The comparative analysis of gastrointestinal toxicity of azithromycin and 3'-decladinosyl azithromycin on zebrafish larvae.

The most commonly reported side effect of azithromycin is gastrointestinal (GI) disorders, and the main acid degradation product is 3'-Decladinosyl azithromycin (impurity J). We aimed to compare the GI toxicity of azithromycin and impurity J on zebrafish larvae and investigate the mechanism causing the differential GI toxicity. Results of our study showed that the GI toxicity induced by impurity J was higher than that of azithromycin in zebrafish larvae, and the effects of impurity J on transcription in the digestive system of zebrafish larvae were significantly stronger than those of azithromycin. Additionally, impurity J exerts stronger cytotoxic effects on GES-1 cells than azithromycin. Simultaneously, impurity J significantly increased ghsrb levels in the zebrafish intestinal tract and ghsr levels in human GES-1 cells compared to azithromycin, and ghsr overexpression significantly reduced cell viability, indicating that GI toxicity induced by azithromycin and impurity J may be correlated with ghsr overexpression induced by the two compounds. Meanwhile, molecular docking analysis showed that the highest -CDOCKER interaction energy scores with the zebrafish GHSRb or human GHSR protein might reflect the effect of azithromycin and impurity J on the expression of zebrafish ghsrb or human ghsr. Thus, our results suggest that impurity J has higher GI toxicity than azithromycin due to its greater ability to elevate ghsrb expression in zebrafish intestinal tract.

A Rapid Assessment Model for Liver Toxicity of Macrolides and an Integrative Evaluation for Azithromycin Impurities.

Impurities in pharmaceuticals of potentially hazardous materials may cause drug safety problems. Macrolide antibiotic preparations include active pharmaceutical ingredients (APIs) and different types of impurities with similar structures, and the amount of these impurities is usually very low and difficult to be separated for toxicity evaluation. Our previous study indicated that hepatotoxicity induced by macrolides was correlated with c-fos overexpression. Here, we report an assessment of macrolide-related liver toxicity by ADMET prediction, molecular docking, structure-toxicity relationship, and experimental verification via detection of the c-fos gene expression in liver cells. The results showed that a rapid assessment model for the prediction of hepatotoxicity of macrolide antibiotics could be established by calculation of the -CDOCKER interaction energy score with the FosB/JunD bZIP domain and then confirmed by the detection of the c-fos gene expression in L02 cells. Telithromycin, a positive compound of liver toxicity, was used to verify the correctness of the model through comparative analysis of liver toxicity in zebrafish and cytotoxicity in L02 cells exposed to telithromycin and azithromycin. The prediction interval (48.1∼53.1) for quantitative hepatotoxicity in the model was calculated from the docking scores of seven macrolide antibiotics commonly used in clinics. We performed the prediction interval to virtual screening of azithromycin impurities with high hepatotoxicity and then experimentally confirmed by liver toxicity in zebrafish and c-fos gene expression. Simultaneously, we found the hepatotoxicity of azithromycin impurities may be related to the charge of nitrogen (N) atoms on the side chain group at the C5 position via structure-toxicity relationship of azithromycin impurities with different structures. This study provides a theoretical basis for improvement of the quality of macrolide antibiotics.

Liver toxicity of macrolide antibiotics in zebrafish.

