Download Viability of micro-organisms in novel anticancer drug solutions

EJHP Science • Volume 13 • 2007 • Issue 2 • P. 27-32
© 2007 The European Journal of Hospital Pharmacy Science. All rights reserved 1781-7595 €20
www.ejhp.eu
Viability of micro-organisms in novel anticancer drug
solutions
Astrid Karstens, PhD, Irene Krämer, PhD
ABSTRACT
Study objectives: In determining expiry dates of aseptically prepared drugs, chemical, physical, toxicological and microbiological factors must be considered. This is especially true because many antineoplastic drugs introduced on to the market are
already known to lack antimicrobial activity. The purpose of this study was to evaluate the growth of four different micro-organisms in diluted solutions of novel chemical or biopharmaceutical drugs (i.e. monoclonal antibody drugs) with antineoplastic
activity.
Methods: Drug products were aseptically reconstituted according to the manufacturer’s recommendations and further
diluted if suitable for administration. Inoculations were performed by adding drug solution to bacterial (Staphyloccus aureus,
Enterococcus faecium, Pseudomonas aeruginosa) or fungal (Candida albicans) suspensions in order to produce concentrations
of approximately 104 micro-organisms per millilitre. Pure vehicle solutions inoculated in the same manner with microorganisms, served as positive controls. Inoculated solutions were stored at a raised temperature of 22°C. Samples of the test
solutions were withdrawn up to 120 hours after inoculation, transferred to suitable growth media, and colony-forming units
were then counted.
Results: Most of the drugs did not show any antimicrobial activity. Only one drug solution with significant antimicrobial activity was
identified, namely busulfan injection concentrate. Moderate antibacterial activity was seen in diluted solutions of busulfan. The
streptozocin test solutions exhibited moderate antibacterial activity and moderate antifungal activity. Species-specific
weak antibacterial activity was seen with pemetrexed test solutions against S. aureus strains. A nutritive value of sodium folinate
solution or antibody drug solutions was not detected.
The lack of antibacterial and antifungal properties should be considered when assigning extended expiry dates to ready-touse solutions of antineoplastic drugs. Monoclonal antibody drug solutions showed neither growth-retarding nor growthsupporting properties, and should be handled in the same manner as other antineoplastic drugs. Except for solutions containing
busulfan or streptozocin, end-product sterility testing can be performed without further dilution or inactivation.
Conclusion: Micro-organisms can survive in diluted solutions of antineoplastic drugs and proliferate when transferred to
proper growth media.
KEYWORDS
Mirco-organisms, antineoplastic drugs, monoclonal antibody drugs, microbiological stability
INTRODUCTION
The microbiological quality of preparations for parenteral
use is directly related to the equipment and methods used
during the preparation process. To ensure adequate microContact for correspondence: Irene Krämer
Department of Pharmacy
Johannes Gutenberg-University Hospital
1 Langenbeckstrasse
D-55101 Mainz, Germany
Tel: +49 6131177209
Fax: +49 6131175525
[email protected]
Received 30 October 2006; revised manuscript received: 13 February 2007;
accepted 13 February 2007
European Association of Hospital Pharmacists
biological stability of the preparations, proper environmental conditions for aseptic processing, correct aseptic
techniques and quality assurance programmes are necessary [1-4]. Quality assurance programmes for prepared
sterile products include environmental monitoring, personnel monitoring, process validation and end-product
validation.
Personnel involved with production are responsible for
ensuring that shelf-lives or expiry dates (USP <797>) are
properly assigned to the prepared products. In determining
the appropriate expiry dates, chemical, physical, toxicological and microbiological aspects must be considered [5].
