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Investigation of the Toxic Functional Group of Cephalosporins by Zebrafish Embryo Toxicity Test.

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Arch. Pharm. Chem. Life Sci. 2010, 10, 553–560
553
Article
Investigation of the Toxic Functional Group of
Cephalosporins by Zebrafish Embryo Toxicity Test
Jingpu Zhang2, Jie Meng2, Yaping Li1 and Changqin Hu1
1
2
National Institute for the Control of Pharmaceutical and Biological Products, Beijing, P.R. China
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Beijing, P.R. China
2-mercapto-5-methyl-1,3,4-thiadiazole (MMTD) is the 3’-side chain of cephalosporin including
cefazolin sodium (CFZL) and cefazedone (CFZD). It is not only present in finished products as the
residual precursor, but also produced through drug degradation. Performing the zebrafish embryo
toxicity test, we evaluated the toxicity effects of cefazolin sodium, cefazedone, their synthetic
precursors and intermediates. Our results suggest that the teratogenic effect of cefazedone and
cefazolin sodium on zebrafish embryonic development is associated with the structure of MMTD.
They mainly interfere with the development of tissues and organs derived from embryonic ectoderm
and mesoderm. We further consider the rationality of the quality control limit of MMTD (1.0%) in
the specification. As the acceptable daily intakes (ADIs) of cefazolin is 10 mg/kg per day [16] and the
minimum teratogenic concentration of MMTD is tenfold lower than that of cefazolin sodium,
we recommend that the acceptable daily intakes of MMTD should be 1 mg/(kg day). In general, the
therapeutic dose of cefazolin sodium is 2–4 g/day. Based upon the calculation of MMTD quality
control limits (1.0%), MMTD intake can be 20–40 mg/day, which will be much more than the
acceptable daily intake value of 1 mg/(kg day). Thus, MMTD should be recommended as a specified
impurity and qualified as serious again.
Keywords: Cephalosporins / Impurity / MMTD / Qualification / Zebrafish-embryo toxicity test
Received: January 5, 2010; Accepted: March 3, 2010
DOI 10.1002/ardp.201000005
Introduction
The unique structural and chemotherapeutic properties
exhibited by b-lactam antibiotics attract much attention of
medicinal chemists to design and study new therapeutic
agents with extended biological activity. Cephalosporin, a
potent antibacterial agent with a very low incidence of side
effects, has maintained its glamour during the previous
Correspondence: Changqin Hu, National Institute for the Control of
Pharmaceutical and Biological Products, No. 2, TianTan XiLi, Beijing
100050, P.R. China.
E-mail: hucq@nicpbp.org.cn
Fax: þ86-10 65115148.
Abbreviations: acceptable daily intakes (ADIs); 7-aminocephalosporanic
acid (7-ACA); cefazedone (CFZD); cefazolin sodium (CFZL); days post
fertilization (dpf); 2-mercapto-5-methyl-1,3,4-thiadiazole (MMTD); relative
retention times (RRT); tetrazole-1-acetic acid (TzAA).
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
50 years. As a result of extensive research, four generations
of cephalosporins are in clinical use. The principal strategy in
designing new drugs is the structural modification at C-3 and
C-7 to impart appropriate lipophilicity and basicity [1].
Cephalosporins with a methoxy [2], carbamoyloxy [3], or
heteroaryl ring such as tetrazole [4] or thiazole [5] in the
C-3 side chain have potent antibacterial activity. The structure-bioactivity relationships of cephalosporin side chains
have also been researched [1, 6–8]. However, fewer studies
involved the specified toxic functional groups of
cephalosporins.