Macrolide antibiotics (macrolides) are among the most commonly prescribed antibiotics worldwide and are used for a wide range of infections, but macrolides also expose people to the risk of adverse events include hepatotoxicity. Here, we report the liver toxicity of macrolides with different structures in zebrafish. The absorption, distribution, metabolism, excretion and toxicology (ADMET) parameters of macrolide compounds were predicted and contrasted by utilizing in silico analysis. Fluorescence imaging and Oil Red O stain assays showed all the tested macrolide drugs induced liver degeneration, changed liver size and liver steatosis in larval zebrafish. Through RNA-seq analysis, we found seven co-regulated differentially expressed genes (co-DEGs) associated with metabolism, apoptosis and immune system biological processes, and two co-regulated significant pathways including amino sugar and nucleotide sugar metabolism and apoptosis signaling pathway. We found that only fosab of seven co-DEGs was in the two co-regulated significant pathways. fosab encoded proto-oncogene c-Fos, which was closely associated with liver diseases. The whole-mount in situ hybridization showed high transcription of c-Fos induced by macrolide compounds mainly in the liver region of zebrafish larvae. Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) leakage assays revealed that macrolides exerts significant cytotoxic effects on L02 cells. qRT-PCR and western blot analysis demonstrated macrolides also promoted human c-Fos expression in L02 cells. The c-Fos overexpression significantly reduced cell viability by using CCK-8 assay. These data indicate that hepatotoxicity induced by macrolides may be correlated with c-Fos expression activated by these compounds. This study may provide a biomarker for the further investigations on the mechanism of hepatotoxicity induced by macrolide drugs with different structures, and extend our understanding for improving rational clinical application of macrolides.

Study on Isomeric Impurities in Cefotiam Hydrochloride.

In this study, two isomeric impurities were identified in cefotiam hydrochloride injection preparation and were characterized. Column-switching HPLC-MS and NMR techniques were used to identify the impurity 1 as the Δ3(4) isomers of cefotiam. Using software-based calculations, it was predicted that neither of the isomeric impurities was embryotoxic. This study provides a reference for the production, storage, and quality control of cefotiam and related cephalosporin antibiotics.

In silico ADME and Toxicity Prediction of Ceftazidime and Its Impurities.

To improve the quality control of drugs, we predicted the absorption, distribution, metabolism, excretion, and toxicity (ADMET) of ceftazidime (CAZ) and its impurities via in silico methods. We used three types of quantitative structure-activity relationship and docking software for precise prediction: Discovery Studio 4.0, OECD QSAR Toolbox 4.1, Toxtree, and the pkCSM approach. The pharmacokinetics and toxicity of ceftazidime and impurity A (Δ-2-CAZ) are similar. The biological properties of impurity B (CAZ E-isomer) are different from CAZ. Therefore, we focused on drug stability to analyze impurity B. Impurities D and I have strong lipophilicity, good intestinal absorption, and poor excretion in the body. Impurity D is particularly neurotoxic and genotoxic. It is important to control the content of impurity D. The toxicity of impurity F is low, but the toxicity is enhanced when it becomes the C-3 side chain of CAZ and forms a quaternary amine group. We conclude that the beta-lactam ring of nucleus, the quaternary amine group at the C-3 side chain, and the acetates at the C-7 side chain of CAZ are the main toxic functional groups. Impurities B and D may be the genetic impurity in CAZ and may also have neurotoxicity. This in silico approach can predict the toxicity of other cephalosporins and impurities.

Research on the relationship between cephalosporin structure, solution clarity, and rubber closure compatibility using volatile components profile of butyl rubber closures.

OBJECTIVE: Establish an effective experimental strategy to determine the compatibility of rubber closures for drugs. SIGNIFICANCE: Various types of rubber closures with different compositions are available for drug packaging. Many additives of rubber closures can be released from rubber closures and may affect the quality of drugs and pose a risk to human health. In this study, we aimed to determine the relationship between cephalosporin structure, solution clarity, and rubber closure compatibility using volatile components profile of butyl rubber closures. METHODS: Two opposite polarity gas chromatography (GC) systems and GC-mass spectrometry (MS) were used to achieve rapid qualitative determination of the main volatile components in rubber closures. Simulated adsorption experiment was performed to investigate the adsorption of main volatile components in rubber closures by cephalosporins with different side chain structures, and to determine the effects of adsorption on solution clarity. RESULTS: A volatile components screening library of rubber closures was established and the structures of some volatile component were confirmed. The specific adsorption of the structure of cephalosporins on volatile components from rubber closures was studied. CONCLUSION: Based on the results of this study, rubber closures with good compatibility for cephalosporins with different side chain structures can be selected rapidly. This experimental strategy not only facilitates the screening of suitable rubber closures more effectively, but also enables the quick determination of volatile components adsorbed by drugs.

