excessive uorescent light. This short stability
prompted us to investigate stability assays that could be use-
ful in clinical practice using a centralized preparation unit.
To our knowledge, no other data are available in the
literature concerning the stability of this compound in
reconstituted solution. Thus, we undertook to study the
effects of temperature and light (room or sunlight) on the
stability of temsirolimus over a period of 15 days. The aim of
our study was to reproduce the different conditions of use
and storage encountered at the hospital pharmacy.
Experimental
Reagents
Temsirolimus (Torisel

) was purchased from Wyeth Pharma-

ceuticals (Paris, France). Two different reconstituted stock
solutions were used; each of them contained 25 g/L of
temsirolimus in the diluent. Solution A was used in the
Temsirolimus stability in polypropylene containers 157
OH OH
(S)
(R)
(R)
(R)
O
O
HO
O
26
42
27
37
39
41
(R)
(R)
(R)
O
O
HO
O
N
(S)
O
O
(R)
O
(R)
(R)
(S)
OH
O
O O
(E)
25
17
21 19
23
29
31 N
(S)
O
OH
(S)
O
(R)
(R)
OH
O O
(E)
(R)
(S)
O
(R)
(R)
(S)
O
(R)
HO
O
(E) (E) (E)
15
14
13
1
7
5
3
9
11
33
35
(R)
(S)
O
(R)
(R)
(S)
O
(S) O
(R)
HO
O
(E) (E) (E)
Temsirolimus:
Molecular weight: 1029.6 g/mole
Seco-temsirolimus:
Molecular weight: 1029.6 g/mole
Figure 1. Chemical structure of temsirolimus and seco-temsirolimus. The stereochemistry is represented.
Structure chimique du temsirolimus et du seco-temsirolimus. La strochimie est reprsente.
preparation of the calibration curves and solution B was
used in the preparation of quality control (QC) samples.
These stock solutions were stored at 20
o
C until use (max-
imum 2 months). Temsirolimus was determined to be stable
under these storage conditions. Multilayer polypropylene
containers (Freeex

, 250 mL) were from Fresenius, (Paris,

France). Methanol was obtained from Carlo Erba (Val de
Reuil, France). Deionized water was prepared by a Milli-Q
purication system from Millipore (Molsheim, France).
Preparation of admixtures
The recommended dose of temsirolimus during renal car-
cinoma treatment is 25 mg intravenously infused over
3060 minutes once per week in 250 mL 0.9% sodium
chloride bags. Sufcient amounts of stock solutions were
therefore added to multilayer laminate polypropylene con-
tainers containing 0.9% sodium chloride in order to achieve
drug concentrations of 100 mg/L. All admixtures were pre-
pared under aseptic conditions in a laminar air ow reserved
for cytostatic drug preparation.
Stability assays were performed under various physi-
cal conditions that could be encountered clinically. Thus,
admixtures were stored under each of the following condi-
tions:

4
o
C in the refrigerator;

room temperature (20 2

o
C) under room lighting provid-
ing an overall illumination of 1,200 Klux h;

room temperature under light protection;

and outdoor temperature (10

o
C) with sunlight exposure.
The room temperature was controlled by digital dis-
play. Moreover, a more drastic condition was tested: 20
o
C
exposed to UV light (200 W h m
-2
) at 365 nm.
Immediately upon sample preparation and at specic
time intervals following storage (1, 2, 3, 4, 7, 10 and 14 days
at 4
o
C in the refrigerator and at 20
o
C protected from light;
0.25, 1, 1.25, 2, 2.25, 3, 3.25, 4, 4.25 and 7 days at 20
o
C
with room light exposure; 1, 2, 3, 4, 5, 6 and 7 days at
10
o
C with sunlight exposure; and 15, 30, 45, 60, 75, 90, 105
and 120 min under the UV lamp), 1-mL sample was with-
drawn from each container and analyzed. Before sampling,
each container was manually shaken for 1 min to ensure a
uniform solution. The samples were observed visually and
then frozen at 80
o
C until analysis. Drug concentrations
were determined in triplicate by liquid chromatography with
ultraviolet detection (LC-UV). Each assay was performed in
triplicate.
Temsirolimus stability in the infusion polyvinyl chloride
(PVC) tube was also studied. Reconstituted temsirolimus in
polypropylene bags (protected from light) was infused, at
room temperature, through PVC-lined administration sets
(Intrax Safe Set, BBraun, Boulogne Billancourt), either pro-
tected (wrapped in aluminum, n = 3) or unprotected (n = 3)
from room light. The simulated infusion took 45 min. The
length of the PVC infusion tubes was 1.85 m and they were
DEHP free.
High-performance liquid chromatography
(HPLC) analysis
The stability study has been carried out by HPLC anal-
ysis with UV detection. The instrumentation included a
delivery pump (Model LC-10AT, Shimadzu Corporation,
Croissy Beaubourg, France), a degasser (Model DGU-14A,
Shimadzu), an autosampler (Model SIL-10ADvp, Shimadzu)
tted with a 100 L loop and set at 4

