張雪梅, 唐小海, 張光祥, 冉茂盛, 黃源芳, 彭 琳

(1. 四川師范大學(xué) 生命科學(xué)學(xué)院, 四川 成都 610101; 2. 重慶萊美藥業(yè)股份有限公司, 重慶 401336)

果膠阿霉素的抗腫瘤活性研究

張雪梅1,2, 唐小海1,2, 張光祥1, 冉茂盛2, 黃源芳2, 彭 琳1,2

(1. 四川師范大學(xué) 生命科學(xué)學(xué)院, 四川 成都 610101; 2. 重慶萊美藥業(yè)股份有限公司, 重慶 401336)

探討果膠阿霉素的抗腫瘤活性,將果膠與阿霉素經(jīng)過(guò)縮合反應(yīng)生成果膠阿霉素軛合物(PAC);通過(guò)高壓均質(zhì)和冷凍干燥的方法制備成PAC納米混懸劑;采用CCK-8方法考察PAC對(duì)8種人源性腫瘤細(xì)胞株的生長(zhǎng)抑制作用;以KM小鼠H22腹水為模型,考察PAC對(duì)腹水瘤的抗腫瘤活性、對(duì)免疫器官的影響以及H22腹水小鼠生存期的影響;初步考察PAC對(duì)KM小鼠的急性毒性,并與阿霉素作對(duì)比.PAC對(duì)8種人源性腫瘤細(xì)胞株均有抑制作用,在阿霉素質(zhì)量濃度為4、8 μg/ml時(shí),對(duì)各種腫瘤細(xì)胞的生長(zhǎng)抑制率達(dá)到50%以上.PAC能有效地抑制H22腹水的生長(zhǎng),在阿霉素質(zhì)量分?jǐn)?shù)為2.5、5、10 mg/kg時(shí)產(chǎn)生的腹水量分別為3.32±0.41、0.96±0.17、0 mL.生存期方面,從接種腫瘤后至45 d時(shí),PAC 10 mg/kg組未出現(xiàn)小鼠死亡,PAC 5 mg/kg組和2.5 mg/kg組存活率分別為75%、60%,阿霉素組2.5 mg/kg存活率80%,而對(duì)照組在25 d以內(nèi)全部死亡.PAC對(duì)KM小鼠的LD50為49.32 mg/kg,而阿霉素對(duì)KM小鼠的LD50為14.70 mg/kg.結(jié)論:PAC能有效地抑制細(xì)胞的生長(zhǎng),有良好的抗腫瘤作用.PAC能有效地抑制H22腹水的產(chǎn)生,對(duì)免疫器官的影響小于阿霉素,能明顯延長(zhǎng)腹水癌小鼠的生存期.PAC的LD50較ADM提高了2.36倍,毒性反應(yīng)大大降低,有望開(kāi)發(fā)成一種高效低毒的高分子抗癌藥物.

PAC; ADM; EPR效應(yīng); 抗腫瘤; H22腹水; LD50

阿霉素(ADM) 是從放線菌發(fā)酵液中提取的重要的氨基糖苷類(lèi)抗生素,可抑制DNA和RNA的合成,抗瘤譜較廣,用于治療血液系統(tǒng)癌癥、各種實(shí)體瘤和肉瘤.由于ADM是小分子,給藥后分布到全身各部位,沒(méi)有靶向性,在殺滅癌細(xì)胞的同時(shí)也危害正常細(xì)胞,在達(dá)到抗癌療效的同時(shí)也產(chǎn)生嚴(yán)重的毒副作用,特別是有明顯的心臟毒性和腎毒性[1-3].此外,由于多藥耐藥性以及ADM在體內(nèi)較短的半衰期,大大限制了該藥的臨床應(yīng)用[4-6].為了改變ADM進(jìn)入體內(nèi)后的分布情況,以及減小毒副作用,靶向給藥系統(tǒng)應(yīng)運(yùn)而生,特別是近幾十年得到快速發(fā)展.靶向給藥系統(tǒng)(TDS)系利用載體負(fù)載藥物,通過(guò)局部給藥或全身血液循環(huán)選擇性地使藥物濃集于靶器官、靶組織、靶細(xì)胞以提高療效、降低毒副作用的新型給藥手段.將ADM與高分子聚合物果膠通過(guò)酰胺鍵反應(yīng)相結(jié)合,由于增強(qiáng)通透和滯留效應(yīng)(EPR效應(yīng))以及腫瘤細(xì)胞較強(qiáng)的吞噬作用,該軛合物在腫瘤細(xì)胞大量積累,在溶酶體的作用下釋放出有效成分,用于定向治療腫瘤,以提高其治療效果和降低毒副作用[7-8].該軛合物與傳統(tǒng)的藥物相比,使靶部位的藥物質(zhì)量濃度增加了數(shù)倍,有的甚至達(dá)到數(shù)十倍[9-11],在腫瘤定向治療領(lǐng)域具有很好的應(yīng)用前景.

