Investigation of the Toxic Functional Group of ... - Wiley Online Library

Arch. Pharm. Chem. Life Sci. 2010, 10, 553–560

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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: [email protected] 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,

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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 www.archpharm.com


Tracing cephalosporin toxic group by zebrafish embryo test

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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.


Figure 1. Toxicity of cefazedone sodium and cefazolin sodium in zebrafish embryo test.

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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

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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.


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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), www.archpharm.com

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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 www.archpharm.com


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.



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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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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.

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