处方和粒径减少提高难溶于水的抗疟疾药物的生物利用度

1、Formulation and Particle Size Reduction Improve Bioavailability of Poorly Water-Soluble Compounds with Antimalarial ActivityHongxing Wang, Qigui Li, Sean Reyes, Jing Zhang, Lisa Xie, VictorMelendez, Mark Hickman, andMichael P. KozarDecoquinate癸氧喹酯 (DQ) is highly effective at killing malaria parasite

2、s in vitro; however, it is extremely insoluble in water. In this study, solid dispersion method was used for DQ formulation which created a suitable physical form of DQ in aqueous phase for particle manipulation（固体分散体的方法是：DQ为处方，使其可以在水相中分散溶解）. Among many polymers and surfactants tested, polyvinylpyrr

3、olidone 10（聚乙烯吡咯烷酮10）, a polymer, and L-phosphatidylcholine（L-卵磷脂） or polysorbate【聚山梨醇酯（吐温）】, two surfactants, were chosen as DQ formulation components.The formulation particles were reduced to a mean size between 200 to 400 nm, which was stable in aqueous medium for at least three weeks（处方粒径平均200-4

4、00nm，在水相中至少稳定3周）. Pharmacokinetic (PK) studies showed that compared to DQ microparticle suspension（相比起微粒悬浮）, a nanoparticle formulation（纳米处方） orally dosed to mice showed a 14.47-fold increase in area under the curve (AUC) of DQ plasma concentration （小鼠口服后DQ血药浓度曲线下面积增加了14.17倍）and a 4.53-fold increase

5、 in AUC of DQ liver distribution（肝脏分配AUC增加了4.53倍）. WR 299666, a poorly water-soluble compound with antimalarial activity, was also tested and successfully made into nanoparticle formulation without undergoing solid dispersion procedure. We concluded that nanoparticles generated by using appropriate

6、formulation components and sufficient particle size reduction significantly increased the bioavailability of DQ and could potentially turn this antimalarial agent to a therapeutic drug.1. IntroductionDQ has been marketed as a veterinary medicine for inhibiting the growth of coccidiosis（球虫病） in the d

7、igestive system of poultry for many years without any obvious adverse effects （DQ作为一种兽药，可以抑制家禽消化系统球虫病的发展而没有明显的副作用）1. This compound has been shown experimentally to have efficacy against diarrheal（腹泻） disease caused by Cryptosporidium parvum（隐孢子虫）（对隐孢子虫引起的腹泻有作用） 2, 3. It has also been shown to be hig

8、hly effective against malaria parasites in both the blood and liver stages as shown in some rodent and primate malaria models（在某些啮齿和灵长类疟疾模型中，DQ在血液和肝脏阶段对疟原虫都非常有效） 48. Unlike the classic antimalarial drugs such as chloroquine（氯喹/氯奎宁）, which enters the red blood cells, is selectively accumulated in the

9、 Plasmodium lysosome（疟原虫溶酶体）, inhibits hematin body packaging, and forms highly toxic complex to the parasites by chloroquine and hematin binding, DQ has recently emerged as a potent in vitro and in vivo inhibitor of the Plasmodium liver stage（DQ在疟原虫肝脏阶段体内外均是强效的抑制剂） and acts by selectively and speci

10、fically inhibiting the parasites mitochondrial electron transport chain（DQ选择并特异抑制疟原虫的线粒体电子传递链） 6. DQ exhibited minimal cross-resistance with its analog compound atovaquone（阿托伐醌）, an existing antimalarial drug（DQ和它的同类药物-阿托伐醌显示了最小的交叉抗药性）. Although both DQ and atovaquone inhibit cytochrome bc1 complex（

11、虽然DQ和阿托伐醌都抑制细胞色素bc1复合体）, there is evidence that they have distinctly different modes of binding within the ubiquinol-binding site（泛醌结合的位点） of cytochrome b （但证据说明他们在细胞色素b泛醌结合位点是不同的）6. DQ has also shown to have potent activity against developing gametocytes（配子体）（DQ强力抑制配子体的形成-疟原虫的传播形式）, the parasite tr

12、ansmission form, which is additional evidence that the two drugs interact differently with the ubiquinol-binding site 7. The insolubility of DQ in water has limited its broad use as a systemic treatment agent for infectious diseases such as malaria and cryptosporidiosis（DQ在水的难溶性限制着其成为系统治疗的药物）. Curre

13、ntly this compound has not been developed as an active component for treating or preventing malaria in humans or animals.DQ, a 4-hydroxyquinoline（4-羟基喹啉）, is highly lipophilic and its water solubility is exceptionally low（高亲脂性，水溶性极低） 9. Water insoluble drugs which exhibit excellent in vitro potency