C, a photodiode
array ultraviolet spectrophotometric detector (Model SPD-
M10Avp, Shimadzu), and a multi-instrument data acquisition
and data processing system (interface SS420x Scientic
Software Inc, Class-VP 7, Shimadzu). All analyses were
158 S. Poujol et al.
performed at room temperature (20 2
o
C). Chromato-
graphic conditions were optimized to ovoid peak tailing
and split peaks. This included column type, composition
of the mobile phase, addition of a buffer to the mobile
phase and ow rate. Best peak shape of the analytes and
relative short analysis time were obtained on a XBridge C8
column (XBridge C8 column, 150 4.6 mm, 5 m particle
size, Waters, Milford, MA, USA); the use of a buffer did not
enhance the quality of the peak. The mobile phase consisted
of eluent A, water and eluent B, methanol (degassed and
ltered in a 0.45-m membrane lter [0.45-m membrane
lter, Alltech Associates, Deereld, IL] under negative
pressure). The starting eluent was 25% A and 75% B after
which the proportion of eluent A was decreased linearly
to 20% in 6 min followed by an isocratic step of 20% A for
10 min, then the eluent returned to initial composition of
eluent A (25%) and B (75%) in 1 min and then held for 3 min
in order to re-equilibrate the column. UV detection was
made at 277 nm and the ow rate was 0.6 mL/min. The
injection volume was 50 L.
Extemporaneous dilutions of stock solutions were made
in 0.9% sodium chloride as appropriate to prepare calibra-
tion curves (0.10, 0.25, 0.50, 0.75, 1.0 and 2.5 mg/L). The
peak areas were plotted against theoretical concentrations.
Both intra- (n = 6) and inter-assay (n = 6) repeatability of cal-
ibration curves were studied. Standard calibration curves
were obtained from unweighted least-squares linear regres-
sion analysis of the data. QC samples were prepared in the
same way to provide low, medium and high concentrations:
0.2, 0.625 and 2 mg/L. These QC samples were used dur-
ing the study to determine accuracy and precision of the
method as well as during stability assays to provide the basis
of accepting or rejecting the run.
The method was validated according to the ICH guidelines
for validation of analytical procedures [22]. Linear relation-
ships between the peak area and the analyte concentration
were statistically conrmed (lack-of-t test). For each point
of the calibration standards, the concentrations were back-
calculated from the equation of the linear regression curves.
The acceptance criteria for each back-calculated stan-
dard concentration was 5% deviation from nominal value.
The good agreement between added and back-calculated
concentrations was statistically evaluated. The normal dis-
tribution of the residuals (the difference between nominal
and back-calculated concentrations) was veried. Moreover,
the mean residual values (or mean predictor error) was
computed and compared to zero (Student t-test); the 95%
condence interval was also determined.
The within-day and between-day precision and accuracy
of the method were validated by analyzing QC samples
against a calibration curve. Determinations were per-
formed with six replicates per QC on the same day as
well as each day for six separate days. The percent rel-
ative standard deviation (RSD) served as the measure of
precision. The accuracy was evaluated as [mean found
concentration/nominal concentration] 100. The criteria
for acceptability of data included accuracy within 5%
relative error (RE) from the nominal values and a preci-
sion of within 5% RSD. The lower limit of quantitation
was determined as the temsirolimus concentration giving a
signal-to-noise ratio of 10 and both precision and accuracy,
expressed as percentage error, less than or equal to 5%.
A Hewlett Packard LC system (Model 1100 series, Agi-
lent Technologies, Les Ulis, France) equipped with a MS
quadrupole mass spectrometer detector, was further used
to only analyze the degradation products produced after UV
light exposure. Optimized parameters were as follows:

heated N
2
gas of 350
o
C and 10 L/min was used to evapo-
rate solvent from the electrospray chamber;

compressed N
2
gas of 35 psi was used for nebulisation;

and voltage was set at + 3.0 kV for the capillary.

The sampling cone voltage was set at 100 V. The mass
spectrometer was operated at negative mode. The above-
mentioned analytical LC conditions were also applied to LC-
MS analysis. Liquid chromatography with mass spectrometry
detection (LC-MS) chromatogram obtained before exposure
(total ion current) and temsirolimus mass spectrum were
used as references.
Analysis of data
The percentage of temsirolimus remaining after each time
interval was determined by comparing the concentration
at that time with the initial temsirolimus concentration,
measured before storage at the different conditions. A
decrease of more or equal to 5% of the initial concentra-
tion was considered to represent a signicant loss of drug
[22].
Results
High-performance liquid chromatography
(HPLC) method validation
Under the chromatographic conditions described above,
retention time of temsirolimus was 11.3 min. Represen-
tative LC-UV chromatogram is given in Fig. 2A (black
chromatogram). As for rapamycin, temsirolimus exists in dif-
ferent isomer forms in solution [2,3]. Thus, the second peak
at the retention time (rt) of 13.3 min could correspond to
the temsirolimus isomer. As previously reported by Cai et al.
[3], reversed-phase columns might be able to resolve the
isomers.
Results showed an excellent linear relationship between
peak area and the concentration of the analyte over
the range of 0.12.5 mg/L. Intra-assay reproducibility
was determined for calibration curves prepared the same
day in replicate (n = 6) using the same stock solutions.
The intra-day average slope of the tted straight lines
was 60,8975 (RSD = 0.75%) and the mean intercept was
1991. For calibration curves prepared on different days
(n = 6), the average results were as follows: slope = 65,6017
(RSD = 2.7%), and intercept = 2142. The obtained correla-
tion coefcients were greater than 0.998. RSD and recovery
values around the mean back-calculated concentrations
were 0.714.6% and 96.4102%, respectively. The residuals
(differences between nominal and back-calculated concen-
trations) showed random variations, the number of positive
and negative values being approximately equal. Moreover,
they were normally distributed and centered on zero.
Accuracy and precision of the method are given in Table 1.
The lower limit of quantitation was 0.1 mg/L.
Temsirolimus stability in polypropylene containers 159
Figure 2. A. Liquid chromatography with ultraviolet detection (LC-UV) chromatograms of temsirolimus before (black curve) and 3 days
after room light exposure (20
o
C) (red curve). B. LC-MS) total ion chromatogram of degraded temsirolimus solution after ultraviolet (UV)
light exposure for 2 h.
A. Chromatogrammes CLHP-UV obtenus avant (courbe en noir) et trois jours aprs exposition la lumire articielle (20
o
C) (courbe en