Zebrafish are widely utilized as an excellent model organism for the study of development. Recently, they have
obtained more and more attention and emerged as an attractive vertebrate model for basic medical and pharmaceutical
research. This animal model offers unique advantages for
toxicological evaluation of drug [9–11]. Apart from acute
toxicity, subacute toxicity, chronic toxicity studies of drugs,
554
J. Zhang et al.
Arch. Pharm. Chem. Life Sci. 2010, 10, 553–560
S
H2 N
N
OAc
N
HS
O
N
CH3
S
COOH
MMTD
7-ACA
S
H2 N
S
S
N
O
N
COOH
Cl
N
CH 2 COOH
TDA
O
CH3
N
N CH 2 COOH
Cl
N N
N
TzAA
DPC
Cl
O
N
Cl
CH 2 CONH
O
S
S
N
COOH
S
N N
CH3 N
N CHC 2 ONH
N
N
CFZD
O
S
S
N
COOH
CFZL
zebrafish can be used to assess the toxicity of drug-targeted
organs and the related mechanisms [12, 13].
Cefazolin sodium (CFZL) and cefazedone (CFZD) are firstgeneration cephalosporins with a 3’-side chain of 2-mercapto5-methyl-1,3,4-thiadiazole (MMTD). In the synthesis of cefazolin and cefazedone, 7-aminocephalosporanic acid (7-ACA)
and MMTD are used as precursors to form TDA followed by
connecting with the C-7 side-chain (Scheme 1). Here, both
these compounds were selected as examples. With the aim of
tracing the specified toxic functional groups, the effects of
both cephasporins, their precursors, and their intermediate
products on zebrafish embryonic development were compared by the zebrafish embryo toxicity test.
Results
Comparison of toxic effects of cefazolin and
cefazedone
The influence of CFZD and CFZL on the zebrafish embryo
development is basically the same (Fig. 1a): the teratogenic
rates in the 10 and 50 mg/mL dose groups are less than 5%. In
the 100-mg/mL dose groups, the teratogenic rates of CFZD and
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
S
CH3
N N
Scheme 1. The synthetic route of cefazolin
sodium and cefazedone sodium.
CFZL are above 50% and 97.1%, respectively. The death rates
of embryos three days post fertilization (3-dpf) in 500-mg/mL
treated groups are lower than 9%, but all of the embryos
being 4-dpf to 5-dpf die. All of the 5-dpf to 7-dpf embryos in
the 300-mg/mL treated groups also die. The abnormal phenotypes of the treated groups (Figs. 1b and 1c) are mainly as
follows: i) embryonic surface colors and eye pigment change
from black spots to small shallow spots even non black; the
body surface, particularly head, becomes transparent yellow;
with the increase in drug concentration, the abnormal
phenomena are more severely displayed accompanied by
eye diminishment and a body bending; ii) the yolk sac and
the yolk sac extension structure (YE) changes from transparent to opaque brown; iii) heart defects: the heart has a cordlike shape, a swollen pericardial sac and shows wanness; iv)
the stress reaction is weakend until it disappears completely,
the swimming activity is poor, and part of the embryonic
brain shows hemorrhages; v) the body length shortens and
the anteroposterior axis werps. The notochord of individual
embryos is S-shaped or shows nodules.
According to the above observations, we speculate that
CFZD and CFZL mainly interfere with the development of
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Arch. Pharm. Chem. Life Sci. 2010, 10, 553–560
Tracing cephalosporin toxic group by zebrafish embryo test
555
120
Teratogenic rate
Fatality rate
Reaction rate (%)
100
80
60
40
20
0
WT
10
50
100
300
500
Cefazedone sodium
10
50
100
300
500 (µg/ml)
Cefazolin sodium
(a) Comparison of the toxicity results of cefazedone sodium and cefazolin sodium on zebrafish
embryonic development.The data are 3-dpf statistics. The teratogenic rates do not include the
fatality rate (the same as below). WT means wild type that stands for the untreated group as the
normal control in the experiments.
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Toxicity of cefazedone sodium and
cefazolin sodium in zebrafish embryo test.
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J. Zhang et al.