Microbiological stability implies sterility or resistance to
microbial growth of the aseptically prepared solutions. The
lack of antimicrobial properties should also be considered
• Volume 13 • 2007/2 • www.ejhp.eu • 27
Viability of micro-organisms in novel anticancer drug solutions
Astrid Karstens and Irene Krämer
The European Journal of Hospital Pharmacy Science
Table 1: Characteristics of antineoplastic drug solutions inoculated with micro-organisms
Drug
Manufacturer
Alemtuzumab
Medac Schering
Bortezomib
Janssen-Cilag
Busulfan injection
Pierre Fabre
Pharma
Busulfan
Pierre Fabre
diluted infusion
Pharma
solution
Cetuximab
Merck
Etoposide
Bristol-Myers
phosphate
Squibb
Irinotecan
Aventis Pharma
Liposomal
Essex Pharma
doxorubicin
Sodium folinate
Medac
Pemetrexed
Lilly
Rituximab
Roche
Streptozocin
Trastuzumab
Upjohn
Roche
Lot
Vehicle
42016F
4EBS001
AQ1003
Concentration
(mg/mL)
0.9% NaCl solution
0.03
1.0
6.0
AQ1003
0.9% NaCl solution
0.5
19 h
12
CT16392064
4C84542
0.9% NaCl solution
2.0
0.09
28 d
28 d
Data on file, Merck
13
D4A879
0412213
0.9% NaCl solution
5% dextrose solution
0.35
0.15
28 d
7d
14
M40824AA
FF4S91S
B2018, B299,
B2019
39MCC
B1083
0.9% NaCl solution
0.9% NaCl solution
0.9% NaCl solution
1.5
9.0
1.0
72 h
28 d
48 h
15
16
0.9% NaCl solution
0.9% NaCl solution
3.0
0.4
96 h
24 h
17
18
when assigning extended expiry dates to ready-to-use antineoplastic drug solutions to be administered by infusion [6].
In previous papers [6-8], we have already described the lack
of antimicrobial activity with most of the 40 antineoplastic
drug solutions tested. Significant antibacterial activity was
discovered or confirmed within our specified experimental
conditions for solutions of carboplatin, cisplatin, dacarbazine,
5-fluorouracil, mitomycin, oxaliplatin and treosulfan.
Moderate antifungal activity was seen with amsacrine, carmustine, epirubicin, mitozantrone, pentostatin, and treosulfan
[6-8]. No direct link between chemical structure or pharmacological principles and antimicrobial activity was obvious
[8]. Moreover, no correlation exists between antifungal and
antibacterial activity, and antimicrobial activity proved to be
species-specific [8].
Over the past several years, novel antineoplastic drugs,
especially biopharmaceutical monoclonal antibodies, have
been introduced on to the market for the treatment of
cancer. Little is known about the antimicrobial activity of the
reconstituted and/or diluted solutions. The purpose of this
study was to evaluate the growth of four different microorganisms in diluted solutions of these novel drugs. As in
previous experiments [6-8], the micro-organisms selected
are commonly associated with nosocomial infections,
and the test conditions selected support growth of microorganisms in the drug solutions.
28 •
• Volume 13 • 2007/2 • www.ejhp.eu
Physicochemical
stability at 2-8°C
24 h
28 d
28 d
Reference
10
11
9
METHODS
The drugs were tested in two consecutive sessions (S. aureus
and P. aeruginosa in parallel; E. faecium and C. albicans in
parallel), as indicated in Tables 2-5.
Drugs were aseptically reconstituted according to the manufacturer’s recommendations and further diluted if appropriate.
Dilution solvent and container material were selected in order
to achieve maximal chemical and physical stability. Dilution was
aseptically performed by injecting a calculated aliquot of each
stock solution into a polypropylene bag containing 0.9% sodium
chloride solution (Freeflex, Fresenius Kabi, lot SK721202)
or 5% dextrose solution (Freeflex, Fresenius Kabi, lot SB7210)
to achieve the desired final concentrations. Cetuximab injection solution (2 mg/mL, Merck) was transferred to empty
ethylenvinyl acetate bags (Baxter), without further dilution. The
final concentrations of the diluted solutions were the lowest
concentrations ordered by our physicians and/or concentrations recommended by the manufacturers for administration. In
addition, 5 mL aliquots of busulfan injection concentrate were
prepared in polypropylene syringes (B Braun) [9]. The drug
products, their final concentrations and the in-house physicochemical stability data considered are shown in Table 1.
Physicochemical stability data were determined in our pharmacy department (busulfan stock solution, diluted (busulfan
infusion solutions (Busulfex, Busilvex); irinotecan (Campto)) or
were carefully evaluated from the literature.
