万古霉素产生菌的选育与发酵培养基优化

万古霉素产生菌东方拟无枝酸菌（Amycolatopsis orientalis）5－13经紫外线（30W，253.7nm，距离30cm，照射50s）诱变后，在含万古霉素1000μg/ml的浓度梯度平板上筛选万古霉素抗性变株，得到一株万古霉素抗性变株A.orientalis 6-21，其摇瓶效价较出发菌株提高23％，对万古霉素的抗性提高200％。传代试验表明该变株的高产性能遗传特性较稳定。用正交试验法对万古霉素发酵培养基进行了优化，与原发酵培养基相比，万古霉素的摇瓶发酵效价提高了14.3%。     

高产纳他霉素的褐黄孢链霉菌选育

目的　以褐黄孢链霉菌 (Streptomyces gilvosporeus) S- 71为出发菌株 ,筛选纳他霉素的高产菌株。方法　紫外线对孢子悬浮液照射 4 0 s后 ,分别用链霉素抗性和琼脂块法进行筛选纳他霉素高产菌株 ,之后对高产菌株进行生产稳定性实验。结果　通过链霉素抗性法筛选获得了约 10 %的正选率突变株 ,其中突变株SG- 5 6摇瓶效价单位为 2 4 10μg/ ml,为出发菌株的 14 6 % ;通过琼脂块筛选法获得了约 1%的正选率突变株 ,其中突变株 SG- 2 0 0 2摇瓶效价单位为 2 6 5 0μg/ ml,为出发菌株的 16 1% ,该菌株无链霉素抗性标记。结论　链霉素抗性筛选和琼脂块筛选均可以获得纳他霉素的高产菌株 ,其中链霉素抗性筛选法效率高 ,琼脂块法筛选全面。     

纳他霉素高产菌株的链霉素抗性选育及其发酵工艺的优化

用紫外线对纳他霉素(Natamycin)产生菌褐黄孢链霉菌(Streptomyces gilvosporeus)ATCC13326菌株孢子进行诱变处理后，利用链霉素抗性突变选育得122株链霉素抗性突变株。其中纳他霉素产量高于出发菌株的有13株。突变株SG-56的生产能力达到出发菌株的146％，研究了其发酵性能，优化了培养基组成和发酵工艺条件，纳他霉素产量为2．81g／L，较出发菌株产量提高了170％。     

雷莫拉宁高产菌株的选育

以雷莫拉宁产生菌RM05-55为出发菌株,首先采用紫外诱变复合链霉素抗性筛选,然后进行微波诱变,获得一株雷莫拉宁高产菌株W07-28,其发酵单位较出发菌株提高了59.3%.传代试验表明该菌株的发酵水平较稳定.     

纳他霉素产生菌基因组重排育种

Streptomyces gilvosporeus SG-1原生质体经紫外线诱变并筛选链霉素抗性菌株，获得纳他霉素高产菌株。在上述高产突变株中选择4株作为亲本进行基因组重排育种，筛选得到了高产重排菌株，其中一株重排菌株S．gilvosporeus GS-74的纳他霉素产量为3574mg／L。为产量最高的亲本菌株的153％，比原始出发菌株SG-1提高1.17倍。     

被孢霉高产γ-亚麻酸菌株的选育

目的：选育高产γ-亚麻酸被孢霉菌株。方法：以被孢霉（Mortierellasp．）为出发菌株经过UV诱变处理和甘草酸筛选,获得一株γ-亚麻酸（GLA）高产菌株A02变株。结果：变株摇瓶培养生物量达20．12g／L,较出发菌株提高25．50％,γ-亚麻酸的产量为0．96g／L,较出发菌株提高了81％。传代实验显示,变株A02具有良好的遗传稳定性。结论：甘草酸筛选法是多不饱和脂肪酸产生前育种工作的一个有效方法。     

产N-demethyl ECO-0501工程菌的构建

ECO-0501是一种在东方拟无枝酸菌(Amycolatopsis orientalis)中分离出的一种新型抗菌化合物.N-demethyl ECO-0501作为ECO-0501生物合成途径的小组分在抗菌活性方面优于ECO-0501.ECO-ORF5(ABM47006.1)编码N-甲基转移酶,负责ECO-0501胍基的甲基化修饰.本研究利用PCR-targeting的方法敲除了产生菌东方拟无枝酸菌HCCB10162的ECO-ORF5,得到了一株高产去甲基ECO-0501的突变株A.orientalis HCCB10370,其产量是东方拟无枝酸菌HCCB10162的3.6倍.     