Arch. Pharm. Chem. Life Sci. 2010, 10, 553–560
tissues and organs derived from the embryonic ectoderm e. g.,
nervous system and skin pigment, and from the mesoderm
such as the skeleton axis and the cardio-cerebral vascular
system.
Comparison of toxic effects of the precursors and
intermediate products
According to Scheme 1, the toxic effects of TzAA, DPA, 7-ACA,
MMTD, and TDA on the embryonic development of zebrafish
are compared by tracing the specified functional group of
each of the cephalosporins.
TDA is the intermediate product of CFZL and CFZD. The
death rate of 3-dpf embryos in a 500-mg/mL (TDA)-treated
group is more than 40% (Fig. 2), which is larger than for
CFZL and CFZD (Fig. 1a). The deformity rates of the 100- and
300-mg/mL treated groups reach more than 96% (Fig. 2),
which are similar to those of CFZL and CFZD (Fig. 1a). The
10-mg/mL treated group has no obvious embryonic deformities. With the increase in the concentration of drug, the
teratogenic phenotypes show short embryos, opaque yolk
and shorted yolk extension, ventral bending tails, S-shaped
distorted notochord, neural tube and yellow surface. In some
of the embryos, the ventral veins near to the embryonic
pericardial sac, eye periphery, and the ventral side of tail
shaft have congestion symptoms similar to those of the CFZL
and CFZD groups. The results suggest that TDA has toxic
effects on zebrafish embryonic development similar to those
of CFZL and CFZD.
MMTD also has obvious toxic effects on zebrafish embryo
development (Fig. 2). The deformity rate of 10-mg/mL treated
groups reaches 98%. The teratogenic phenotype is similar to
CFZL and CFZD as follows: transparent and yellow surface, no
melanin spots, colorless eye, opaque embryonic yolk sac and
extension structure, swollen pericardial sac, no redness of
the heart (wan heart), slow heart rate, short body length,
bending anteroposterior axle, no stress response and low
swimming activity. However, there is a difference from both
formerly described groups: all of these embryos treated with
a MMTD dose over 300 mg/mL of have severely S-shaped and
distorted notochords, resulting in larval body axis distortion
and losing the ability to swim. The embryo development
stagnates at 2-dpf and all larvae die in the first four days
post-fertilization (4-dpf) (Fig. 3). The teratogenic rates and
mortality rates of TzAA, DPA, and 7-ACA in all experimental
groups are no more than 10%, preliminarily suggesting that
they have no apparent toxic effects on zebrafish embryonic
development (Fig. 2).
The above experiments suggest that the teratogenic effect
of CFZD and CFZL on zebrafish embryonic development is
strongly associated with the structure of MMTD.
Toxic effects of a forced-degradation solution of
cefazedone
b-Lactams are intrinsically unstable in water because of their
high susceptibility to hydrolysis through both acid- and basemediated catalysis. The attack of the hydroxyl group on the
ester function bonded to the 30 -carbon is the fastest step in an
alkaline medium [15], and is responsible for groups leaving
C(30 ). For further evaluations of the toxic effects of MMTD in
the cephalosporins, the various impurities in the CFZD degradation solution were employed directly in the zebrafish
embryo toxicity test. Comparing these solutions with the
120
Teratogenic rate
Reaction rate (%)
100
Mortality rate
80
60
40
20
0
WT
1
5
10 100 300 500 10 100 300 500 10
MMTD
50 100 300 500 10 100 300 500 10
TDA
7-ACA
DPA
50 100 300 500 (µg/ml)
TAA
All of the data are 3-dpf statistics, in which the teratogenic rate does not include the. mortality rate. WT means wild
type that stands for the untreated group as the normal control in the experiments.
Figure 2. Comparisons of the toxicity for the embryo of MMTD, TDA, 7-ACA, DPA, and TzAA in zebrafish.