European Association of Hospital Pharmacists
Viability of micro-organisms in novel anticancer drug solutions
Astrid Karstens and Irene Krämer
The European Journal of Hospital Pharmacy Science
Table 2: Viability of S. aureus in drug solutions and control solutions
Drug/control solution
S. aureus (CFU log/mL)
0 min
15 min 30 min
60 min
4.7
4.8
4.8
4.7
4.8
4.7
4.7
4.7
5% Dextrose solution
4.8
4.8
4.7
4.7
4.7
4.7
4.8
4.7
Alemtuzumab
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
Bortezomib*
4.7
4.7
4.7
4.7
Busulfan concentrate*
4.7
4.4
3.1
3.1
Busulfan diluted
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
Cetuximab
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
Etoposide phosphate
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
Irinotecan
4.8
4.7
4.7
4.7
4.7
4.7
4.8
4.7
Liposomal Doxorubicin
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
Sodium folinate
4.8
4.7
4.7
4.7
4.8
4.8
4.8
4.7
Pemetrexed
4.7
4.8
4.7
4.7
4.8
4.7
4.7
4.7
Rituximab
4.8
4.8
4.7
4.7
4.7
4.7
4.7
4.7
Streptozocin
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
Trastuzumab
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
CFU = colony-forming units, h = hour, min = minute, *one test solution
0.9% NaCl solution
2h
4.8
4.7
4.7
4.7
4.7
4.7
4.7
0
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
3h
4.7
4.7
4.7
4.7
4.7
4.7
4.7
0
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4h
4.7
4.7
4.7
4.7
4.7
4.7
4.7
0
4.6
4.6
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
24 h
4.7
4.7
4.7
4.7
4.7
4.7
4.3
0
0
0
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
0
0
4.7
4.7
48 h
4.6
4.6
4.6
4.6
4.6
4.7
4.7
0
0
0
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.6
4.6
4.7
4.7
0
0
4.6
4.6
120 h
2.4
2.3
1.8
1.6
2.8
2.8
1.9
0
0
0
2.7
2.7
1.3
1.6
2.6
2.5
2.2
2.3
2.6
2.5
0
0
2.8
2.6
0
0
2.6
2.4
Table 3: Viability of E. faecium in drug solutions and control solutions
Drug/control solution
0.9% NaCl solution
5% dextrose solution
Alemtuzumab
Busulfan diluted
Cetuximab
Etoposide phosphate
Irinotecan
Liposomal doxorubicin
E. faecium (CFU log/mL)
0 min
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
15 min
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
30 min
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
60 min
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
2h
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
3h
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
4h
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
24 h
5.4
5.4
5.4
5.4
5.4
5.4
2.0
1.9
5.4
5.4
5.4
5.4
5.4
5.4
48 h
5.4
5.4
5.4
5.4
5.4
5.4
0
0
5.4
5.4
5.4
5.4
5.4
5.4
120 h
5.4
5.4
5.3
5.4
5.4
5.4
0
0
5.4
5.4
5.4
5.4
5.4
5.3
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.1
5.1
5.4
5.4
5.4
5.4
5.4
5.4
2.3
2.5
5.4
5.4
5.4
5.4
5.4
5.4
0
0
5.4
5.4
5.4
5.4
5.4
5.4
0
0
5.4
5.4
5.4
5.4
5.4
5.4
0
0
5.4
5.4
Sodium folinate
5.4
5.4
5.4
5.4
Rituximab
5.4
5.4
5.4
5.4
Streptozocin
5.4
5.4
5.4
5.4
Trastuzumab
5.4
5.4
5.4
5.4
CFU = colony-forming units, h = hour, min = minute
European Association of Hospital Pharmacists
• Volume 13 • 2007/2 • www.ejhp.eu • 29
Viability of micro-organisms in novel anticancer drug solutions
Astrid Karstens and Irene Krämer
The European Journal of Hospital Pharmacy Science
The preparation of inocula of S. aureus (ATCC 6538), E.