ARTP诱变结合抗性筛选选育西索米星高产菌株

目的 利用等离子体诱变,结合抗生素抗性筛选,获得西索米星高产菌株。方法 首先通过链霉素抗性筛选,获得一株比出发菌株产抗能力高的S4;随后以S4为出发菌株,经等离子体处理40s,借助巴龙霉素抗性筛选,得到双重抗性菌株。最后对突变株进行再次诱变,并结合高浓度链霉素抗性筛选。结果 获得一株高产突变株M. inyoensis SAAP22,比出发菌株效价提高了38.18%,具有稳定的遗传性能。结论 通过等离子体诱变,结合抗生素抗性筛选,可有效提升伊尼奥小单孢菌的西索米星合成能力,所得高产菌株具有潜在应用价值。     

维吉尼亚链霉菌物理诱变及外源添加氯化镧对VGM产量的影响

目的 利用物理诱变结合链霉素抗性筛选维吉尼亚霉素(VGM)高产菌株维吉尼亚链霉菌,并考察外源添加稀土元素氯化镧对维吉尼亚链霉菌发酵的影响。方法 一方面,以链霉素为筛选剂建立孢子致死突变标志的微波及紫外诱变两种筛选模型,采用管碟法获得抑菌圈直径较大的作为初筛菌株,再通过摇瓶复筛进一步确定高产菌株。另一方面,在发酵培养基中外源添加3~30mg/L的氯化镧,考查其对链霉菌合成VGM的影响。结果 比较两种筛选模型,基于链霉素的微波诱变效果较好,正突变率高达32.83%;并获得一株高产菌株MW 178(146.72±11.80)μg/mL,较出发菌株(21.24±1.34)μg/mL提高了约6倍。在发酵初始时,外源添加10mg/L氯化镧,VGM的产量由146.72μg/mL提高到180.01μg/mL,提高了约22.69%。结论 采用微波诱变结合链霉素抗性筛选的方法可以获得VGM高产突变株;在发酵初始时,外源添加适量氯化镧可以显著提高VGM产量。     

原生质体诱变及高通量筛选选育链霉素高产菌株

目的 通过诱变和筛选方法的研究，提高灰色链霉菌(Streptomyces griseus)生产链霉素的水平。方法 优化灰色链霉 菌的原生质体的生成和再生条件，并对得到的原生质体进行紫外诱变，然后利用微孔板高通量方式对获得菌株进行筛选。结果 经过诱变选育获得一株菌NP-11703，其链霉素产量在100L罐上比出发菌株提高了21.8%。结论 用紫外诱变原生质体，可以有 效提高灰色链霉菌产链霉素的能力。结合高通量筛选模型的应用，可大大加快高产菌株的筛选效率。     

The antiobesity action of (S)-(+)-1-(4-chlorophenylthiomethyl)-N-methylethylamine fumarate (AO-124)

The antiobesity effects of (S)-(+)-1-(4-chlorophenylthiomethyl)-N-methylethylamine fumarate (AO-124) were examined in rats and dogs. AO-124 suppressed food intake dose dependently in normal, Zucker fatty and VMH-obese rats, and beagle dogs. Its anorectic activity was not altered by pretreatment with methysergide, a serotonin receptor blocker. AO-124 also reduced the hyperphagia induced by 2-deoxy-D-glucose but not that induced by insulin, noradrenaline or muscimol, suggesting that the anoretic mechanism of AO-124 may be implicated in a glucostatic regulatory system of feeding. In addition, AO-124 decreased insulin secretion in response to an oral, but not an intravenous, glucose load. Such a suppression in insulin secretion may be explained by slow absorption of glucose from the intestine: AO-124 delayed the gastric emptying time of glucose and inhibited the active transport of glucose as observed in the everted small intestine. Two week administration of AO-124 to Zucker fatty rats resulted in a significant reduction of plasma insulin levels, body weight gain, and body lipid without exerting any changes in body protein. These findings indicate that AO-124 may be useful as an antibesity agent on the basis of its unique mechanisms of action.     