rouge) du temsirolimus. B. Chromatogramme CLHP-SM (courant ionique total) obtenu aprs exposition du temsirolimus aux ultraviolets
durant deux heures. CLHP-UV: chromatographie liquide haute performance et dtection dans lultraviolet ; CLHP-SM: chromatographie
liquide haute performance et dtection par spectromtrie de masse.
Stability study
At the end of each study period, to ensure specicity and
selectivity of the analyses, photodiode array analyses of
Table 1 Within-day and between-days precision and
accuracy of the method.
Variabilit intra- et inter-jour de la dlit et exactitude de la
mthode.
Temsirolimus concentrations
(mg/L)
Precision
(RSD, %)
Accuracy (%)
Within-day (n = 6)
0.2 3.5 99.9
0.625 3.2 99.9
2 0.5 97.3
Between-days (n = 6)
0.2 2.24 99.9
0.625 2.63 99.9
2 2.62 101.5
RSD: relative standard deviation.
the spectra showed the purity of the temsirolimus peak. No
peaks for unidentied degradation products, by-products or
impurities overlapped with the temsirolimus peak. We found
that the stability of temsirolimus is light and temperature
dependent. Results are given Table 2 and presented in Fig. 3.
After storage at 20
o
C under room light exposure, 92.5% of
the initial temsirolimus concentration was recovered after
24 h; after the extended exposure period of 7 days, the drug
concentration fell below 60% (Table 2). Drug concentrations
followed a linear decline; the rate of degradation was 0.25%
per hour. Thus, temsirolimus loss of 5% or more occurred
after 20 h. A chromatogram obtained 3 days after room light
exposure is presented in Fig. 2A (red curve); losses were
about 20%. When protected to room light, at 4 and 20
o
C,
losses were decelerated; the decrease in drug concentration
was 1.0 and 1.56% per day, respectively. Thus, temsirolimus
remains stable for 4 days at 4
o
C and 3 days at 20
o
C. Under
daylight exposure, a substantial decrease in drug concen-
tration was observed; after 1 h, losses were higher than
10%. This decrease followed a mono-exponential decline;
the corresponding half-life value was 4.3 h.
We found no signicant degradation of the temsirolimus
solution through the tubing (with or without light exposure)
during the simulated infusion.
160 S. Poujol et al.
Table 2 Stability of temsirolimus added to 0.9% sodium chloride injection into multilayer polypropylene bags.
Stabilit de la solution de temsirolimus dans des poches pour perfusion en polypropylne contenant une solution de chlorure de sodium
0,9 %.
Storage time, days Initial concentration remaining, %
20 2
o
C 20 2
o
C 4 0.2
o
C 10 4
o
C
Exposed to room light Protected from light Exposed to daylight
0.25 99.5 2.2
1 92.5 2.4 98.3 4.7 101.2 1.2 87.0 3.4
1.25 92.3 1.0
2 89.9 3.0 101.3 3.1 102.3 3.0 79.7 1.4
2.25 88.2 3.4
3 79.1 3.4 94.7 2.6 99.0 0.25 61.5 4.3
3.25 78.6 1.9
4 76.0 3.4 94.7 2.1 99.7 4.3 51.4 0.5
4.25 73.6 3.7
5 43.1 2.2
6 36.2 1.4
7 59.5 3.6 88.4 5.9 98.0 4.1 34.8 0.4
10 84.1 1.8 89.9 1.9
14 80.0 1.6 91.8 3.2
Data are expressed in percentage of the initial drug concentration.
100
110
80
90
50
60
70
30
40
P
e
r
c
e
n
t