faecium (ATCC 6057), P. aeruginosa (ATCC 15442) and
C. albicans (ATCC 10231) were carried out as previously
described [8]. In general, inoculations were performed by
Table 4: Viability of P. aeruginosa in drug solutions and control solutions
Drug/control solution
P. aeruginosa (CFU log/mL)
0 min
15 min
30 min
5.1
5.2
5.1
5.1
5.1
5.2
5% dextrose solution
5.1
5.1
5.1
5.2
5.2
5.1
Alemtuzumab
5.0
5.0
5.1
5.0
5.0
5.1
Busulfan diluted
5.0
5.0
5.2
5.1
5.1
5.2
Cetuximab
5.0
5.0
5.0
5.1
5.1
5.1
Etoposide phosphate
5.2
52
5.1
5.2
5.2
5.1
Irinotecan
5.2
5.2
5.1
5.2
5.2
5.1
Liposomal doxorubicin
5.2
5.2
5.1
5.2
5.2
5.1
Sodium folinate
5.2
5.2
5.1
5.2
5.2
5.1
Pemetrexed
5.2
5.2
5.2
5.2
5.2
5.2
Rituximab
5.2
5.2
5.2
5.3
5.2
5.2
Streptozocin
5.3
5.3
5.2
5.2
5.2
5.2
Trastuzumab
5.2
5.2
5.1
5.2
5.2
5.1
CFU = colony-forming units, h = hour, min = minute
0.9% NaCl solution
60 min
5.1
5.1
5.2
5.2
5.1
5.1
5.1
5.1
5.1
5.1
5.1
5.1
5.1
5.1
5.1
5.1
5.1
5.1
5.2
5.2
5.2
5.2
5.2
5.2
5.1
5.1
2h
5.2
5.1
5.1
5.1
5.1
5.1
5.0
5.0
5.1
5.1
5.1
5.1
5.1
5.1
5.1
5.1
5.1
5.1
5.1
5.1
5.1
5.1
4.6
4.6
5.1
5.1
3h
5.1
5.1
5.1
5.2
5.1
5.1
4.7
4.7
5.1
5.1
5.1
5.1
5.1
5.1
5.1
5.1
5.1
5.1
5.1
5.1
5.0
5.1
4.2
4.2
5.1
5.1
4h
5.1
5.1
5.1
5.2
5.1
5.0
4.3
4.3
5.1
5.1
5.1
5.1
5.2
5.1
5.1
5.1
5.1
5.1
5.1
5.1
5.0
5.1
4.2
4.2
5.1
5.1
24 h
5.1
5.1
5.1
5.1
5.0
5.1
1.6
0
5.1
5.1
5.1
5.1
5.1
5.2
5.1
5.1
5.1
5.1
5.1
5.1
5.0
5.1
0
0
5.0
5.1
48 h
4.8
4.8
4.8
4.8
4.4
4.8
0
0
4.8
4.8
4.8
4.8
4.8
4.8
4.9
4.8
4.8
4.8
4.8
4.8
4.8
4.8
0
0
4.8
4.8
120 h
4.9
4.9
4.3
4.3
4.9
4.8
0
0
4.9
4.9
4.5
4.5
4.7
4.6
5.0
5.0
4.8
4.9
4.9
4.9
4.9
4.9
0
0
4.9
4.9
Table 5: Viability of C. albicans in drug solutions and control solutions
Drug/control solution
0.9% NaCl solution
5% dextrose solution
Alemtuzumab
C. albicans (CFU log/mL)
0 min
5.2
5.2
5.2
5.2
5.2
5.2
15 min
5.2
5.2
5.2
5.2
5.2
5.2
Busulfan concentrate*
30 min
5.2
5.2
5.2
5.2
5.2
5.2
60 min
5.2
5.2
5.2
5.2
5.2
5.2
5.2
3.1
2.8
2.3
Busulfan diluted
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
Cetuximab
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
Etoposide phosphate
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
Irinotecan
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
Liposomal doxorubicin
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
Sodium folinate
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
Rituximab
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
Streptozocin
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
Trastuzumab
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
CFU = colony-forming units, h = hour, min = minute, * one test solution
30 •
• Volume 13 • 2007/2 • www.ejhp.eu
2h
5.2
5.2
5.2
5.2
5.2
5.2
3h
5.2
5.2
5.2
5.2
5.2
5.2
4h
5.2
5.2
5.2
5.2
5.2
5.2
24 h
5.2
5.2
5.2
5.2
5.2
5.2
48 h
5.2
5.2
5.2
5.2
5.2
5.2
120 h
4.4
4.3
4.3
4.2
5.1
5.1
0
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
0
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
0
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
0
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
0
0
5.2
5.2
0
5.2
5.2
5.2
5.2
5.1
5.1
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
0
0
5.2
5.2
0
4.8
4.8
4.7
4.8
2.8
2.6
4.8
4.8
5.2
5.2
4.3
4.5
4.7
4.7
0
0
5.1
5.0
European Association of Hospital Pharmacists
Viability of micro-organisms in novel anticancer drug solutions
Astrid Karstens and Irene Krämer
The European Journal of Hospital Pharmacy Science
adding 9 mL of a drug solution to 1 mL of bacteria or C. albicans suspension to produce concentrations of approximately