Development of sesquiterpenes from Alpinia oxyphylla as novel skin permeation enhancers.

To improve the drug permeation into and/or across the skin, essential oils extracted from Alpinia oxyphylla (AO) were evaluated using in vitro and in vivo permeation techniques with Wistar rats as the animal model. Hydrocarbons and oxygenated sesquiterpenes were the major components in the lower-polarity fraction (AO-1) and higher-polarity fraction (AO-2), respectively. Permeation of indomethacin was significantly enhanced after treatment with AO-1 and AO-2 in the in vitro and in vivo studies. AO-2 generally showed a higher ability to promote drug permeation compared to AO-1. The increment of skin/vehicle partitioning may be the predominant mechanism for this enhancing activity. Both transepidermal water loss (TEWL) and colorimetric evaluation showed limited irritation to skin by AO essential oils at the macroscopic level. Human skin fibroblasts were used to investigate the in vitro screening of skin toxicity. AO-1 slightly increased prostaglandin E(2) (PGE(2)) formation from skin fibroblasts. A striking result was observed with AO-2, which greatly inhibited the release of PGE(2). Moreover, both AO essential oils had no statistically significant effect on PGE(2) release by human lung epithelial cells. The results of this study indicate that skin disruption and inflammation do not necessary correspond to the enhancing efficiency of the enhancers tested.     

Comparison of the effects of losigamone and its isomers on maximal electroshock induced convulsions in mice and on three different patterns of low magnesium induced epileptiform activity in slices of the rat temporal cortex

Summary Losigamone (AO-33) is a recemate of a tetronic acid derivative. The effects of losigamone and its three isomers (AO-242, AO-294 and AO-23) were compared on maximal electroshock (MES) induced convulsions in mice and on different patterns of extracellularly recorded, low Mg ²⁺ induced epileptiform activity in slices of the rat temporal cortex. Lowering Mg ²⁺ induced recurrent short discharges in areas CA3 and CA1 while ictaform events that lasted for many seconds were induced in the entorhinal cortex. In the hippocampus the activity stayed stable over a number of hours. In contrast, the ictaform events in the entorhinal cortex changed their characteristics after one to two hours to recurrent discharges of 0.8 to 10 s. Afterdischarges and interictal events were absent. 50 M AO-242 showed a similar efficacy to 50 M AO-33 in reducing and blocking epileptiform discharges in areas CA1 and CA3 while 50 M AO-294 and 50 M AO-23 had weaker effects than 50 M AO-33. Concentrations of 50 M and 100 M AO-242 showed a similar efficacy to AO-33 on ictaform events in the entorhinal cortex. Late recurrent discharges were also blocked by AO-33 and AO-242 although at higher concentrations (300 M). The in vitro observations are with respect to order of efficacy in accordance with the in vivo data obtained in the maximal electroshock test in mice. The order of potency in the MES test was AO-242>AO-33AO-294 AO-23. The results show that the erythro-isomer AO-23, although active, is much less potent than AO-33. Of the two optical isomers of losigamone the (+) isomer AO-242 is more active than the (–) form AO-294. Send offprint requests to C. L. Zhang at the above address     

Interactions between two enantiomers of losigamone and conventional antiepileptic drugs in the mouse maximal electroshock model--an isobolographic analysis

The aim of this study was the isobolographic evaluation of interactions between two enantiomers of losigamone, AO-242 [(+)-5(R)-alpha(S)-5-(2-chlorophenylhydroxymethyl)-4-methoxy-2(5H)-furanone] and AO-294 [(-)-5(S)-alpha(R)-5-(2-chlorophenylhydroxymethyl)-4-methoxy-2(5H)-furanone], and valproate, carbamazepine, phenytoin, or phenobarbital in the maximal electroshock test in mice. Both enantiomers interacted additively with conventional antiepileptic drugs at all studied fixed dose ratios (1:3, 1:1, 3:1). Furthermore, AO-242, AO-294 and antiepileptics applied alone, as well as combinations of enantiomers and antiepileptics did not affect motor performance in the chimney test. Significant impairment of long-term memory (passive-avoidance task) was noted only in the case of valproate alone, given at the dose equal to its median effective dose (ED(50)) against maximal electroshock. All other antiepileptics and their combinations with AO-242 or AO-294 did not impair memory of mice. Enantiomers did not affect the brain concentrations of antiepileptic drugs, indicating a pharmacodynamic nature of the observed interactions. In conclusion, the present results suggest both AO-242 and AO-294 as promising candidate drugs in the add-on therapy of refractory epilepsy.     

