Separation and Characterization of Unknown Impurities and Isomers in Cefminox Sodium and Study of the Forming Mechanisms of Impurities by Liquid Chromatography Coupled with Ion Trap/Time-Of-Flight Mass Spectrometry.

Thirteen unknown impurities and isomers in cefminox sodium were separated and characterized by liquid chromatography coupled with high-resolution ion trap/time-of-flight mass spectrometry (LC-IT-TOF-MS) with the positive mode of electrospray ionization (ESI) method. New HPLC-gradient elution method was developed for the detection of impurities in cefminox sodium. And the ESI ion trap multiple-stage tandem mass spectrometry had been applied successfully to the direct investigation of impurities and isomers in cefminox sodium. The fragmentation patterns and structural assignment of these impurities were studied. Full scan liquid chromatography-mass spectrometry (LC-MS) was first performed to obtain the m/z value of the protonated molecules and formulas of all detected peaks, LC-MSn (n = 1-6) were then carried out on the compounds of interest. Structures of 13 degradation products in cefminox sodium were deduced based on the high-resolution MSn (n = 1-6) data, assisted by the UV spectra and stress testing. And the forming mechanisms of degradation products in cefminox were also studied. The method of LC-IT-TOF-MSn (n = 1-6) was worthy of widespread use and application for the further improvement of official monographs in pharmacopoeias with the advantages of stability and repeatability.

Kinetic study on cephamycin C degradation.

Cephamycin C (CepC) is a ß-lactam antibiotic that belongs to the cephalosporin class of drugs. This compound stands out from other cephalosporins for its greater resistance to ß-lactamases, which are enzymes produced by pathogenic microorganisms that present a major mechanism of bacterial resistance to ß-lactam antibiotics. Cephamycin C is produced by the bacterium Streptomyces clavuligerus. Knowledge about the stability of the compound under different values of pH is important for the development of the process of production, extraction, and purification aimed at obtaining higher yields. Therefore, the stability of cephamycin C under different pH levels (2.2, 6.0, 7.0, 7.6, and 8.7) at 20 °C was evaluated in this study. Ultrafiltered broth from batch fermentations of S. clavuligerus was used in the trials. The results indicated that cephamycin C is a more stable compound than other ß-lactam compounds such as penicillin and clavulanic acid. A higher degradation rate was observed at very acidic or basic pH levels, while this rate was lower at quasi-neutral pH levels. After 100 h of trial, the initial CepC showed 46 % degradation at pH 2.2, 71 % degradation at pH 8.7, and varied from 15 to 20 % at quasi-neutral pH levels.

Preliminary studies for cephamycin C purification technique.

A study was made for purification of cephamycin C from fermentation of Streptomyces clavuligerus. Initially, the culture broth was clarified by microfiltration and ultrafiltration, after which the resulting permeates were subjected to nonspecific adsorption and ion-exchange chromatography on resin columns. The antibiotic activity was measured by the biological method at each stage by assaying its activity against the Escherichia coli ESS, super sensitive to ß-lactam antibiotic. The purification processes were assessed in relation to the variables affecting each step. The purification efficiency by nonspecific adsorption was monitored by UV spectrophotometry, while the ion-exchange adsorption fractions were assessed by NMR spectroscopy. Some of the fractions obtained during purification were also analyzed by mass spectrometry (LC/MS and LC/MS/MS) to identify the cephamycin C molecule. These preliminary results proved the process feasibility.

Simultaneous determination of aminomethylbenzoic acid, cefminox sodium and etamsylate in human urine by capillary electrophoresis.

A sensitive and selective capillary electrophoresis method is developed, for the first time, for effective separation and simultaneous determination of aminomethylbezoic acid (PAMBA), cefminox sodium (CMNX) and etamsylate (ETM). The electrophoresis conditions were investigated and optimized. A 25 mM phosphate solution (pH 8.5) was used as a buffer and the peak area was determined with UV detection at 216 nm wavelength under 18 kV separation voltage. Under optimal conditions, the three drugs can be separated effectively. Good linearity was achieved in 3.13-150 microg/mL for PAMBA, 6.25-150 microg/mL for CMNX and 3.13-150 microg/mL for ETM, with the correlation coefficients of >0.999. The limit of detection (LOD) for PAMBA, CMNX and ETM was 1.04, 2.08 and 1.04 microg/mL, respectively. Their recoveries in human urine were in the range from 90.2% to 101% with the RSD (n=5) of 0.7-3.1%. The proposed method is simple, rapid and accurate, and provides the sensitivity and linearity necessary for analysis of the test drugs in human urine at clinically relevant concentrations.