104 micro-organisms per millilitre. With the exception of
bortezomib and busulfan, duplicate test suspensions were
prepared per micro-organism for each drug, Because of
financial restrictions, only 3.5 mL of bortezomib reconstituted solution (directly withdrawn from the vial) and 5 mL of
busulfan injection concentrate were added to the microorganism suspensions. For the same reason, bortezomib
reconstituted solution, busulfan injection concentrate and
pemetrexed diluted solutions were added to selected microorganism suspensions only. Sodium chloride solution 0.9%
inoculated in the same manner with micro-organisms,
served as positive control. Dextrose solution 5% inoculated
with micro-organisms in the same way, served as positive
control for the liposomal doxorubicin test solution (Essex
Pharma). The storage of test solutions (22°C, light-protected), sampling (at 0, 15, 30 and 60 minutes and 2, 3, 4, 24,
48 and 120 hours after inoculation), and the quantitative
determination of colony-forming units (CFU) were performed as described in our previous publications [7, 8].
Samples were transferred to tryptic soy agar and incubated
at 36°C for 24 hours.
RESULTS
The viability of S. aureus, E. faecium, P. aeruginosa and C.
albicans in the drug solutions tested and in control solutions
are shown in Tables 2-5. Most of the drugs did not show any
antimicrobial activity. The selected micro-organism strains
remained viable up to 120 hours after inoculation in the control solutions and in the drug solutions of alemtuzumab,
bortezomib (S. aureus), cetuximab, etoposide phosphate,
irinotecan, liposomal doxorubicin, sodium folinate, rituximab
and trastuzumab (Tables 2-5). The decreasing viability of S.
aureus after 48 hours was also observed in our previous
experiments and is most likely to be caused by exhaustion of
nutritive substances. A nutritive value of sodium folinate or
the solutions containing monoclonal antibodies was not
detected. There was only one drug solution with significant
antimicrobial activity that was identified, namely busulfan
injection concentrate. The loss of viability of S. aureus and C.
albicans inoculated into busulfan injection concentrate was
seen two hours after inoculation. Because busulfan injection
concentrate (Busilvex, Busulfex) is a non-aqueous drug
solution containing the drug substance dissolved in N,Ndimethylacetamide (33% w/w) and polyethylene glycol 400
(67% w/w), this result was expected. The thesis of autosterility caused by the drug formulation is supported by weaker
antibacterial activity and missing antifungal activity of
diluted busulfan solutions (see Tables 2-5). The loss of
European Association of Hospital Pharmacists
viability of S. aureus, E. faecium and P. aeruginosa strains
occurred 24 or 48 hours after inoculation of the diluted
busulfan solutions.
The streptozocin test solutions exhibited moderate antibacterial activity against each of the bacteria species tested (Tables 24) and moderate antifungal activity against C. albicans (Table 5).
The loss of viability of micro-organisms occurred 24 hours after
inoculation. Because solutions for streptozocin infusion are
known to be physicochemically stable for more than 48 hours
when stored at room temperature [19], antimicrobial activity of
streptozocin solutions is attributed to the drug substance itself
and not to resulting degradation products.
Species-specific weak antibacterial activity was seen with
pemetrexed test solutions against S. aureus strains (Table 2).