Antiviral activity of diarylheptanoid stereoisomers against respiratory syncytial virus in vitro and in vivo

We previously showed that (5S)-5-hydroxy-7-(4-hydroxyphenyl)-1-phenylhept-3-one (AO-0011) and (5S)-5-methoxy-1,7-diphenylhept-3-one (AO-0016) isolated from Alpinia officinarum exhibited stronger anti-influenza virus activity and anti-respiratory syncytial virus (RSV) activity, respectively, than the other isolated diarylheptanoids. In this study, we synthesized an enantiomer (AO-0503) and racemate (AO-0504) of AO-0011 and an enantiomer (AO-0514) of AO-0016. The anti-RSV activities of the three stereoisomers (AO-0503, AO-0504, and AO-0514) and AO-0011 were examined in vitro and in vivo to evaluate the stereoisomeric effect on anti-RSV activity. In a plaque reduction assay using human epidermoid carcinoma cells, all four diarylheptanoids significantly exhibited anti-RSV activity, and AO-0514 and AO-0016 exhibited stronger anti-RSV activity than AO-0503, AO-0504, and AO-0011. In a murine RSV infection model, all four diarylheptanoids with anti-RSV activity in vitro were also significantly effective in reducing virus titers in the lungs of RSV-infected mice. In the histopathological analysis of RSV-infected lungs, the oral administration of even AO-0514, which showed the lowest reduction of virus titers in the lungs, was significantly effective in reducing the infiltration of lymphocytes and in reducing the interferon-γ level, which is a marker of severity of pneumonia due to RSV infection, in bronchoalveolar lavage fluids prepared from RSV-infected mice. Although the stereoisomeric effects of diarylheptanoids on anti-RSV activity varied moderately, all four diarylheptanoids examined were suggested to ameliorate pneumonia and have a potential anti-RSV activity in vivo. They are possibly mother compounds for the development of an anti-RSV drug in the future.     

Isobolographic analysis of interactions among losigamone analog AO-620 and two conventional antiepileptic drugs

In the present study, we investigated pharmacodynamic interactions among AO-620, a losigamone analog, and two conventional antiepileptic drugs, valproate (VPA) and phenobarbital (PB). Experiments were conducted in the maximal electroshock test in mice. Isobolographic analysis of the obtained data revealed pure additive interactions between AO-620 and PB applied at three dose ratios of 1:1, 1:3 and 3:1. Antagonism was observed when AO-620 was co-administered with VPAat the ratio of 3:1, while additive interactions were seen in two remaining proportions (1:3 and 1:1). Surprisingly, the interaction pattern of AO-620 appeared quite different from that of losigamone.     

Antioxidants in some pharmaceuticals, cosmetics and food from the European market.

A specific and sensitive reverse phase HPLC method (AO-1) for the quantitative determination of ten phenolic antioxidants and propyl paraben in fatty products (food, pharmaceuticals and cosmetics) has been developed. Extraction with acetonitrile from a hexane dilution of the products; acetic acid-water-acetonitrile-methanol solvent system and detection at 280 nm and quantification by internal standard procedure (reference compound: DMP, dimethylphenol) were used. Good resolutions for all peaks, especially BHT and DG, and no interferences with others common additives were achieved. Mean recoveries, by addition of standards to sample at 20, 50 and 100 micrograms/g, were 96-101% (BHT, 84-86%); minimal quantifiable levels were 0.5-1.6 micrograms/g. Two simple isocratic methods for the confirmation of data obtained by AO-1 were also developed: AO-2 for seven phenolic antioxidants and propyl paraben with DMP as internal standard, AO-3 for four lipophilic antioxidants with di-tert-butylphenol (DTBP) as internal standard.     