許多惡性腫瘤,如胃癌、肝癌、卵巢癌易發(fā)生腹膜轉(zhuǎn)移癌后形成癌性腹水.目前常用治療腹膜轉(zhuǎn)移癌的治療方法為手術(shù)加全身化療,但不能有效地提高患者的生存質(zhì)量.腹腔注射高分子前藥,一方面可以減少化療藥物本身對(duì)腹膜的刺激性,另一方面由于腹膜-血漿屏障的存在,可以防止腹膜對(duì)大分子藥物的吸收,使腹腔內(nèi)維持高質(zhì)量濃度的藥物質(zhì)量濃度,外周血管則保持相對(duì)較低的藥物質(zhì)量濃度,因此增加了藥物對(duì)游離癌細(xì)胞的細(xì)胞毒作用[12],能有效改善患者的生存期和生活質(zhì)量,是一種值得推薦的治療方案.

1 材料和方法

1.1 藥品試劑、儀器與細(xì)胞株 1) 藥品試劑:DMEM高糖培養(yǎng)基、青霉素鏈霉素溶液、PBS緩沖液、新生小牛血清(賽默飛世爾生物化學(xué)制品北京有限公司);CCK-8檢測(cè)試劑(DOJINDO);注射用鹽酸ADM(深圳萬(wàn)樂(lè)藥業(yè)有限公司);PAC(實(shí)驗(yàn)室制備);2) 儀器:生物倒置顯微鏡(上海光學(xué)儀器廠);培養(yǎng)箱(SANYO);離心機(jī)(XiangYi);超凈工作臺(tái)(蘇靜安泰);水浴鍋(上海躍進(jìn)醫(yī)療器械有限公司);酶標(biāo)儀(Thermo FC);3) 細(xì)胞株與動(dòng)物:SMMC-7721;Bel-7402;MCF-7/ADR;HCT-116;Hela;SiHa;SKOV3;HO-8910;K562;H22.以上細(xì)胞由四川大學(xué)生物治療國(guó)家重點(diǎn)實(shí)驗(yàn)室惠贈(zèng).KM小鼠(成都達(dá)碩生物科技有限公司).

1.2 方法

1.2.1 PAC納米混懸劑的制備 將果膠與ADM通過(guò)縮合劑發(fā)生酰胺化反應(yīng)生成PAC軛合物.用紫外分光光度法測(cè)定其載藥量為15%.再采用高壓均質(zhì)法制備PAC納米混懸劑,測(cè)得其粒徑為150 nm左右,通過(guò)冷凍干燥方法制備成制劑[13].

1.2.2 CCK-8方法檢測(cè)PAC的體外抗腫瘤活性 取對(duì)數(shù)生長(zhǎng)期的各種腫瘤細(xì)胞制成單細(xì)胞懸液,計(jì)數(shù)并調(diào)整細(xì)胞體積濃度大約為105個(gè)/mL,取96孔板,每孔加入100 μL的細(xì)胞懸液,大約5 000個(gè)細(xì)胞,放入37 ℃,體積分?jǐn)?shù)5%CO2的孵箱,12 h后,將細(xì)胞分成3組:ADM組、PAC組、陰性對(duì)照組.ADM組加入質(zhì)量濃度梯度的藥物(8、4、2、1、0.5、0.25 μg/mL).PAC組根據(jù)載藥量計(jì)算,加入質(zhì)量濃度梯度的ADM質(zhì)量濃度(8、4、2、1、0.5、0.25 μg/mL),每個(gè)質(zhì)量濃度設(shè)置4個(gè)復(fù)孔.設(shè)置不加藥物的陰性對(duì)照組以及不加細(xì)胞和不加藥物的空白對(duì)照組.放入37 ℃、體積分?jǐn)?shù)5% CO2孵箱,孵育48 h.輕輕吸掉孔內(nèi)的液體,每孔加入90 μL的新鮮培養(yǎng)基,再加入10 μL的CCK-8檢測(cè)試劑,放入37 ℃、5% CO2孵箱孵育2 h后,在酶標(biāo)儀450 nm處檢測(cè)光密度(OD值).計(jì)算藥物對(duì)細(xì)胞的抑制率為