Chapter 16. Enzymology of beta-lactam compounds with cephem structure produced by actinomycete.

Cephamycins are beta-lactam antibiotics with a cephem structure produced by actinomycetes. They are synthesized by a pathway similar to that of cephalosporin C in filamentous fungi but the actinomycetes pathway contains additional enzymes for the formation of the alpha-aminoadipic acid (AAA) precursor and for the final steps specific to cephemycins. Most of the biochemical and genetic studies on cephemycins have been made on cephemycin C biosynthesis in the producer strains Streptomyces clavuligerus ATCC27064 and Amycolatopsis lactamdurans NRRL3802. Genes encoding cephamycin C biosynthetic enzymes are clustered in both actinomycetes. Ten enzymatic steps are involved in the formation of cephamycin C. The precursor alpha-AAA is formed by the sequential action of lysine-6-aminotransferase and piperideine-6-carboxylate dehydrogenase. Steps common to cephalosporin C biosynthesis include the formation of the tripeptide L-delta-alpha-aminoadipyl-L-cysteinyl-D-valine (ACV) by ACV synthetase, the cyclization of ACV to form isopenicillin N (IPN) by IPN synthase, the epimerization of IPN to penicillin N by isopenicillin N epimerase, the ring expansion of penicillin N to a six member cephem ring by deacetoxycephalosporin C synthase (DAOCS) and the hydroxylation at C-3' by deacetylcephalosporin C hydroxylase. However, in actinomycetes, the epimerization step is different from that in cephalosporin-producing fungi, and the expansion of the ring and its hydroxylation are performed by separate enzymes. Specific steps in cephamycin biosynthesis include the carbamoylation at C-3' by cephem carbamoyl transferase and the introduction of a methoxyl group at C-7 by the joint action of a C-7 cephem-hydroxylase and a methyltransferase. All the enzymes of the pathway have been purified almost to homogeneity and the DAOC synthase and 7-hydroxycephem-methyltransferase (CmcI) of S. clavuligerus have been crystallized giving insights into the mode of action of these enzymes. The cefE gene of S. clavuligerus, encoding DAOCS, has been extensively used to expand the penicillin ring in filamentous fungi in vivo using DNA recombinant technology.

Identification of transcriptional activators for thienamycin and cephamycin C biosynthetic genes within the thienamycin gene cluster from Streptomyces cattleya.

Two regulatory genes, thnI and thnU, were identified in the thienamycin (thn) gene cluster from Streptomyces cattleya. ThnI resembles LysR-type transcriptional activators and ThnU belongs to the SARP family of transcriptional activators. Their functional role was established after independent inactivation by gene replacement together with transcriptional analysis involving reverse transcription polymerase chain reaction (RT-PCR). Deletion of thnI abolished thienamycin production showing its involvement in thienamycin biosynthesis. Gene expression analysis applied to the thn gene cluster demonstrated that ThnI is a transcriptional activator essential for thienamycin biosynthesis that regulates the expression of nine genes involved in thienamycin assembly and export (thnH, thnJ, thnK, thnL, thnM, thnN, thnO, thnP and thnQ). Unexpectedly, the thnU disrupted mutant was not affected in thienamycin production but turned out to be essential for cephamycin C biosynthesis. Transcript analysis applied to early and late structural genes for cephamycin C biosynthesis (pcbAB and cmcI), revealed that ThnU is the transcriptional activator of these cephamycin C genes although they are not physically linked to the thn cluster. In addition, it was shown that deletion of thnI has an upregulatory effect on pcbAB and cmcI transcription consistent with a significant increase in cephamycin C biosynthesis in this mutant.

Industrial production of beta-lactam antibiotics.