Microarray Expression Profiling and Raman Spectroscopy Reveal Anti-Fatty Liver Action of Berberine in a Diet-Induced Larval Zebrafish Model.

Background: The prevalence of non-alcohol fatty liver disease (NAFLD) is increasing in children and adolescents who are mostly resulted from overfeeding. Previous studies demonstrate that berberine (BBR), a compound derived from plant, has beneficial effects on NAFLD in adults but poorly understood in the pediatric population. This study employed a larval zebrafish model to mimic the therapeutic effects of BBR in the pediatric population and the mechanisms underlying its hepatoprotection. Methods: High-cholesterol diet (HCD)-fed zebrafish exposed to BBR at doses of 0, 1, 5, and 25 µM. After the larvae were treated with BBR for 10 days, its effect on hepatic steatosis was evaluated. We introduced Raman imaging and three-dimensional (3D) molecular imaging to detect changes in the biochemical composition and reactive oxygen species (ROS) levels of zebrafish liver. Gene expression microarray was performed to identify differentially expressed genes (DEGs) followed by gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and functional category analysis. Results: BBR (5 and 25 µM) administration prevented HCD-induced liver lipid accumulation in larval zebrafish. The result was further confirmed by the pathological observation. Raman mapping indicated that the biochemical composition in the liver of BBR-treated group shifted to the control. The quantitative analysis of 3D imaging showed that the ROS level was significantly decreased in the liver of BBR-treated larvae. In the livers of the BBR group, we found 468 DEGs, including 172 genes with upregulated expression and 296 genes with downregulated expression. Besides, GO enrichment, KEGG pathway, and functional category analysis showed that various processes related to glucolipid metabolism, immune response, DNA damage and repair, and iron were significantly enriched with DEGs. The expression levels of the crucial genes from the functional analysis were also confirmed by quantitative PCR (qPCR). Conclusion: BBR can significantly improve hepatic steatosis in HCD-fed zebrafish larvae. Its mechanisms might be associated with the regulation of lipid metabolism, oxidative stress, and iron homeostasis. Raman imaging in larval zebrafish might become a useful tool for drug evaluation. Mainly, the gene expression profiles provide molecular information for BBR on the prevention and treatment of pediatric NAFLD.

Characterization of Solid-State Drug Polymorphs and Real-Time Evaluation of Crystallization Process Consistency by Near-Infrared Spectroscopy.

Herein, we aimed to develop a strategy for evaluating the consistency of pharmaceutically important crystallization processes in real time, focusing on two typical cases of polymorphism. Theoretical analysis using a combination of 13C solid-state nuclear magnetic resonance spectroscopy with other polymorphism analysis techniques identified a number of marker signals, the changes of which revealed the presence of two or more structural orientations (lattices and/or molecular conformations) in both cefazolin sodium pentahydrate (α-CEZ-Na) and cephathiamidine (CETD). The proportions of these forms were shown to be batch-dependent and were defined as critical quality attributes (CQAs) to evaluate process consistency. Subsequently, real-time analysis by chemometrics-assisted near-infrared spectroscopy (NIR) was used to obtain useful information corresponding to CQAs. The pretreated spectra of representative samples were transformed by first derivative and vector normalization methods and used to calculate standard deviations at each wavelength and thus detect significant differences. As a result, vibrational responses of H2O, CH3, and CH2 moieties (at 5,280, 4,431, and 4,339 cm-1, respectively) were shown to be sensitive to the CQAs of α-CEZ-Na, which allowed us to establish a highly accurate discrimination model. Moreover, signals of H2O, CONH, and COOH moieties (at 5,211, 5,284, and 5,369 cm-1, respectively) played the same role in the case of CETD, as confirmed by theoretical results. Thus, we established a technique for the rapid evaluation of crystallization process consistency and deepened our understanding of crystallization behavior by using NIR in combination with polymorphism analysis techniques.