1.2.3 PAC對(duì)H22腹水瘤的抗腫瘤活性 取液氮保存的H22細(xì)胞株一支,放入37 ℃的水浴鍋,使其快速融化,加入生理鹽水重懸,離心1 000 r/min,3 min.棄掉上層液體,細(xì)胞加入1 mL的生理鹽水重懸.取正常的雌性KM小鼠,腹腔注射0.2 mL的細(xì)胞懸液,待小鼠長(zhǎng)出腹水后,傳1～2代后,在無(wú)菌條件下取腹水,離心1 000 r/min,3 min,棄掉上層液體,沉淀的細(xì)胞用生理鹽水重懸,混勻后用臺(tái)盼藍(lán)進(jìn)行細(xì)胞計(jì)數(shù),確認(rèn)活細(xì)胞數(shù)大于95%,根據(jù)計(jì)數(shù)結(jié)果調(diào)整細(xì)胞濃度為1×107個(gè)/mL.取100只體重在18～22 g的正常雌性小鼠,每只腹腔接種0.1 mL的細(xì)胞懸液.3 d后,稱取小鼠體重,將小鼠分成5組:PAC 10 mg/kg組、PAC 5 mg/kg組、PAC 2.5 mg/kg組、ADM 2.5 mg/kg組和陰性對(duì)照組(相同體積的生理鹽水),按照體重腹腔給藥,間隔3 d第二次按照上述方法給藥.給藥期間,觀察小鼠的毛發(fā)、食欲、精神狀態(tài)等情況.第二次給藥3 d后,每組小鼠隨機(jī)取10只小鼠稱體重后全部脫脊椎處死,無(wú)菌條件下收取腹水,計(jì)算腹水的重量.解剖取小鼠的胸腺、脾臟稱重,計(jì)算小鼠的胸腺、脾臟指數(shù).每組剩余10只飼養(yǎng),考察動(dòng)物的生存期.動(dòng)物的飼養(yǎng)條件:溫度25 ℃,濕度60%.飼養(yǎng)條件為SPF級(jí)(無(wú)特定病原體),整個(gè)實(shí)驗(yàn)研究過(guò)程遵從實(shí)驗(yàn)動(dòng)物飼養(yǎng)管理和使用指南.

1.2.4 PAC的急性毒性 將120只KM小鼠隨機(jī)分成12組,每組10只,腹腔注射PAC混懸液,給藥劑量分別為ADM質(zhì)量分?jǐn)?shù)19.69、26.25、35.00、46.67、62.22、82.96 mg/kg,劑間比為0.75.腹腔注射ADM溶液,給藥劑量分別為8.00、10.00、12.50、15.63、19.53、24.41 mg/kg,劑間比為0.8.給藥后觀察14 d,記錄各組動(dòng)物食欲、精神狀態(tài)以及死亡數(shù),計(jì)算LD50(半數(shù)致死量,簡(jiǎn)稱LD50).

2 結(jié)果

2.1 PAC對(duì)8種人源性腫瘤細(xì)胞的抑制作用 PAC對(duì)8種人源性腫瘤細(xì)胞均有較好的抑制作用,并與質(zhì)量濃度呈正相關(guān).對(duì)SMMC-7721、Bel-7402、HCT-116、SiHa、SKOV3、K562、HO-8910的生長(zhǎng)抑制作用與ADM相當(dāng)(P>0.05),在質(zhì)量濃度為ADM質(zhì)量濃度8、4 μg/mL時(shí),對(duì)腫瘤細(xì)胞的生長(zhǎng)抑制率達(dá)到50%以上.對(duì)耐ADM細(xì)胞株MCF-7/ADR的生長(zhǎng)抑制作用好于ADM(P<0.05).各種藥物質(zhì)量濃度-抑制率曲線見(jiàn)圖1.

