New Aminoglycoside Antibiotics Vertilmicin The Pharmacodynamic Study Of Recombinant Aminoglycoside Bifunctional Modifying Enzyme Stability

Aminoglycoside antibiotics are important bactericidal agents used for the treatment of serious infections in our country, which are highly potent, broad-spectrum antibiotics and have the advantages of good water solubility, absence of hypersensitivity, inexpensiveness. Antibiotics such as Sagamicin, Netilmicin, Etimicin, Amikacin and Spectinomycin have become important agents in the treatment of infections in our country. Vertimicin is a new aminoglycoside semisynthetic antibiotic, which is the 1-N-ethyl derivative of Verdamycin, one of the natural products of Micromonospora olivoasterospora subsp. Wuxiensis M-41.In the first part of this work, the pharmacodynamic characteristics of Vertimicin were studied, including its MICs to 1185 clinical isolates, MBC, KCs, incubation conditions' effects experiments and the in vivo mouse systemic infection test, mouse transurethral infection test and rabbit burn infection test. The MIC test demonstrated that Vertimicin had relatively potent broad-spectrum antibacterial activity, the MIC₅₀s of Vertimicin against most Gram-negative organisms were 0.25-1 mg/L, the MIC₅₀s against Gram-positive organisms MSSA and S. epidermidis were 0.125 and 0.25 mg/L respectively, MRSA and E. faecalis were both 4 mg/L. The susceptible rate of Vertimicin to 1185 clinical isolates was 75.4%, similar to that of Netilmicin (73.1%), lower than that of Amikacin (82.2%) and higher than those of Verdamycin (65.4%), Gentamicin (61.3%), Etimicin (64.9%) and Tobramycin (66.0%). Vertimicin still had some antibacterial activity to Gentamcin resistant strains, its susceptible rate to 405 Gentamicin resistant isolates was 41.2%, lower than that of Amikacin (60.0%) and higher than those of Netilmicin (34.8%), Verdamycin (9.6%), Etimicin (18.0%) and Tobramycin (18.0%). The MBC and KCs experiments revealed that Vertimicin had bactericidal activity against E. coli, K. pneumonia, S. aureus and S. epidermidis, and the activity was concentration dependent. Low pH and divalent cations had negative effects on the activity of Vertimicin, while inoculum size and serum had no apparent effects.Vertimicin given by s. c. or iv had relatively good protection to the mice systemically infected with E. coli, K. pneumonia, Ps. aeruginosae and S. aureus, the in vivo activities were similar to those of Netilmicin, P＞0.05. Its protection to E. coli, K. pneumonia and S. aureus infected mice was apparent better than those of Etimicin and Gentamicin, P＜0.01, the ED₅₀ of Vertimicin against Ps. aeruginosae infected mice was lower than those of Etimicin and Gentamicin without statistical significance, P＞0.05.Vertimicin had good activity to mice transurethrally infected with E. coli, the renal colony counts for the Vertimicin treated groups were significantly lower than that of the control, and the negative ratios of kidney stamping were significantly higher than that of the control, P＜0.01. The activity of Vertimicin was better than that of Gentamicin.Vertimicin had good activity to skin-burned rabbit infected with E. coli. Vertimicin treatment significantly decreased the skin bacteria counts when compared to the control, P＜0.05 or P＜0.01. Vertimicin 1mg/mL treatment had significantly better results than those of Verdamycin and Gentamicin, P＜0.01, slightly better than those of Etmicin and Netilmicin without statistical significance, P＞0.05.The main mode of bacterial resistance to aminoglycosides is through the modification of antibiotics by the produced aminoglycoside modifying enzymes, and decreasing the affinity of the antibiotics for the ribosomal subunit. The most important modifying enzyme in Gram-positive strains is the aminoglycoside bifuncfional modifying enzyme AAC(6')-APH(2"). The enzyme simultaneously comprises activities of 6'-N-acetyltransferase and 2"-O-phosphotransferase, and modifies almost all kinds of aminoglycosides, leading to disappearance of the synergistic killing effect with cell wall-active agents such as penicillin.In the second part of this work, Vertimicin's stabilities to the aminoglycoside bifunctional modifying enzyme were studied after cloning the aminoglycoside bifunctional modifying enzyme AAC(6'), APH(2") and AAC(6')-APH(2") in E. coli to prepare the crude enzyme extracts and set the in vitro metabolizing model of the recombinant aminoglycoside bifunctional modifying enzyme. By comparasion