Compilation of a Near-Infrared Library for Construction of Quantitative Models of Oral Dosage Forms for Amoxicillin and Potassium Clavulanate.

The accuracy of quantitative models for near-infrared (NIR) spectroscopy is dependent upon calibration samples with concentration variations. Conventional sample-collection methods have shortcomings (especially time-consumption), which creates a "bottleneck" in the application of NIR models for Process Analytical Technology (PAT) control. We undertook a study to solve the problem of sample collection for construction of NIR quantitative models. Amoxicillin and potassium clavulanate oral dosage forms (ODFs) were used as examples. The aim of this study was to find an approach to construct NIR quantitative models rapidly using a NIR spectral library based on the idea of a universal model. The NIR spectral library of amoxicillin and potassium clavulanate ODFs was defined and comprised the spectra of 377 batches of samples produced by 26 domestic pharmaceutical companies, including tablets, dispersible tablets, chewable tablets, oral suspensions, and granules. The correlation coefficient (rT) was used to indicate the similarities of the spectra. The calibration sets of samples were selected from a spectral library according to the median rT of the samples to be analyzed. The rT of the samples selected was close to the median rT. The difference in rT of these samples was 1.0-1.5%. We concluded that sample selection was not a problem when constructing NIR quantitative models using a spectral library compared with conventional methods of determining universal models. Sample spectra with a suitable concentration range in NIR models were collected rapidly. In addition, the models constructed through this method were targeted readily.

Isolation, identification and in silico toxicity predictions of two isomers from cefotiam hydrochloride.

Two structural isomers of cefotiam in cefotiam hydrochloride for injection were observed, and the structures of the isomers were determined by mass spectrometry and various 1D and 2D NMR techniques. The thermo-isomerization mechanism of cefotiam was also discussed. Thermo-isomerization occurred not only in cefotiam but also in cephalosporins containing a 1-alkyl-1H-tetrazole-5-thiol side chain at C-3. Furthermore, the toxic effects of the two impurities of cefotiam hydrochloride were predicted and it is thought that they could be more toxic than cefotiam. The results reported in this article may be important for quality control and stability studies of this class of drugs.

Application of Solid-State NMR to Reveal Structural Differences in Cefazolin Sodium Pentahydrate From Different Manufacturing Processes.

Solid-state Nuclear magnetic resonance, thermogravimetric analysis, X-ray diffraction, and Fourier-transform infrared spectroscopy were combined with theoretical calculation to investigate different crystal packings of α-cefazolin sodium obtained from three different vendors and conformational polymorphism was identified to exist in α-cefazolin sodium. Marginal differences observed among cefazolin sodium pentahydrate 1, 2, and 3 were speculated as being caused by the proportion of conformation 2.

Embryo and Developmental Toxicity of Cefazolin Sodium Impurities in Zebrafish.

Cefazolin sodium is an essential drug that is widely used in clinical therapy for certain infective diseases caused by bacteria. As drug impurities are considered to be one of the most important causes of drug safety issues, we studied embryotoxicity, cardiotoxicity, and neurotoxicity of nine cefazolin sodium impurities in zebrafish embryo and larvae for the objective control of impurity profiling. LC-MS/MS was employed to analyze the compound absorbance in vivo, and the structure-toxicity relationship was approached. Our results suggested that the structure of MMTD (2-mercapto-5-methyl-1, 3, 4-thiadiazole) is the main toxic functional group for embryo deformities; the 7-ACA (7-aminocephalosporanic acid) structure mainly affects motor nerve function; and both the MMTD and 7-ACA structures are responsible for cardiac effects. Impurity G (7-ACA) presented with the strongest toxicity; impurity A was most extensively absorbed to embryo and larvae; and impurity F (MMTD) exhibited the strongest apparent toxic effect; Therefore, impurities F and G should be monitored from the cefazolin sodium preparations.