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2010, 10, 553–560
Tracing cephalosporin toxic group by zebrafish embryo test
557
Figure 3. Effects of MMTD and TDA on the
zebrafish embryonic development (3-dpf).
pre-degradation solution, we find that the zebrafish embryo
toxicity of various degradation solutions is lower than that of
CFZD (Fig. 4). No teratogenic phenotypes different from CFZD
were found.
The degradation solutions were analyzed by HPLC (Fig. 5).
The CFZD content in the forced acid degradation solution is
about 90% of that of pre-degradation; there are two
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
degradation impurities in the solution. The relative retention
times (RRT) are about 0.25 for impurity 1, 0.33 for impurity 2,
and 1.0 for CFZD; when calculating as CFZD, the contents of
the impurities are 0.29% and 0.42%, respectively. The CFZD
content in the forced alkaline degradation solution is about
70% of pre-degradation; there are five degradation impurities
in the solution. The RRTs are about 0.19, 0.24, 0.75 (MMTD),
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Arch. Pharm. Chem. Life Sci. 2010, 10, 553–560
Reaction rate (%)
120
Teratogenic rate
Mortality rate
100
80
60
40
20
0
WT 10 50 100 300 500 10 50 100 300 500 10 50 100 300 500 10 50 100 300 500 (µg/ml)
Cefazedone sodium
Alkaline degradation Acid degradation
Oxidative degradation
WT means wild type that stands for untreated group as the normal control in the experiments.
Figure 4. Comparison of zebrafish embryo toxicity of cefazedone alkaline degradation solution, acid degradation solution, and oxidative
degradation solution to that of cefazedone sodium.
Figure 5. Typical HPLC chromatograms of forced (a) oxidation, (b) acid, and (c) alkaline degradation solutions of cefazedone sodium.
0.913 and 1.172; the contents calculated as CFZD are 0.86%,
9.27%, 4.15% (MMTD), 1.01% and 10.07%, respectively. The
CFZD content in the forced oxidation degradation solution is
about 80% of pre-degradation; there are two degradation
impurities in the solutions; the RRTs are 0.572 and 0.672;
the contents calculated as CFZD are 1.75% and 6.27%,
respectively.
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
We further analyzed the data in Fig. 4 in combination with
the HPLC results. The toxicity of CFZD forced oxidative degradation solution is significantly reduced compared with predegradation, suggesting that the toxic effects of the oxidative
degradation impurities can be ignored in the normal products. The toxic effects of CFZD forced acid degradation
solution mainly reflect the toxic effects of CFZD because only
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Arch. Pharm. Chem. Life Sci. 2010, 10, 553–560
approximately 10% CFZD is degraded in the degradation
process and the resulting impurity content is low. The toxic
effects of CFZD forced alkaline degradation solution are
comparable to the pre-degradation solution. Because CFZD
degrades about 30%, the results demonstrate that some toxic
impurities may exist in the degradation solution. HPLC
analysis shows that there is approx. 4% of the MMTD in
the degradation solution. The teratogenic effect of MMTD
is tenfold higher than that of CFZD. Therefore, MMTD should
be the major toxic degradation impurity in the alkaline
degradation solution.
Our results suggest that the toxic effects generated by
the degradation impurities are not significantly greater
than those of cefazedone per se, and in the various
degradation impurities of CFZD, MMTD is a major toxic
impurity.
Discussion
At present, CFZL is widely used in clinical treatment and
adopted by United States Pharmacopoeia 32th (USP32),
British Parmacopoeia 2010 (BP2010), and European
Pharmacopoeia edition 6.0 (EP6.0). Our study shows that
MMTD is not only the specified toxic functional group in
CFZL, but also a major toxic impurity. The teratogenic effect
of CFZL in zebrafish embryonic development is mainly
related to the structure of MMTD. The minimum teratogenic
concentration of MMTD is tenfold lower than that of CFZL. In
BP2010, EP6.0, and USP32, the control limits of MMTD in
CFZL are 1.0%, meaning that the toxic effect of MMTD in CFZL
can be equivalent to about 10% of the toxic effect of CFZL
according to the limit of 1.0%. Recently, Cunningham et al.