The industrial production of beta-lactam antibiotics by fermentation over the past 50 years is one of the outstanding examples of biotechnology. Today, the beta-lactam antibiotics, particularly penicillins and cephalosporins, represent the world's major biotechnology products with worldwide dosage form sales of approximately 15 billion US dollars or approximately 65% of the total world market for antibiotics. Over the past five decades, major improvements in the productivity of the producer organisms, Penicillium chrysogenum and Acremonium chrysogenum (syn. Cephalosporium acremonium) and improved fermentation technology have culminated in enhanced productivity and substantial cost reduction. Major fermentation producers are now estimated to record harvest titers of 40-50 g/l for penicillin and 20-25 g/l for cephalosporin C. Recovery yields for penicillin G or penicillin V are now >90%. Chemical and enzymatic hydrolysis process technology for 6-aminopenicillanic acid or 7-aminocephalosporanic acid is also highly efficient (approximately 80-90%) with new enzyme technology leading to major cost reductions over the past decade. Europe remains the dominant manufacturing area for both penicillins and cephalosporins. However, due to ever increasing labor, energy and raw material costs, more bulk manufacturing is moving to the Far East, with China, Korea and India becoming major production countries with dosage form filling becoming more dominant in Puerto Rico and in Ireland.

Crystal structures of the Bacillus licheniformis BS3 class A beta-lactamase and of the acyl-enzyme adduct formed with cefoxitin.

The Bacillus licheniformis BS3 beta-lactamase catalyzes the hydrolysis of the beta-lactam ring of penicillins, cephalosporins, and related compounds. The production of beta-lactamases is the most common and thoroughly studied cause of antibiotic resistance. Although they escape the hydrolytic activity of the prototypical Staphylococcus aureus beta-lactamase, many cephems are good substrates for a large number of beta-lactamases. However, the introduction of a 7alpha-methoxy substituent, as in cefoxitin, extends their antibacterial spectrum to many cephalosporin-resistant Gram-negative bacteria. The 7alpha-methoxy group selectively reduces the hydrolytic action of many beta-lactamases without having a significant effect on the affinity for the target enzymes, the membrane penicillin-binding proteins. We report here the crystallographic structures of the BS3 enzyme and its acyl-enzyme adduct with cefoxitin at 1.7 A resolution. The comparison of the two structures reveals a covalent acyl-enzyme adduct with perturbed active site geometry, involving a different conformation of the omega-loop that bears the essential catalytic Glu166 residue. This deformation is induced by the cefoxitin side chain whose position is constrained by the presence of the alpha-methoxy group. The hydrolytic water molecule is also removed from the active site by the 7beta-carbonyl of the acyl intermediate. In light of the interactions and steric hindrances in the active site of the structure of the BS3-cefoxitin acyl-enzyme adduct, the crucial role of the conserved Asn132 residue is confirmed and a better understanding of the kinetic results emerges.

Inactivation of Aeromonas hydrophila metallo-beta-lactamase by cephamycins and moxalactam.

Incubation of moxalactam and cefoxitin with the Aeromonas hydrophila metallo-beta-lactamase CphA leads to enzyme-catalyzed hydrolysis of both compounds and to irreversible inactivation of the enzyme by the reaction products. As shown by electrospray mass spectrometry, the inactivation of CphA by cefoxitin and moxalactam is accompanied by the formation of stable adducts with mass increases of 445 and 111 Da, respectively. The single thiol group of the inactivated enzyme is no longer titrable, and dithiothreitol treatment of the complexes partially restores the catalytic activity. The mechanism of inactivation by moxalactam was studied in detail. Hydrolysis of moxalactam is followed by elimination of the 3' leaving group (5-mercapto-1-methyltetrazole), which forms a disulfide bond with the cysteine residue of CphA located in the active site. Interestingly, this reaction is catalyzed by cacodylate.

Applications of Lewis acids for the efficient syntheses of diltiazem, cephems, and taxoids.

An efficient method is described for preparation of diltiazem hydrochloride (Herbesser(R)), a marketed calcium antagonist widely used for the treatment of ischemic heart disease. In the reaction of 2-nitrothiophenol (1) with trans-3-phenylglycidic esters (2) carrying various substituents on the benzene ring, both reactivity and stereoselectivity of the oxirane ring-opening of the glycidates were markedly influenced by the electronic nature of the substituents. As a result of our investigation on the catalytic effect of various Lewis acids in the reaction of 2a with 1, tin compounds were found to be effective catalysts for the cis-opening and readily produced the threo-nitro ester (3a-t), a key intermediate for the synthesis of diltiazem. Isolation of the crystalline complex from the reaction of 1 and SnCl(4); and its efficient catalytic activity similar to that of SnCl(4) suggests that the transition state involves co-coordination of tin derivatives both with 1 and the epoxy oxygen of 2a to result in highly specific cis-opening. We have also amplified this chemistry into other fields, leading to applications in the syntheses of cephem and taxoid templates.

New aspects of genes and enzymes for beta-lactam antibiotic biosynthesis.