[Drug Discrimination Method Based on Near Infrared Reflectance Spectrum and Balance Cascading Sparse Representation Based Classification].

The combination of near infrared spectrum and pattern recognition methods has a wide application prospect in rapid and nondestructive supervision and management of drugs. The traditional identification methods regard the smallest error rate as the goal while the imbalance of classes is ignored. This makes the positive class is overwhelming covered by the negative class and reduces its effect for the classifier, so that the classification results tend to recognize the negative class correctly, which severely affects the identification accuracy. In this paper, we mainly studied the class imbalance problems of true or false drugs via infrared spectral data of its, and then propose a balance cascading and sparse representation based classification method (BC-SRC) by combining the Balance Cascading with SRC. We sampling majority samples from the majority class for several times, which has the same size as minority samples and the majority samples we sampled can contain all the majority class samples entirely (sampling times is ceiling the result of majority samples number divide minority samples number). We can get sets of results, and then obtain the final predict labels form those results. Experiments of three databases achieved on Matlab2012a shows that the method is effective. From the experimental results, it can be seen that the method is superior to the commonly used Partial Least Squares (PLS), Extreme Learning Machine (ELM) and BP. Particularly, for the imbalanced databases, when the imbalance factor is greater than 10, the proposed method has more stable performance with higher classification accuracy than the existing ones mentioned above.

Rapid Analysis of the Quality of Amoxicillin and Clavulanate Potassium Tablets Using Diffuse Reflectance Near-Infrared Spectroscopy.

The cycle-closed dimer of amoxicillin influences its critical quality and is an important impurity in amoxicillin and clavulanate potassium tablets. The quality of the tablets could be rapidly evaluated using the impurity as an indicator. Here, we report a quantitative model to determine the cycle-closed dimer in samples from different manufacturers using diffuse reflectance near-infrared (NIR) spectroscopy by partial least squares regression for one y variable (PLS1) and hierarchical cluster analysis. Because the contents of the (active pharmaceutical ingredients) APIs (amoxicillin and clavulanate potassium) and water are also the important indexes of the tablet quality, three other quantitative models were used to confirm the API data and water content. All of the four models facilitate rapid and complete control of the tablet quality. In addition, quantitative models were validated in terms of specificity, linearity, accuracy, repeatability, and intermediate precision according to the International Conference on Harmonisation guidelines by evaluating the characteristics of the NIR spectra. These results confirmed that the models were satisfactory.

[The control of the critical quality attributes of amoxicillin and clavulanate potassium tablet].

The critical attribute was analyzed in clavulanate potassium tablet of amoxicillin according to the principle QbD. By investigation of the drug impurity profile, the cycle-closed dimer and penicilloic acid of amoxicillin were considered to be the critical impurities, and the sources and the degradation pathways of these two impurities were discussed. The research confirmed that crystal form was the critical attribute of drug substance. The drying process in the tablet granulation was regarded as the critical process parameter. The tablet formulation was also another factor in the impurity generation. This study provides a new idea for the evaluation of drug quality.

[Study on the Selection of Parameters for Evaluating Drug NIR Universal Quantitative Models].

In order to find out the optimum combination of the evaluation parameters for the selection of the best drug near infrared (NIR) universal quantitative model during model optimization, 13 common evaluation parameters of NIR quantitative models were collected and arranged from commercial chemometrics software or References based on the requirements of validation of quantitative analytical procedures of ICH (International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use). Then all these parameters of 92 drug NIR universal quantitative models were calculated and analyzed. By studying the correlation of these parameters, the optimum combination of evaluation parameters for drug NIR universal quantitative models was determined. And the value range of these parameters in the optimum combination was also obtained. Root mean square error of cross-validation(RMSECV)/root mean square error of prediction (RMSEP), average relative deviation (ARD) and ratio of (standard error of) prediction (validation) to (standard) deviation (RPD) were used as the key parameters to evaluate the model accuracy. Most of RMSECV/RMSEP was within 3%, and the value of RMSECV was roughly equivalent to the average absolute deviation of the corresponding model. Most of RPD was more than 2. The value of ARD was related to the type of universal models (such as the drug preparation and packing) and the content range which the test sample belonged to. Determination coefficient (R2) was used as the key parameter to evaluate the model linearity and most of its values were from 80% to 100%. The ratio of RMSEP to RMSECV was selected as the key evaluation parameter of model robustness and its value was usually within 1.5. The standard deviation of repeated measurement data was chosen to evaluate model precision. And it was an important parameter for standardizing operation of NIR instruments and studying the feasibility of model transfer in different instruments. However, the parameter for NIR universal quantitative models received much less attention in previous studies and it was difficult to give a value range for this parameter at present. All the results can not only provide evidence for evaluation of drug NIR universal quantitative models for the model builders or users, but also supply basic data to establish and improve the parameter evaluation system of drug NIR universal quantitative models.