2.2 PAC對(duì)H22腹水癌細(xì)胞的抑制作用 PAC10、5、2.5 mg/kg 3個(gè)劑量組產(chǎn)生的腹水量分別為0、0.96±0.17、3.32±0.41,ADM 2.5 mg/kg產(chǎn)生的腹水為0.86±0.41 mL,而陰性組產(chǎn)生的腹水量為9.82±2.34 mL,3個(gè)劑量組均能有效的抑制腹水的產(chǎn)生,且呈現(xiàn)出量效關(guān)系.PAC 10 mg/kg組、PAC 5 mg/kg組和PAC 2.5 mg/kg與對(duì)照組相比腹水的重量明顯減少,3個(gè)劑量組與對(duì)照組相比有明顯差別(P<0.05),ADM 2.5 mg/kg組與PAC 5 mg/kg組效果相當(dāng)(P>0.05).3個(gè)劑量組中,PAC 2.5 mg/kg和PAC 5 mg/kg組對(duì)免疫器官的影響小于ADM 2.5 mg/kg(P<0.05).從腹水瘤小鼠的生存期方面看,PAC能明顯延長(zhǎng)荷H22小鼠的生存期,陰性組小鼠在腫瘤接種15 d后由于腹水逐漸增多開(kāi)始出現(xiàn)死亡,且在25 d內(nèi)已經(jīng)全部死亡.PAC 10 mg/kg組未出現(xiàn)小鼠死亡,PAC 5 mg/kg、PAC 2.5 mg/kg、ADM 2.5 mg/kg組中有個(gè)別小鼠由于腹水量較大無(wú)法承受而死亡,分別死亡3、4、2只,記錄各組小鼠死亡時(shí)間作生存時(shí)間曲線.實(shí)驗(yàn)結(jié)果見(jiàn)圖2和表1.

2.3 PAC的急性毒性 在給藥后5～6 d,ADM組小鼠出現(xiàn)嗜睡、慵懶、立毛、體重減輕的現(xiàn)象,個(gè)別小鼠出現(xiàn)粘液便,隨著ADM劑量的加重動(dòng)物死亡數(shù)也逐漸增加.采用bliss法計(jì)算LD50為14.70 mg/kg(95%可信限為12.17～116.99 mg/kg).PAC組僅在46.67、62.22、82.96 mg/kg 3個(gè)劑量組出現(xiàn)立毛、嗜睡、體重減輕現(xiàn)象,采用bliss法計(jì)算LD50為49.23 mg/kg(95%可信限為42.17～57.94 mg/kg).病理結(jié)果可見(jiàn):ADM組12.5 mg/kg及其以上組出現(xiàn)胃腸粘膜損壞、局部有炎細(xì)胞浸潤(rùn).心肌細(xì)胞的細(xì)胞核消失、呈片狀壞死.而PAC組在46.67 mg/kg及其以上組出現(xiàn)胃腸有輕度病理?yè)p害、心肌細(xì)胞有點(diǎn)狀壞死,病理?yè)p害明顯減輕.PAC將ADM的LD50提高了大約2.36倍,而CCK-8的檢測(cè)結(jié)果顯示,PAC在降低了藥物毒性后,同時(shí)保持了其抗腫瘤活性.各實(shí)驗(yàn)小組小鼠的死亡情況見(jiàn)表2和3.

表 1 PAC對(duì)H22腹水以及免疫器官的影響

注:和NS比較,*P<0.05,**P<0.01;和ADM比較,#P<0.05,##P<0.01.

表 2 PAC各組給藥劑量以及小鼠死亡數(shù)

劑量/(mg/kg)動(dòng)物總數(shù)/只死亡動(dòng)物數(shù)/只10.6910026.2510035.0010046.6710462.2210482.961010

表 3 ADM各組給藥劑量以及小鼠死亡數(shù)

3 討論

腫瘤一直以來(lái)都威脅著人類(lèi)的身體健康,惡性腫瘤居全球死亡原因的第二位[14].目前治療腫瘤的方法常為手術(shù)加化療,而傳統(tǒng)的化療藥物常常都不具備選擇性,在殺滅腫瘤細(xì)胞的同時(shí),引起大量正常組織和細(xì)胞的死亡.臨床上,為了更好地控制腫瘤,往往需要多次化療,因此大多數(shù)癌細(xì)胞會(huì)對(duì)藥物產(chǎn)生多藥耐藥性.許多體外顯示有良好抗腫瘤活性的化療藥物,由于多藥耐藥性的產(chǎn)生,治療效果大大降低,影響化療效果[15].為了提高療效,加大化療藥物的用藥,又會(huì)引起嚴(yán)重的副反應(yīng),如骨髓移植、免疫力下降、惡心嘔吐等[16].