[16] evaluated the hazards to human health of a wide range of
chemical substances in water in the environment and determined that the acceptable children’s daily intake (acceptable
daily intakes, ADIs) of cefazolin is 10 mg/(kg day). Therefore,
we consider that the ADIs of MMTD should be 1 mg/(kg day).
In general, the therapeutic dose of CFZL is 2–4 g/day. Based
upon the calculation of MMTD quality control limits (1.0%),
MMTD intake should be 20–40 mg/day, which will be much
greater than the ADIs value of 1 mg/(kg day). Thus, MMTD
should be recommended as a specified impurity and qualified
as serious.
MMTD, a main impurity in cephalosporins with the 30 -side
chain of 2-mercapto-5-methyl-1,3,4-thiadiazole, exists in the
compound in form of a precursor and can be produced in
the process of degradation. Our results show that MMTD is
not only the specified toxic functional group, but also a
major toxic impurity. The hazards of MMTD in cephalosporins should be paid great attention.
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Tracing cephalosporin toxic group by zebrafish embryo test
559
Experimental
Samples
CFZL and its C-7 side-chain tetrazole-1-acetic acid (TzAA) were
from Shenzhen Jiuxin Pharmaceutical Co., Ltd. China. CFZD, its
C-7 side-chain 3,5-dichloro-4-pyridone-N-acetic acid (DPA) and the
synthetic intermediate 7-amino-3-[[(5-methyl-1,3,4-thiadiazole-2yl)thio]methyl]-8-oxo-5-sulfur mixed-1-nitrogen miscellaneous
bicyclo[4,2,0]sim-2-ene-2-carboxylic acid (TDA) were from
Xinfeng Pharmaceutical Co., Ltd., South Korea. 7-ACA reference
substance and MMTD reference substance were from National
Institute for the Control of Pharmaceutical and Biological
Products (NICPBP).
Solution preparation
Test solution
All test samples were dissolved in water and diluted to 10 mg/mL
stock solutions, and, if necessary, an appropriate amount of
Na2CO3 solution was added to help to dissolve. Subsequently,
the solutions were diluted with artificial seawater (‘‘Instant
Ocean’’ sea salts, Tropical marine, Tianjin Casic Marine
Biotechnology Co., Ltd, China) into 10, 50, 100, 300, and
500 mg/mL test solutions.
Forced-degradation solution
6 mL sodium hydroxide solution (0.1 mol/L) were added to
60 mL CFZD stock solution and the mixture was placed in a
container for 2 h. The solution was adjusted to neutral pH with
0.1 mol/L hydrochloric acid solution and then diluted with water
to a concentration of 9 mg/mL, which was used as the alkaline
degradation solution.
Then, 6 mL HCl solution (0.1 mol/L) were added in 60 mL CFZD
stock solution and the mixture was placed in a container for 2 h.
The solution was adjusted to neutral pH with 0.1 mol/L hydrochloric acid solution and then diluted with water to 9 mg/mL.
This solution was used as the acid degradation solution.
The oxidative degradation solution was prepared by mixing
six drops of 30% hydrogen peroxide solution into 60 mL CFZD
stock solution for 2 h followed by dilution with water to
9 mg/mL. In the zebrafish embryo toxicity experiments,
the various forced-degradation solutions were diluted with
artificial seawater to concentrations as needed.
Zebrafish embryo toxicity test
Zebrafish (Danio rerio Tuebingen) were fed in the Institute of
Medicinal Biotechnology, Chinese Academy of Medical
Sciences, Beijing Union Medical College. 30 embryos of 50%
epiboly stage (mid-gastrula) were immersed in 3–4 mL of the
test solutions in a dish with a diameter of 20 mm till three days
post-fertilization (3-dpf) for each group. Three or four doses of
administered groups were set for each compound. The zebrafishfeeding liquid – artificial seawater for bathing the 30 embryos
was used as normal control. The embryos were incubated in a
standard environment [14] and the development of embryos was
observed daily. The abnormal embryonic development rates,
mortality rates, and survival number of embryos were statistically analyzed from the third day on. Each experiment was
repeated three times.