Factors Influencing the HPLC Determination for Related Substances of Azithromycin.

The European Pharmacopoeia, the U.S. Pharmacopoeia, and the Pharmacopoeia of the People's Republic of China all prescribe a high-performance liquid chromatography-ultraviolet method within a C18 column system for the separation of Azithromycin (AZT) and its related substances. The identification of impurities in the AZT complex was performed according to the relative retention time (RRT) between each impurity and AZT. However, the RRT values of impurities often vary on different types of C18 packing materials and at different column temperatures, which could affect the accurate and fast identification of impurities. In our study, five different commonly used C18 columns as well as nine different column temperature set points were assessed for the analysis of AZT and its related substances. A factorial design was applied to analysis the relationships between column types/column temperatures and RRT value of each impurity. The results showed that the change rates of the RRT values of impurities were different on different columns and at different column temperature set points. Therefore, the current method adopted by the three Pharmacopeias, in which the RRT values were used to identify the related substances, is not suitable to identify the ones in the AZT chromatographic system.

Guidelines and strategy of the International Conference of Harmonization (ICH) and its member states to overcome existing impurity control problems for antibiotics in China.

In the present report, we review the technical guidelines and principles on impurity research and control for antibiotics established by various agencies, including the International Conference of Harmonization (ICH), the US Food and Drug Administration (FDA), the European Medicines Agency (EMA) and the China Food and Drug Administration (CFDA). Progresses with the US Pharmacopoeia (USP), the European Pharmacopoeia (EP) and the Chinese Pharmacopoeia (ChP) to control impurities in antibiotics are also presented. Next, our discussion is focused on analyzing the CFDA's requirements on impurity research and control for antibiotics, and the implementation of ICH, FDA and other technical guidelines for generic drugs impurity control in China. Existing problems are further reviewed, in order to improve the overall process for the control of antibiotic purity.

Antibiotic toxicity and absorption in zebrafish using liquid chromatography-tandem mass spectrometry.

Evaluation of drug toxicity is necessary for drug safety, but in vivo drug absorption is varied; therefore, a rapid, sensitive and reliable method for measuring drugs is needed. Zebrafish are acceptable drug toxicity screening models; we used these animals with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in a multiple reaction monitoring mode to quantify drug uptake in zebrafish to better estimate drug toxicity. Analytes were recovered from zebrafish homogenate by collecting supernatant. Measurements were confirmed for drugs in the range of 10-1,000 ng/mL. Four antibiotics with different polarities were tested to explore any correlation of drug polarity, absorption, and toxicity. Zebrafish at 3 days post-fertilization (dpf) absorbed more drug than those at 6 h post-fertilization (hpf), and different developmental periods appeared to be differentially sensitive to the same compound. By observing abnormal embryos and LD50 values, zebrafish embryos at 6 hpf were considered to be suitable for evaluating embryotoxicity. Also, larvae at 3 dpf were adapted to measure acute drug toxicity in adult mammals. Thus, we can exploit zebrafish to study drug toxicity and can reliably quantify drug uptake with LC-MS/MS. This approach will be helpful for future studies of toxicology in zebrafish.