However, no antibacterial activity was observed against P.
aeruginosa strains (Table 4).
DISCUSSION
The test conditions of the inoculation experiments were originally chosen to simulate the worst possible conditions for
patients and optimal circumstances for the growth of microorganisms [6]. Therefore, all test solutions were stored at
22°C, although according to their physicochemical instability, storage under refrigeration is required [6]. In all cases,
samples were withdrawn up to five days after inoculation,
regardless of the physicochemical stability of the drug solutions. Bortezomib, etoposide phosphate and irinotecan test
solutions, as well as busulfan injection concentrate, are
known to be physicochemically stable for at least five days at
room temperature. Shelf-lives of the other drug products
tested are shorter than five days when stored at room temperature. However, resulting degradation products did not
affect viability of the micro-organisms in our experiments.
Viability of micro-organisms in the drug solutions is also
encouraged by the low drug concentrations selected, which
probably fell below the minimal inhibitory concentration [8].
The viability pattern of micro-organisms in the control solutions reflects the known viability of the selected species in
nutrient-poor solutions over the test period.
For the first time, antimicrobial activity of ready-to-use solutions of
antibody drugs (alemtuzumab, cetuximab, rituximab, trastuzumab) was investigated. Because dilution is not recommended by
the manufacturer, cetuximab was inoculated without further dilution. The antibody drug solutions exhibited neither antibacterial
nor antifungal activity; they even lacked growth-supporting properties for the micro-organisms. Therefore, microbiological instability of biopharmaceutical drug solutions for the treatment of
cancer is comparable with the microbiological instability of
• Volume 13 • 2007/2 • www.ejhp.eu • 31
Viability of micro-organisms in novel anticancer drug solutions
Astrid Karstens and Irene Krämer
The European Journal of Hospital Pharmacy Science
chemical antineoplastic drug solutions. The same is true for
ready-to-use-solutions of calcium folinate, and, according to this
study, also for sodium folinate solutions.
None of the tested ready-to-use drug solutions exhibited
exceptional antimicrobial activity. Although the antimicrobial
activity of antineoplastic agents is for the most part unpredictable [8], moderate antimicrobial activity was expected and
recorded for streptozocin solutions. Streptozocin is a naturally
occurring nitrosourea. The drug is derived from Streptomyces
achromogenes and has broad antibacterial properties as well as
antitumour activity. Our test method proved to be suitable in
confirming the antibacterial activity of streptozocin. In addition,
antifungal activity against C.albicans was detected. This contrasts with the lack of antifungal activity observed with diluted
mitomycin-C drug solutions. Mitomycin-C also derives from a
streptomyces species and has alkylating and antibacterial activity [8].
Bortezomib, the first in class of proteasome inhibitors, showed
neither antimicrobial activity nor growth-supporting properties
against the S. aureus strains tested. As no further experiments
were performed, and antimicrobial activity is often species-specific and independent from the chemical structure or mechanism of action, no further conclusions can be drawn from this
result.
The strong antibacterial and antifungal activity of Busilvex injection concentrate is mostly caused by the formulation of the drug
with organic solvents only, and not by the drug substance itself.
Busulfan itself seems to have negligible antimicrobial activity
according to the results seen with the diluted busulfan solutions.
This suggestion is also supported by our test results observed
for solutions of amsacrine. Diluted solutions of amsacrine for
infusion also contain dimethylacetamide and exhibited weak
antibacterial activity, but moderate antifungal activity [8].
CONCLUSIONS
As was established in our earlier studies, micro-organisms can
survive in diluted solutions of antineoplastic drugs and proliferate when transferred to proper growth media. Solutions containing antineoplastic drug antibodies showed neither growthretarding nor growth-supporting properties, and are to be handled in the same manner as other antineoplastic drugs. In pharmacy departments, solutions for parenteral administration are to
be prepared under strict aseptic conditions and appropriate
quality assurance programmes are to be established in order to
guarantee the sterility of drug preparations. Except for busulfan
and streptozocin, end-product sterility testing can be performed without further dilution or inactivation. The lack of antibacterial and antifungal properties should be considered when
assigning extended expiry dates to ready-to-use solutions of
most antineoplastic drugs. Such reconstituted solutions should
be kept refrigerated whenever possible to inhibit the growth of
any contaminating organisms.