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J. Zhang et al.
Arch. Pharm. Chem. Life Sci. 2010, 10, 553–560
Table 1. Gradient-elution program used in HPLC analysis.
Time
Mobile phase A
Mobile phase B
(min)
(%)
(%)
0
3
33
38
43
55
90
90
60
60
90
90
10
10
40
40
10
10
HPLC analysis of forced-degradation solution
UltiMate 3000 LCi Series HPLC System (Dionex Co., US) and
Capcell PAK C18 MG II chromatographic column (4.6 mm
(I. D.) 250 mm, 5 mm) were employed. The detection wavelength was set at 278 nm; the injection amount was 10 mL.
The mobile phase A was 0.02 mol/L ammonium dihydrogen
phosphate buffer solution, the pH value was adjusted to 5.0 with
0.1 mol/L sodium hydroxide solution. Mobile phase B was acetonitrile; the run was subjected to a linear-gradient elution
(Table 1) with a flow rate of 1.0 mL/min.
This work is supported by The National Natural Science Foundation of
China (N0. 30772681).
The authors have declared no conflict of interest.
References
[1] G. S. Singh, Mini Rev. Med. Chem. 2004, 4, 93–109.
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[2] K. Fujimoto, S. Ishihara, H. Yanagisawa, J. Ide, et al.,
J. Antibiotics 1987, 40, 370–384.
[3] S. Negi, M. Yamanaka, I. Sugiyama, Y. Komatsu, et al.,
J. Antibiotics 1994, 47, 1507–1525.
[4] H. Sadaki, T. Imazumi, T. Inaba, T. Hirakawa, et al., Yakugaku
Zasshi 1986, 106, 129–146.
[5] K. Sakagami, K. Atsumi, A. Tamura, T. Yoshida, et al.,
J. Antibiotics 1990, 43, 1047–1050.
[6] E. Caselli, R. A. Powers, L. C. Blasczcak, C. Y. E. Wu, et al.,
Chem. Biol. 2001, 8, 17–31.
[7] J. R. Hwu, K. S. Ethiraj, G. H. Hakimelahi, Mini Rev. Med. Chem.
2003, 3, 305–313.
[8] R. D. Beger, Drug Discov. Today 2006, 11, 429–435.
[9] T. P. Barros, W. K. Alderton, H. M. Reynolds, A. G. Roach,
S. Berghmans, Br. J. Pharmacol. 2008, 154, 1400–1413.
[10] A. L. Rubinstein, Expert Opin. Drug Metab. Toxicol. 2006, 2, 231–
240.
[11] A. C. MacRae, R. T. Peterson, Chem. Biol. 2003, 10, 901–
908.
[12] H. C. Ou, D. W. Raible, E. W. Rubel, Hearing Res. 2007, 233,
46–53.
[13] D. M. Hentschel, K. M. Park, L. Cilenti, A. S. Zervos, et al., Am. J.
Physiol. Renal Physiol. 2005, 288, F923–F929.
[14] M. Westerfield (Ed.), The Zebrafish Book. A guide for the laboratory
use of zebrafish (Danio rerio), 4th ed., Univ. of Oregon Press,
Eugene, OR, USA 2000. http://zfin.org/zf_info/zfbook/
zfbk.html
[15] B. Vilanova, F. Munoz, J. Donoso, J. Frau, F. G. Blanco,
J. Pharm. Sci. 1994, 83, 322–327.
[16] V. L. Cunningham, S. P. Binks, M. J. Olson, Regul. Toxicol.
Pharmacol. 2009, 53, 39–45.
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