r
e
c
o
v
e
r
y
10
20
0
14 12 10 8 6 4 2 0
Time (days)
Figure 3. Stability of temsirolimus (concentration, 100 mg/L) at ( ) 4
o
C in the refrigerator; ( ) 20
o
C protected from light; ( ) 20
o
C
with room light exposure; and ( ) 10
o
C with sunlight exposure.
Stabilit du temsirolimus (concentration 100 mg/L) ( ) 4
o
C dans le rfrigrateur ; ( ) 20
o
C protg de la lumire ; ( ) 20
o
C avec
exposition la lumire articielle ; et ( ) 10
o
C avec exposition la lumire solaire.
No change in visual appearance or clarity was observed
in the temsirolimus solution at any point of the study for all
study temperatures.
Experimental stressed-condition
From the LC-MS full-scan spectra, temsirolimus was evi-
denced by the deprotonated molecule, [M-H]-, at m/z
1028.6. Exposed to UV light (at a wavelength of 365 nm),
an exponential and rapid decrease in temsirolimus concen-
tration was observed; after 45 min, half of the drug was
lost. Results are presented in Fig. 4. We observed a strong
decrease in the peak area of temsirolimus, along with the
appearance of new peaks corresponding to degradation
products. The LC-MS analysis of this solution allowed iden-
tication of seven degradation products. These compounds
Temsirolimus stability in polypropylene containers 161
Figure 4. Stability of temsirolimus (concentration, 100 mg/L) at
20
o
C under ultraviolet (UV) light exposure (365 nm).
Stabilit du temsirolimus (concentration 100 mg/L) 20
o
C lors
dexposition articielle aux rayons ultraviolets (365 nm).
could be formed from seco-temsirolimus or temsirolimus.
Indeed, according to Cai et al. [3], seco-temsirolimus is
a nonspecic degradation product of temsirolimus formed
by hydrolysis of the macrocyclic lactone ring followed by
dehydration of C25/C26 (Fig. 1). Degradation products were
more polar than temsirolimus: retention time (rt), 11 min,
[M-H]
-
at m/z 1028.6. They were named accordingly: DP1
(rt, 4.2 min; m/z, 879.6), DP2 (rt, 5.1 min; m/z, 877.6), DP3
(rt, 5.3 min; m/z, 877.6), DP4 (rt, 6.3 min; m/z, 1156.6), DP5
(rt, 7.2 min; m/z, 897.5), DP6 (rt, 8.9 min; m/z, 1140.6) and
DP7 (rt, 9.3 min; m/z, 1140.6). DP2, DP4, DP5 and DP6 are
the most abundant; the peak area of each compound over
the peak area of temsirolimus (before UV light exposure)
ranged from 15 to 19%. For each component, mass-to-charge
value led to tentative structural assignments. Peaks DP1,
DP2, DP3 and DP5 could be produced through removal of
the pipecolic acid moiety (C1C9). Such a cleavage has
been reported previously for rapamycin [23]. Products elut-
ing with retention times of 6.3, 8.9 and 9.3 min (DP4, DP6
and DP7) showed the deprotonated molecule at m/z 1156.6,
1140.6 and 1140.6, respectively; they were likely formed
by addition of oxygen atoms. For the exact identication of
these unidentied degradation products, more experimental
work should be performed.
Conclusions
A stability-indicating HPLC assay method to determine
the chemical stability of temsirolimus injections has been
developed and validated, which will be appropriate for
temsirolimus stability studies. Light is the most important
factor inuencing stability of the drug; sunlight can have
a dramatic effect on the stability of diluted solutions in
polypropylene containers. The second factor that inuences
the rate of temsirolimus degradation is the temperature.
Ready-to-use temsirolimus infusion solutions could there-
fore be stored, protected from light, 4 days at 4
o
C and 3 days
at 20
o
C, variations in drug concentration were less than 5%
of the initial concentration. Within these intervals, the solu-
tions can be safely used, reducing wastage and unnecessary
expenses. The degradation rate under articial light is suf-
ciently low to authorize the absence of opaque infusion
sets. However, the exposition to sunlight must be absolutely
avoided.
Disclosure of interest
The authors declare that they have no conicts of interest
concerning this article.
Funding: This study was not sponsored.
References
[1] Sehgal SN, Molnar-Kimber K, Ocain TD, Weichman BM.
Rapamycin: a novel immunosuppressive macrolide. Med Res
Rev 1994;14:122.