ACKNOWLEDGEMENT
The technical assistance of Sabine Teske-Keiser at the
Institute for Microbiology and Hygiene, Johannes
Gutenberg-University, Mainz is acknowledged.
REFERENCES
1. Bundesverband Deutscher Krankenhausapotheker (ADKA) e.V.
Aseptische Herstellung und Prüfung applikationsfertiger Parenteralia.
Krankenhauspharmazie 2003;6:195-201.
2. United States Pharmacopeial Convention: <797>. Pharmaceutical
compounding - sterile preparations. In: United States Pharmacopeia
(USP), 28rd rev./national formulary, 23th edition, Rockville MD, US,
2005. p2461-77.
3. NHS Quality Control Committee. The quality assurance of aseptic
preparation services (2nd ed). London: Department of Health; 1996.
4. Nederlandse Verenigung van Ziekenhuisapothekers: GMP
Ziekenhuisfarmacie, GL’s Gravenhage, 1996.
5. Krämer I. Stabilität applikationsfertiger Zytostatikazubereitungen. PZ
Prisma 1996; 3: 163-71.
6. Krämer I. Viability of micro-organisms in novel antineoplastic and
antiviral drug solutions. J Oncol Pharm Pract 1998; 4: 32-7.
7. Krämer I, Wenchel HM. Wachstumsverhalten ausgewählter
Mikroorganismen in Zytostatika-Zubereitungen. Krankenhauspharmazie 1988;11:439-42.
8. Krämer I, Wenchel HM. Viability of microorganisms in antineoplastic
drug solutions. Eur J Hosp Pharm 1991;1:14-9.
9. Karstens A, Krämer I. Stability of busulfan injection solution
(Busilvex, Busulfex) in B/Braun Injekt syringes. Pharmazie 2006;
61: 845-50.
10. Thiesen J, Krämer I. Physikalisch-chemische Stabilität, Kompatibilität
und Inkompatibilität parenteral applizierbarer Zytostatika, Virustatika
und Supportivtherapeutika, Stabil-Liste 2004.
32 •
• Volume 13 • 2007/2 • www.ejhp.eu
11. Friess D, Nguyen HC, Lipp HP. HPLC-Stabilitätsuntersuchungen zu
rekonstituierten Bortezomib-Lösungen. Krankenhauspharmazie
2005;6:206-10.
12. Karstens A, Krämer I. Chemical and physical stability of diluted busulfan infusion solutions. Eur J Hosp Pharm-Sci 2007;13:40-47.
13. Zhang Y, Trissel LA. Physical and chemical stability of etoposide
phosphate solutions. J Am Pharm Assoc 1999;39:146-50.
14. Thiesen J, Krämer I. Physicochemical stability of irinotecan injection
concentrate and diluted infusion solutions in PVC bags. J Oncol
Pharm Pract 2000; 6: 115-21.
15. Rondelot G, Serrurier C, Zenier H, Vigneron J, May I. Stability of
pemetrexed 25 mg/mL in glass vial and 5 mg/mL diluted in 0.9%
sodium chloride and stored in PVC containers. [Poster abstract] 11th
Congress of the European Association of Hospital Pharmacists
(EAHP), 22-24 March 2006; Geneva, Switzerland. E4, p.26, 2006.
16. American Hospital Formulary Service: American Society of HealthSystem Pharmacists. Rituximab. p.1181 In: AHFS Drug Information,
2006.
17. Trissel LA, Kleinmann LM, Davignon JP, Cradock JC. Investigational
Drug Information. Drug Intelligence & Clin Pharm 1987;12:404-5.
18. Electronic Medicines Compendium. Summary of product characteristics for trastuzumab. Roche Products Limited. www.emc.medicines.org.uk (accessed 19 February 2007).
19. Root T. Streptozocin. In: Allwood M, Wright P, editors. The cytotoxics handbook. 3rd ed. Oxford: Radcliffe Medical Press, 1990.
p382-3.
European Association of Hospital Pharmacists