争光霉素A_5和争光霉素A_2的抗肿瘤作用与毒性研究

争光霉素是由浙江平阳分离的链霉菌产生的抗肿瘤物质,是由十多种组分组成的复合物。理化性质研究表明,争光霉素A_2、A_5、B_2等组分与日本报道的博莱霉素(Bleomycin)相应的组分A_2、A_5、B_2等相同。目前国外临床使用的博莱霉素是以A_2为主要组分的复合物。我们对A_5与A_2组分进行了比较研究,结果表明,两者的生物活性有明显差别,主要表现为:(1)对小鼠食管癌(SGA-73)的抑制作用,A_6比A_2强;(2)在小鼠食管癌组织中的浓度(微生物测定法),A_5比A_2高;(3)对小鼠肺的损害作用,A_5比A_2轻;(4)对小鼠的急性毒性,A_5比A_2低。上述研究结果提示,争光霉素A_5是具有一定特点的新抗癌药。最近,单一组分的争光霉素A_5定名为平阳霉素。     

争光霉素A6和它在争光霉素复合物的地位

争光霉素A₅已鉴别为Bleomycin A₆,在争光霉素复合物中所占比例一般在10%左右,在某些批样中可高达15%以上。文献报告Bleomycin A₆在天然产的Bleomyein复合物中只有痕量。通过向发酵培养基中加入特定组分的末端胺可大大提高其特定组分在复合物中的含量比而其它组分的产生则不同程度地被抑制。但Bleomyein A₆例外,即使向培养基中加入其末端胺精胺(0.3mg/ml),在所产生的复合物中大大增多的组分是Bleomyein A₆,而Bleomycin A₆仍只有痕量。这表明争光霉素产生菌有和Bleomycin产生菌明显不同的特点。     

不同组分的57Co(钴57)——争光霉素在肿瘤动物中的分布特性及其与抑瘤和毒性关系的探讨

⁵⁷Co标记的七种争光霉素组分(A₂+B₂混合品、A₂、A₄、A₅、A₆、A₅₀₃₃和B₂)在实体型艾氏腹水癌小鼠的分布实验中,发现七种组分都亲瘤和抑瘤。争光霉素A₅和A₆的亲瘤性最高。用等毒性剂量,A₅、A₄和A₂具有最高的抑瘤性。小鼠肝、肾和肿瘤对各种标记的争光霉素组分的摄取以A₅₀₃₃为最低,A₆为最高,因此目前看来,以A₆进行临床试用的前景似比其他的组分为差。A₅₀₃₃在肺中集聚少,排出较快,急性毒性又最低,按相等的LD₅₀剂量给药其抑瘤率也较高,从减少肺纤维化这点考虑,它可能是值得进一步研究的。A₅亲瘤抑瘤较高,肾和肝摄取较高,但较A₆为低,排出较慢,在体内保留时间较长,也是一种有发展前途的单一争光霉素组分。     

用流式细胞光度术(FCM)研究争光霉素对肿瘤及正常细胞周期的影响

用流式细胞光度术发现七种争光霉素组分(A₂+B₂混合品,A₂,A₂,A₅,A₆,A₅₀₃₃和B₂)都使中国仓鼠卵巢细胞(CHO)阻断在G₂期,而对G₁→S→G₂的进行无影响。这种G₂期阻断呈剂量依赖性。不同组分的G₂阻断效应不同,在40 μg/ml时,以A₄和A₅最强,其次为A₆,A₂,A₂+B₂混合物和B₂,而A₅₀₃₃无影响。用等毒性剂量,所有组分都使小鼠艾氏腹水癌产生多倍化细胞。由于争光霉素的G₂期阻断效应与抑瘤存在着平行关系,我们认为争光霉素的抑瘤作用与癌细胞被阻断在G₂期有关。     

平阳霉素的分离和鉴别

轮枝链霉菌平阳新变种(Str.Verticillus Vat.Pingyangensis n.Var.)所产生的抗肿瘤抗菌素是由十余种组分组成的复合物,其中两个主要组分分别与博莱霉素A₂和B₂完全相同。在生产过程中通过菌种选育和发酵工艺的改进,产品的组成发生了变化,主要组分变成了博莱霉素A₅,已正式命名为平阳霉素。它在复合物中所占比例一般约为60%。本文报告平阳霉素的分离、理化性质和鉴别。     

平阳霉素(博菜霉素A5)对同步化的CHO细胞的敏感周期时相的研究

平阳霉素(博莱霉素A₅)作用于同步化的各个周期时相的CHO细胞。结果用集落形成率来表示。研究表明平阳霉素对M期细胞最为敏感,其敏感顺序为M>G₂>S>G₁。实验使用的细胞同步化方法是用有丝分裂选择法收集M期细胞;用2 mMTdR双阻断法获得S期同步化细胞,再由M期和S期细胞分别向后推移1.5～2小时和7小时以获得G₁期和G₂期的同步细胞,并附以有丝分裂指数(MI)和标记指数(LI)检测各周期时相细胞的同步化程度。     