Antimicrobial activity of AO-128, a novel inhibitor of alpha-D-glucosidase, against anaerobic bacteria]

The in vitro antimicrobial activity of AO-128, an inhibitor of alpha-D-glucosidase, was evaluated against anaerobic bacteria of 45 reference strains (12 genera, 44 species). AO-128 inhibited no strains tested at a concentration of 1,600 micrograms/ml. The results strongly suggested that this compound would not have any influence on the human intestinal microflora, a majority of which is composed of anaerobic bacteria.     

Perspectives of losigamone in epilepsy treatment

Patients with drug resistant epilepsy represent about 40% of the whole population of epileptic patients. These patients require more than one antiepileptic drug. In animal models of epilepsy, it is possible to determine which combinations produce supra-additive anticonvulsive effects with minimal or even no adverse reactions. The experimental data can be helpful for predicting effective drug combinations in patients with refractory epilepsy. Losigamone is a new antiepileptic drug with an unknown mechanism of action. The drug belongs to the group of beta-methoxy-butenolides, and exists as a racemic mixture of two enantiomers (AO-242 and AO-294). The drug is eliminated by oxidation. Cytochrome CYP2A6 appears to be the main isoenzyme responsible for the metabolism of losigamone. In vitro, losigamone exerts anticonvulsant activity in the picrotoxin model in CA1 and CA3 hippocampal areas, the low Ca(2+) model in CA1 area and the low Mg(2+) model in the entorhinal cortex and hippocampus. In vivo, the drug exhibits significant efficacy against maximal electroshock-induced seizures in rodents and pentetrazole-induced clonic convulsions in mice. Potency of losigamone varies with the respective seizure test, animal species used in experiments and route of drug administration. Toxicity studies do not indicate any teratogenic risk of the drug, at least in animals. In clinical trial, losigamone proved to have satisfactory effectiveness and good tolerance in the treatment of partial and secondary generalized seizures. The enantiomer AO-242 seems to be more potent than AO-294 or racemate.     

Influence of losigamone on the pharmacokinetics of a combined oral contraceptive in healthy female volunteers

OBJECTIVES: The influence of the new antiepileptic drug losigamone (CAS 112856-44-7/123783-52-8) on the pharmacokinetics of a combined oral contraceptive containing ethinylestradiol (CAS 57-63-6) and levonorgestrel (CAS 797-63-7) was investigated in 16 healthy women. METHODS: This phase I study consisted of 3 periods with an uncontrolled first period and a double-blind, placebo-controlled, cross-over design in the second and third period. All subjects received a single dose of 200 mg losigamone (1 tablet) in period 1 (on day 14) as well as multiple doses of losigamone (3 tablets = 600 mg per day) or placebo for 15 days in periods 2 and 3. During all three periods an oral contraceptive containing 30 microg ethinylestradiol and 150 microg levonorgestrel was given. Single-dose pharmacokinetics was investigated on day 14 of period 1. Multiple-dose pharmacokinetic investigations were performed on day 15 of periods 2 and 3. The samples were assayed to derive pharmacokinetic data of ethinylestradiol and levonorgestrel. In addition, the concentrations of losigamone racemate (AO-33) and its enantiomers AO-242 and AO-294 were determined in these samples. RESULTS: The mean values of the pharmacokinetic parameters AUC and Cmax of ethinylestradiol and levonorgestrel after multiple-dose treatment with losigamone or placebo were quite similar and met the criteria for bioequivalence. The 90% confidence intervals of the log-transformed ratios of the geometric means of the primary pharmacokinetic variables were included in the respective acceptance ranges of 80% to 125% (AUC) and 70% to 143% (Cmax). CONCLUSIONS: The study demonstrated that multiple doses of losigamone did not influence the multiple dose kinetics of ethinylestradiol and levonorgestrel. The single- and multiple-dose kinetics of 200 mg losigamone and its enantiomeres did not differ from each other in a significant way. The combination of losigamone and the combined oral contraceptive was well tolerated and no serious adverse events occurred. It can be stated that the antiepileptic drug losigamone and the combined contraceptive do not interact each others metabolism.