為了改善傳統(tǒng)化療藥物的不足,醫(yī)學(xué)研究者一直在尋找合適的治療方法,高效低毒的靶向化療藥物是抗癌藥物發(fā)展的方向和熱點(diǎn).研究發(fā)現(xiàn)腫瘤新生血管生成的速度很快,血管壁結(jié)構(gòu)存在缺陷,還伴有淋巴引流系統(tǒng)不完善,對(duì)高分子顆粒有較強(qiáng)的通透性和滯留效應(yīng)[17].將果膠與ADM制成一種抗腫瘤的高分子前藥,由于通透性、滯留效應(yīng)以及腫瘤細(xì)胞的吞噬作用,使ADM的質(zhì)量濃度在靶器官富集,減少用藥劑量,大大降低了ADM的毒副作用.PAC注射到體內(nèi)后,改變了藥物在體內(nèi)的分布,主要分布在網(wǎng)狀內(nèi)皮系統(tǒng)器官如肝臟、肺、脾,使心臟的藥物質(zhì)量濃度明顯降低,減少了藥物對(duì)實(shí)質(zhì)性器官的損害[13].由于PAC的LD50較ADM大大提高,臨床上有望增加藥物用量,提高抗腫瘤效果.體外實(shí)驗(yàn)顯示PAC能有效抑制腫瘤細(xì)胞的生長(zhǎng),對(duì)耐藥株細(xì)胞的抑制作用要好于ADM,提示高分子藥物由于進(jìn)入細(xì)胞方式的改變,可以克服由于多次化療而引起的部分多藥耐藥性,為臨床對(duì)化療藥物耐藥的患者的研究提供了重要的意義.PAC能有效地抑制H22癌腹水的產(chǎn)生,延長(zhǎng)腹水癌小鼠的生存期,為臨床上癌性腹水的治療提供了一條新路徑.

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(編輯 鄭月蓉)

Study on Anti-tumor Activity of Pectin-adriamycin Conjugate

ZHANG Xuemei1,2, TANG Xiaohai1,2, ZHANG Guangxiang1,

RAN Maosheng2, HUANG Yuanfang2, PENG Lin1,2

( 1. College of Life Science, Sichuan Normal University, Chengdu 610101, Sichuan; 2. China Chongqing Lummy Pharmaceutical Co., Ltd., Chongqing 401336

Pectin-adriamycin conjugate(PAC), was made by amidation condensation between pectin and adriamycin, and its anti-tumor activity was studied. PAC nano suspension was made by high pressure homogenization and freeze drying. The in vitro antitumor effect of PAC against eight kinds of tumor cells were evaluated by CCK-8 method. H22 ascitic tumor bearing mice were used to investigate the effects of PAC on tumor, ascites, thymus, spleen and survival rate. Then the acute toxicity of PAC was detected, using adriamycin as comparison. PAC could effectively inhibit the growth of eight tumor cell lines. The growth inhibition rates of PAC on these tumor cells reached more than 50% when adriamycin equivalent concentration of PAC between 4-8 μg/mL. H22 ascites tumor was significantly inhibited by PAC. As the adriamycin equivalent doses were 2.5,5 and 10 mg/kg , the volumes of the ascites were 3.32±0.41, 0.96±0.17 and 0 mL respectively. The negative effects of PAC on the immune organs was obviously smaller than adriamycin. After 45 d observation, the survival time of the mice from each groups was different. The survival rates were 60%,75% and 100% at 2.5,5 and 10 mg/kg of adriamycin equivalent dose while none of the mice in the untreated group were alive in 25 d. The LD50 of PAC on KM mice was 49.32 mg/kg, while the LD50 of adriamycin was 14.70 mg/kg. Therefore, PAC could effectively inhibit the cells growth and H22 ascites, and prolong the survival time of mice with ascites carcinoma, showing a good antitumor activity. The LD50 of PAC was increased 2.36 times compared to adriamycin and the toxicity was greatly reduced. The PAC could be expected to be developed into polymeric prodrug with high efficiency and low toxicity.

PAC; adriamycin; anti-tumor; EPR effect; H22 ascites mice; LD50

2015-11-17

科技部新藥創(chuàng)制“科技重大專(zhuān)項(xiàng)”(2011ZX09102-001)

R979.1

A

1001-8395(2016)06-0905-06

10.3969/j.issn.1001-8395.2016.06.024

*通信作者簡(jiǎn)介：唐小海(1963—),男,教授,主要從事靶向抗腫瘤藥物的研究,E-mail:pharmmateceo@aliyun.com.cn