Penicillins, cephalosporins and cephamycins are peptide antibiotics synthesized by condensation of L-alpha-aminoadipic acid, L-cysteine and L-valine to form the tripeptide delta(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (Aad-Cys-Val) by a non-ribosomal peptide synthetase. The genes pcbAB and pcbC, common to all penicillin and cephalosporin producers, that encode the Aad-Cys-Val synthetase and isopenicillin N (IPN) synthase respectively, have been cloned and the encoded enzymes studied in detail. The IPN synthase has been crystallized and its active center identified, providing evidence for the molecular mechanism of cyclization of the tripeptide Aad-Cys-Val to isopenicillin N. The late genes of the penicillin and cephalosporin pathways have also been characterized although some of the molecular mechanisms catalyzed by the encoded enzymes (e.g. IPN acyltransferase) are still obscure. In cephamycin-producing organisms, biosynthesis of the alpha-aminoadipic acid precursor proceeds in two steps catalyzed by lysine 6-aminotransferase and piperideine-6-carboxylic acid dehydrogenase. The gene lat for the first of these enzymes is located in the cephamycin gene cluster, providing an interesting example of association of genes encoding enzymes for the formation of a precursor with genes involved in assembly of the antibiotics. Novel enzymes involved in methoxylation at C-7 and carbamoylation at C-3' of the cephem nucleus were isolated from Nocardia lactamdurans and Streptomyces clavuligerus. The methoxylation system is encoded by two linked genes cmcI-cmcJ and their products (proteins P7 and P8) form a complex that is required for hydroxylation at C-7 and for the subsequent methylation of the 7-hydroxycephem derivative to form the methoxyl group. Carbamoylation at the C-3'-hydroxyl group of the cephem nucleus is catalyzed by a specific carbamoyltransferase encoded by the gene cmcH. Finally, genes for a beta-lactamase (bla), a penicillin-binding protein (pbp) and a transmembrane protein (cmcT) that appears to be involved in cephamycin exportation, are clustered together with the biosynthetic genes in the cephamycin clusters of S. clavuligerus and N. lactamdurans. Availability of the cloned genes allows metabolic engineering of the beta-lactam biosynthetic pathways such as a channelling precursors and directed removal of bottlenecks in the beta-lactam biosynthetic pathways. Several new beta-lactam antibiotics have been discovered in gram-positive and gram-negative bacteria that will provide new genes for combinatorial synthesis of new molecules.

Analysis of binary mixtures of cephalothin and cefoxitin by using first-derivative spectrophotometry.

A method for the analysis of two-component mixtures of cephalothin and cefoxitin using zero-crossing first-derivative spectrophotometry is described. This technique permits the quantification of these drugs with closely overlapping spectral bands without any separation step. Linear calibration graphs of first-derivative values at 235.00 and 236.75 nm for cephalothin and cefoxitin, respectively, with negligible intercepts were obtained versus concentration in the range 4.0-32.0 micrograms ml-1 for both antibiotics. This paper presents a systematic examination of the experimental data by applying an exhaustive statistical analysis to demonstrate the validity of the method. The results of the determination of these antibiotics in mixtures of injectable dosage forms are also presented, together with their determinations in physiological serum and glucosed physiological serum.

Genes for beta-lactam antibiotic biosynthesis.

The genes pcbAB, pcbC and penDE encoding enzymes involved in the biosynthesis of penicillin have been cloned from Penicillium chrysogenum and Aspergillus nidulans. They are clustered in chromosome I (10.4 Mb) of P. chrysogenum, but they are located in chromosome II of Penicillium notatum (9.6 Mb) and in chromosome VI (3.0 Mb) of A. nidulans. Expression studies have shown that each gene is expressed as a single transcript from separate promoters. Enzyme regulation studies and gene expression analysis have provided useful information to understand the control of gene expression leading to overexpression of the genes involved in penicillin biosynthesis. Cephalosporin genes have been studied in Cephalosporium acremonium and also in cephalosporin-producing bacteria. In C. acremonium the genes involved in cephalosporin biosynthesis are separated in at least two clusters. Cluster I (pcbAB-pcbC) encodes the first two enzymes of the cephalosporin pathway which are very similar to those involved in penicillin biosynthesis. Cluster II (cefEF-cefG), encodes the last three enzymatic activities of the cephalosporin pathway. It is unknown, at this time, if the cefD gene encoding isopenicillin epimerase is linked to any of the two clusters. In cephamycin producing bacteria the genes encoding the entire biosynthetic pathway are located in a single cluster extending for about 30 kb in Nocardia lactamdurans, and in Streptomyces clavuligerus. The cephamycin clusters of N. lactamdurans and S. clavuligerus include a gene lat which encodes lysine-6-aminotransferase an enzyme involved in formation of the precursor alpha-aminoadipic acid. The N. lactamdurans cephamycin cluster includes, in addition, a beta-lactamase (bla) gene, a penicillin binding protein (pbp), and a transmembrane protein gene (cmcT) that is probably involved in secretion of the cephamycin. Little is known however about the mechanism of control of gene expression in the different beta-lactam producers. The availability of most of the structural genes provides a good basis for further studies on gene expression. This knowledge should lead in the next decade to a rational design of strain improvement procedures. The origin and evolution of beta-lactam genes is intriguing since their nucleotide sequences are extremely conserved despite their restricted distribution in the microbial world.