维拉帕米增强博莱霉素A5抗癌活性的机制

本文利用流式细胞术(FCM)和动物整体水平的同位素标记药物示踪方法探讨了维拉帕米(VP)增强博莱霉素A₅(BLM)抗癌活性的机制。实验发现VP显著增强BLM对S-180和HEP-2细胞的G₂期阻断效应。VP改变了⁵⁷Co-BLM在荷瘤小鼠部分器官中的分布,增加厂该药在肿瘤中的积聚。VP增強BLM抗癌活性可能是通过增加药物在肿瘤中的积聚,增强药物的G₂期阻断效应以及其它作用实现的。     

用自旋捕捉技术检测争光霉素A6产生的活泼自由基

本文用自旋捕捉技术与ESR相结合的方法,研究了争光霉素A₆-Fe²⁺复合物产生的活泼自由基。结果发现用NOS自旋捕捉剂可检测到该体系所产生的超氧阴离子自由基。在水溶剂中用PBN自旋捕捉到了羟基自由基。根据PBN-OH自旋加合物在水溶剂和甲醇溶剂中的超精细分裂常数,进一步确证了羟基自由基的生成。     

争光霉素几种不同组分的抗菌谱及其对小鼠移植性食管癌效果的比较

争光霉素是由链霉菌产生的抗肿瘤抗生素。该产生菌是从浙江平阳的土壤分离得到的。经形态与培养特征等研究表明,该菌株为一新变种.定名为平阳链霉菌(Streptom-yces pingyangensis n.SP)。其产生的抗肿瘤物质为多组分的复合物,各种组分具有共同的主核(糖肽),彼此间的差别在于侧链(末端胺)不同。对争光霉素各组分的理化性质研究表明,其A_2、A_5、B_2等组分与日本报导的博莱霉素相应的组分A_2 、A_5、B_2等相同。我们对争光霉素单一组分A_2、A_5、A_6、B_2 等的抗菌谱及其对小鼠移植性食管癌的组织分布及抑瘤作用进行了比较研究,本文报告研究结果。 材料方法与结果 实验使用的争光霉素各组分是去铜的冷冻干燥粉末,易溶于水,均为单一组分物质。争光霉素A_2、A_4、A_5、A_6、B_2制品是我所化学室与天津河北药厂协作从该厂产品中分     

A2B型腺苷受体拮抗剂的研究进展

A_2B腺苷受体参与激活肥大细胞和炎症细胞的活性功能，选择性A_2B腺苷受体作为潜在的药物靶点正逐渐成为一个充满希望的研究热点。本文对近年来国内外有关A_2B腺苷受体拮抗剂的文献资料进行了分析和归纳。首先介绍了选择性A_2B腺苷受体拮抗剂药理学作用和开发意义；然后对选择性A_2B腺苷受体拮抗剂的开发现状、构效关系及拮抗剂与受体相互作用情况作了深入讨论；最后，对选择性A_2B腺苷受体拮抗剂研发前景进行了展望。     

博来霉素A6抗人体肝癌的实验研究

用克隆形成测定法检查博来霉素A₆对人肝癌、鼻咽癌和胃癌等细胞系的杀伤作用,发现对肝癌BEL-7402细胞有高度活性,半数杀伤浓度(IC₅₀)为6×10^-11mol/L。用流式细胞光度术研究表明,博来霉素A₆对肝癌BEL-7402细胞有G₂期阻断作用。在裸鼠可以耐受的剂量下,博来霉素A₆对移植性人肝癌的肿瘤生长有显著抑制作用,抑制率为75%。研究结果提示博来霉素A₆有可能用于肝癌化疗。     

3-氨基苯甲酰胺增强平阳霉素的抗瘤作用

3 AB是聚(ADP-R)合成酶抑制剂,能在体外和体内协同地增加平阳霉素对S₁₈₀的抑制作用,而本身不具有抗瘤与细胞毒作用。这种加强作用与3 AB的剂量有关,剂量加强因子为4。在体内实验中加用3 AB后,平阳霉素对小鼠S₁₈₀的抑制率由原来的32.3%,41.4%,37.4%和66.0%分别增至60.1%,72.4%,68.3%和77.8%,我们还在体内探讨了量效关系及给药方式的影响。     

Differential regulation of adenosine A1 and A2A receptors in serotonin transporter and monoamine oxidase A-deficient mice