[2] http://www.emea.europa.eu/docs/en GB/document library/EPAR -
Scientic Discussion/human/000799/WC500039915.pdf.
(Assessed 2010 September 27).
[3] Cai P, Tsao R, Ruppen ME. In vitro metabolic study of tem-
sirolimus: preparation, isolation, and identication of the
metabolites. Drug Metab Dispos 2007;35:155463.
[4] Schmelzle T, Hall MN. TOR, a central controller of cell growth.
Cell 2000;103:25362.
[5] Fingar DC, Richardson CJ, Tee AR, Cheatham L, Tsou C, Blenis
J. mTOR controls cell cycle progression through its cell growth
effectors S6K1 and 4E-BP1/eukaryotic translation factor 4E.
Mol Cell Biol 2004;24:20016.
[6] Hudson CC, Liu M, Chiang GG, Otterness DM, Loomis DC, Kaper
F, et al. Regulation of hypoxia-inducible factor 1alpha expres-
sion and function by the mammalian target of rapamycin. Mol
Cell Biol 2002;22:700414.
[7] Harding MW. Immunophilins, mTOR, and pharmacodynamic
strategies for a targeted cancer therapy. Clin Cancer Res
2003;9:28826.
[8] Rini B, Kar S, Kirkpatrick P. Temsirolimus. Nat Rev Drug Discov
2007;6:599600.
[9] Rubio-Viqueira B, Hidalgo M. Targeting mTOR for cancer treat-
ment. Curr Opin Investig Drugs 2006;7:50112.
[10] Yu K, Toral-Barza L, Discafani C, Zhang WG, Skotnicki J, Frost P,
et al. mTOR, a novel target in breast cancer: the effect of CCI-
779, an mTOR inhibitor, in preclinical models of breast cancer.
Endocr Relat Cancer 2001;8:24958.
[11] Thomas GV, Tran C, Mellinghoff IK, Welsbie DS, Chan E, Fueger
B, et al. Hypoxia-inducible factor determines sensitivity to
inhibitors of mTOR in kidney cancer. Nat Med 2006;12:1227.
[12] Del Bufalo D, Ciuffreda L, Trisciuoglio D, Desideri M, Cognetti
F, Zupi G, et al. Antiangiogenic potential of the Mam-
malian target of rapamycin inhibitor temsirolimus. Cancer Res
2006;66:554954.
[13] Chan F, Samlowski EE, Samlowski WE. Temsirolimus: a review
of its use in the treatment of advanced renal cell carcinoma.
Clin Med Ther 2009;1:16774.
[14] Raymond E, Alexandre J, Faivre S, Vera K, Materman E, Boni
J, et al. Safety and pharmacokinetics of escalated doses of
weekly intravenous infusion of CCI-779, a novel mTOR inhibitor,
in patients with cancer. J Clin Oncol 2004;22:233647.
[15] Atkins MB, Hidalgo M, Stadler WM, Logan TF, Dutcher JP, Hudes
GR, et al. Randomized phase II study of multiple dose lev-
els of CCI-779, a novel mammalian target of rapamycin kinase
inhibitor, in patients with advanced refractory renal cell carci-
noma. J Clin Oncol 2004;22:90918.
162 S. Poujol et al.
[16] Witzig TE, Geyer SM, Ghobrial I, Inwards DJ, Fonseca R, Kurtin
P, et al. Phase II trial of single-agent temsirolimus (CCI-779) for
relapsed mantle cell lymphoma. J Clin Oncol 2005;23:534756.
[17] Rizzieri DA, Feldman E, Dipersio JF, Gabrail N, Stock W, Strair
R, et al. A phase 2 clinical trial of deforolimus (AP23573, MK-
8669), a novel mammalian target of rapamycin inhibitor, in
patients with relapsed or refractory hematologic malignancies.
Clin Cancer Res 2008;14:275662.
[18] Gadducci A, Tana R, Cosio S, Fanucchi A, Genazzani AR. Molec-
ular target therapies in endometrial cancer: from the basic
research to the clinic. Gynecol Endocrinol 2008;24:23949.
[19] Yao JC, Phan AT, Chang DZ, Wolff RA, Hess K, Gupta S, et al. Ef-
cacy of RAD001 (everolimus) and octreotide LAR in advanced
low- to intermediate-grade neuroendocrine tumors: results of
a phase II study. J Clin Oncol 2008;26:43118.
[20] MacKenzie AR, von Mehren M. Mechanisms of mammalian target
of rapamycin inhibition in sarcoma: present and future. Expert
Rev Anticancer Ther 2007;7:114554.
[21] Chan S, Scheulen ME, Johnston S, Mross K, Cardoso F, Dit-
trich C, et al. Phase II study of temsirolimus (CCI-779), a novel
inhibitor of mTOR, in heavily pretreated patients with locally
advanced or metastatic breast cancer. J Clin Oncol 2005;23:
531422.
[22] Bardin C, Astier A, Vulto A, Sewell G, Vigneron J, Trittler R,
et al. Guidelines for the practical stability studies of anti-
cancer drugs: a European consensus conference. Ann Pharm
Fr 2011;69:22131.
[23] Ilichev YV, Alquier L, Maryanoff CA. Degradation of rapamycin
and its ring-opened isomer: role of base catalysis. ARKIVOC
2007;xii:11031.