[Clinical value of cefmetazole and other cephamycin antibiotics]. / Klinicheskoe znachenie tsefmetazola i drugikh cefamitsinovykh antibiotikov.

The review is concerned with the description of the antibiotics belonging to cephamycins. The data on the chemical structure of cefmetazole and other cephamycins and the mechanisms of their action are presented. The peculiarities of the binding to penicillin-binding proteins and the stability to the action of various beta-lactamases are discussed. The spectrum of the antibacterial activity of cefmetazole against gram-positive and gram-negative aerobes and anaerobes, as well as the antibiotic pharmacokinetics is described. The literature data on the clinical efficacy of cefmetazole in the treatment of infectious diseases of various etiology and localization and the data on the drug tolerance and the incidence of adverse reactions are summarized. The indications to the use of cefmetazole and other cephamycins are substantiated.

A gene encoding lysine 6-aminotransferase, which forms the beta-lactam precursor alpha-aminoadipic acid, is located in the cluster of cephamycin biosynthetic genes in Nocardia lactamdurans.

A gene (lat) encoding lysine 6-aminotransferase was found upstream of the pcbAB (encoding alpha-aminoadipylcysteinyl-valine synthetase) and pcbC (encoding isopenicillin N synthase) genes in the cluster of early cephamycin biosynthetic genes in Nocardia lactamdurans. The lat gene was separated by a small intergenic region of 64 bp from the 5' end of the pcbAB gene. The lat gene contained an open reading frame of 1,353 nucleotides (71.4% G + C) encoding a protein of 450 amino acids with a deduced molecular mass of 48,811 Da. Expression of DNA fragments carrying the lat gene in Streptomyces lividans led to a high lysine 6-aminotransferase activity which was absent from untransformed S. lividans. The enzyme was partially purified from S. lividans(pULBS8) and showed a molecular mass of 52,800 Da as calculated by Sephadex gel filtration and polyacrylamide gel electrophoresis. DNA sequences which hybridized strongly with the lat gene of N. lactamdurans were found in four cephamycin-producing Streptomyces species but not in four other actinomycetes which are not known to produce beta-lactams, suggesting that the gene is specific for beta-lactam biosynthesis and is not involved in general lysine catabolism. The protein encoded by the lat gene showed similarity to ornithine-5-aminotransferases and N-acetylornithine-5-aminotransferases and contained a pyridoxal phosphate-binding consensus amino acid sequence around Lys-300 of the protein. The evolutionary implications of the lat gene as a true beta-lactam biosynthetic gene are discussed.

Formulary evaluation of second-generation cephamycin derivatives using decision analysis.

Use of decision analysis in the formulary evaluation of the second-generation cephamycin derivatives cefoxitin, cefotetan, and cefmetazole is described. The rating system used was adapted from one used for the third-generation cephalosporins. Data on spectrum of activity, pharmacokinetics, adverse reactions, cost, and stability were taken from the published literature and the FDA-approved product labeling. The weighting scheme used for the third-generation cephalosporins was altered somewhat to reflect the more important aspects of the cephamycin derivatives and their potential role in surgical prophylaxis. Sensitivity analysis was done to assess the variability of the final scores when the assigned weights were varied within a reasonable range. Scores for cefmetazole and cefotetan were similar and did not differ significantly after sensitivity analysis. Cefoxitin scored significantly lower than the other two drugs. In the absence of data suggesting that the N-methyl thiotetrazole side chains of cefmetazole and cefotetan cause substantial toxicity, these two drugs can be considered the most cost-efficient members of the second-generation cephamycins.