The serotonin (5HT) transporter (5HTT) removes 5HT from the synaptic cleft and is thus critical to the control of serotonergic neurotransmission. Mice with a targeted inactivation of the 5HTT represent a novel and unique tool to study serotonergic system functioning. Because the release of 5HT is regulated by adenosine, we investigated 5HTT-deficient mice for possible adaptive changes of adenosine A₁ and A_2A receptors. A₁ and A_2A receptors were studied by means of quantitative autoradiography using the radioligands [³H]8-cyclopentyl-1,3-dipropylxanthine and [³H]CGS 21680, respectively. A comparison of 5HTT knockout versus control mice revealed upregulation of A₁ receptors in the dorsal raphe nucleus (DRN, +21%), but not in any of the serotonergic projection areas, and downregulation of A_2A receptors in basal ganglia. The adaptive changes of A₁ and A_2A receptors in 5HTT-deficient mice are likely to represent a compensatory neuroprotective effect mediated by the adenosinergic modulatory system. For comparison, these receptors were also studied in monoamine oxidase A (MAOA) knockout mice and in 5HTT/MAOA double knockout mice. 5HTT/MAOA double knockout mice showed adaptive changes of adenosine A₁ and A_2A receptors similar to 5HTT knockout mice, while investigation of MAOA-deficient mice revealed an upregulation of A_2A receptors, which may relate to a role of both MAOA and adenosine A_2A receptors in anxiety.     

The coexistence of adenosine A1 and A2 receptors in guinea-pig aorta

The effects of adenosine, 5'-(N-ethyl)carboxamidoadenosine (NECA), 2-chloroadenosine (2-CA), No-cyclohexyladenosine (CHA) and N6(R-2-phenylisopropyl)-adenosine (R-PIA) on the tone of phenylephrine-constricted guinea-pig isolated aorta have been examined. For aortic relaxation the analogues exhibited the following rank order of potency: NECA > adenosine > 2-CA > R-PIA > CHA. This is consistent with previous reports that relaxation of this tissue is mediated by the adenosine A₂ receptor. An unexpected finding was that R-PIA, 2-CA and CHA all induced contractions at concentrations lower than were required for relaxation, giving a biphasic dose-response curve. Neither NECA nor adenosine contracted the aorta. This is consistent with activation of vascular A₁ receptors. An A₁-selective concentration of the antagonist l,3-dipropyl-8-cyclopentyl xanthine abolished the contraction elicited by R-PIA in the guinea-pig aorta. This further suggests that the contraction is mediated by a₁ receptors.     

Mechanisms of inhibition of phospholipase A2

Differential scanning calorimetry (d.s.c.) and assays of phospholipase A₂ activity were used as tools to distinguish between drugs which interact with the phospholipids and those which interact directly with the enzyme. Cholesterol lowered the transition temperature (tc) and reduced the heat absorbed at transition and inhibited phospholipase A₂ activity on liposomes prepared from dipalmitoyllecithin-cholesterol. Subsequent addition of filipin to these liposomes overcame the inhibitory effect. Cholesterol therefore inhibits phospholipase A₂ by interacting with the membrane phospholipids. Mepacrine and phentermine did not interact with dipalmitoyl-lecithin (DPL) as determined by d.s.c., but reduced the rate of hydrolysis induced by purified phospholipase A₂ by a direct interaction with the enzyme. The anaesthetics, ethrane, halothane and trichloroethylene, inhibited phospholipase A₂ more than 90 per cent and were found to interact with DPL to modify membrane fluidity and lower the transition temperature. However, they also appeared to interact directly with the enzyme because the inhibitory effect was not overcome either by assaying phospholipase A₂ at the new tc or by a ten-fold increase in Ca²⁺ concentration. The anti-inflammatory steroids hydrocortisone, dexamethasone and betamethasone did not affect the rate of hydrolysis of DPL liposomes induced by phospholipase A₂ even at 2:1 w/w steroid/lipid. Furthermore, these steroids were found to be without any effect on membrane fluidity as examined by d.s.c. and microviscosimetry.     

Phospholipase A2 electrophoretic variants in reptile venoms

The venoms of poisonous reptiles provide rich sources of phospholipase A₂. We have investigated the potential usefulness of this enzyme in the characterization of venoms and in biochemical taxonomy of venomous reptiles using a recently developed electrophoretic technique. Venoms from Elapidae (18 species examined), especially Australian elapids (6), exhibited high phospholipase A₂ activity and complex electrophoretic patterns. Dendroaspidae venoms (3) exhibited low or non-detectable phospholipase A₂ activity in simple patterns. Viperidae venoms (5) yielded phospholipase A₂ patterns with intermediate complexity and activity. Sea snake venoms (3) exhibited low phospholipase A₂ activity and patterns of variable complexity. Crotalidae venoms (16) exhibited low or no detectable phospholipase A₂ activity in simple patterns. The single colubrid venom examined yielded low phospholipase A₂ activity and a simple electrophoretic pattern. Helodermatidae venoms (2) showed high activity with characteristic, complex patterns. In general, the venoms of snakes widely regarded as being more advanced exhibited phospholipase A₂ patterns of lower complexity and activity. The complexity of the patterns obtained precludes extensive usefulness of the technique for taxonomy at the family and subfamily level, but it may make the technique useful for taxonomy at the subspecies level.