of AAC(6')-APH(2") and AAC(6'), APH(2")'s modifying functions to the antibiotics, we studied gene fusion's effect on the modifying activities of the bifunctional enzyme. In the experiment, we cloned the bifunctional modifying enzyme gene of the E. faecalis HH22 into protein expressing vector pET-30a(+), and constructed the bifunctional modifying enzyme expressing plasmids pET-30a-aac(6') (demonstrating acetyltransferase activity, and the corresponding crude enzyme extract is AAC(6')), pET-30a-aph(2") (demonstrating phosphotransferase activity, and the corresponding crude enzyme extract is APH(2")) and pET-30a-aac(6')-aph(2") (demonstrating bifunctional modifying enzyme activity, and the corresponding crude enzyme extract is AAC(6')-APH(2")). From the strains of E. coli BL21 (DE3) transformed with the three plasmids, we can easily get the crude enzyme extracts through inductive cultivation and sonication.Using the crude enzyme extracts, we set the in vitro metabolizing model of recombinant aminoglycoside bifunctional modifying enzyme to study aminoglycosides' stability to the enzyme. In this model, the crude enzyme extracts were incubated with the test aminoglycoside antibiotics under certain conditions, and the stabilities of the antibiotics were evaluated by the HPLC tests of the antibiotics' peaks changes and the appearance of the metabolizing products' peaks. The method has the advantages of simpleness, convenience, accuracy, automatization and more important, the distinguishment of the parent antibiotics' and different metabolizing products' peaks, which should provide more information for drug design. The defect of the model is the lack of the criteria for stability evaluation, and two or more antibiotics should be studied to assess the stability difference.Using the above model, Vertimicin's stabilities to AAC(6'), AAC(6')-APH(2")'s acetyltransferase activities and APH(2"), AAC(6')-APH(2")'s phosphotransferase activities were studied and compared to those of Verdamycin and Netilmicin. The results demonstrated that Vertimicin was the most stable antibiotic of the three as to AAC(6'), AAC(6')-APH(2")'s acetyltransferase activities, the modified rates were 27.02% and 27.20% respectively, lower than those of Netilmicin (98.64% and 94.17%) and Verdamycin (76.94% and 59.17%). When it comes to APH(2"), AAC(6')-APH(2")'s phosphotransferase activities, Vertimicin's stabilities were higher than those of Verdamycin and lower than those of Netilmicin. In APH(2"), C system, Vertimicin's modified rate was 49.60%, lower than that of Verdamycin (93.11%) and higher than that of Netilmicin (19.46%); In AAC(6')-APH(2"), B system, Vertimicin's modified rate was 49.99%, lower than that of Verdamycin (92.95%) and higher than that of Netilmicin (24.29%).One distinct characteristic of aminoglycoside bifunctional modifying enzyme is its bifunction, containing both acetyltransferase activity and phosphotransferase activity, and attempts to separate the two activities by conventional protein separation techniques have not been successful. What's more, the molecular weight of the enzyme is about double size of the average molecular weight for the monofunctional enzymes. These lead to the speculation that the aminoglycoside bifunctional modifying enzyme is a gene fusion product of two individual resistance determinants, which protect the strains from almost all kinds of aminoglycosides. Comparasion of the acetylating HPLC chromatographs of the antibiotics incubated with AAC(6')-APH(2") and AAC(6') revealed that in AAC(6')-APH(2") results, the two metabolizing products' peaks of Verdamycin changed in different direction compared to AAC(6'), with one decreased and the other increased, similar phenomenon can be seen with the metabolizing products' peaks of Netilmicin (decreased) and Vertimicin (increased), suggesting there was some difference in the acetyltransferase activities of the crude enzyme extracts, and gene fusion had effect on the acetyltransferase activity. Comparasion of the phosphorylating HPLC chromatographs of the antibiotics incubated with AAC(6')-APH(2") and APH(2") demonstrated that phosphorylating functions to the three antibiotics decreased when comparing the HPLC results of AAC(6')-APH(2") with that of APH(2"). But as the modifying functions to the three antibiotics changed in the same direction, effects of target proteins' concentration can't be excluded, and the impact of gene fusion on the phosphotransferase activity can't be determined.