14、may be converted to useful therapeutic agents if sufficient improvement of bioavailability can be achieved（在体外有着强效作用的水难溶性药物在提高其生物利用度后，也许可以转化成有用的治疗药物）. Drug microparticles and nanoparticles have been shown to significantly improve drug permeability, bioavailability and enhance drug exposure for oral

15、and parenteral（胃肠外的） dosage forms（微粒和纳米粒药物显著提高药物穿透性，生物利用度和提高药物在口服和胃肠外的暴露）. This is based on previous data showing that reduction of drug crystals in size from 10 microns to 100nm particles generates a 100-fold increase in surface area to volume ratio （10微米-100nm的微粒增加了100倍的表体比）10. This increase in su

16、rface area has a profound impact on the dissolution rate and absorption of the molecule（增加的表面积对分子的溶出度和吸收有着重要影响）. The increased dissolution rate can significantly improve the performance of poorly watersoluble compounds（提高不溶于水的化合物的能力）.There are several techniques that are known to produce drug microp

17、articles and nanocrystals and to improve the dissolution rate of compounds with poor oral bioavailability 11. One of the current methods used to generate drug nanoparticles is by high pressure homogenization（高压均质法） (HPH) 12. Typically, drug microparticles and nanocrystals are generated in a liquid d

18、ispersion medium（药物微粒和纳米晶粒产生于液体分散介质） (e.g., by precipitation（沉淀） or a disintegration（崩解） process). The product obtained from this process is a suspension of small drug particles in a liquid stabilized by a surfactant or polymer（在这过程中可以获得在液体稳定的表面活性剂或聚合物的微小的药物悬浮颗粒） 1315. In contrast to micronized powd

19、ers, drug nanocrystals can be administered using very different administration routes（纳米药物和微粉相比，其给药途径不同） 10. Solid dispersion is also an important approach for improvement of bioavailability of poor water-soluble drugs （固体分散体也是一种提高水不溶性药物的重要方式）15, 16. It is a commonly used method for formulation of i

20、nsoluble drug molecules scattered in other water-soluble materials in a solid state prior to the addition of water（这种方式就是不容的药物先分散在其他以固体形式存在的水溶的材料中，然后加入水）. Recent reports in the literature have demonstrated the benefits in improved bioavailability by formulating drugs with reduced particle size. The

21、antimalarial agent, halofantrine（卤泛群）, was formulated into various solid dispersions, and the reformulated dispersions showed a five- to seven-fold increase in absolute bioavailability（卤泛群在固体分散体中的绝对生物利用度增加了5-7倍） 17. Other examples of drugs that have benefited from the reduction of drug particle size

22、s leading to increased bioavailability have also been reported （其他药物都因药物粒径减少而提高生物利用度）1821.In this study, we have made DQ in nanoparticle formulations by solid dispersion（使用固体分散体制备DQ纳米粒）, use of formulation carriers, and particle size reduction to increase DQ aqueous compatibility and bioavailability

23、. The formulation carriers included polymers such as PVP, specifically PVP 10, and surfactants such as PC or polysorbate 80 (Tween 80) or both. The particle size of DQ formulation was aimed to achieve nanometer range to improve its bioavailability（DQ处方目标是达到纳米级别）. A water insoluble compound, WR299666

24、, previously synthesized by our institute, has powerful prophylactic activity against Plasmodium falciparum in vitro and in mice against Plasmodium berghei via intramuscular injection. It was made in various particle sizes as an example for comparison with DQ to evaluate different techniques used to

25、 produce microparticle and nanoparticle drugs. Various preparations of these compoundswere administered to mice by PO (manually intragastric（胃肠内的） method) and their pharmacokinetic profiles studied. The drug concentrations in the plasma and the liver were determined by LCMS/ MS (Liquid Chromatograph

26、y/Mass Spectrometry) assay and analyzed with PKmodeling software.2. Materials and Methods2.1. Reagents and Animals. DQ, polyvinylpyrrolidone (PVP10 and providence), dextrose（右旋葡萄糖）, egg L-phosphatidylcholine (PC), polysorbate (Tween 80), and hydroxyethyl cellulose（羟乙基纤维素） (HEC) were purchased from S

27、igma, Saint Louis, Missouri. Poloxamer 407 was obtained from Spectrum ChemicalMFG Corporation (New Brunswick, NJ). Methanol, n-butyl chloride（1-氯丁烷）, ethanol, and all other reagents used in the study were of the highest purity commercially available. WR299666 was synthesized in this institute, and t

28、he structure is shown in Figure 2. Male ICR mice obtained from Charles River Laboratories were used in this study. On arrival, the animals were acclimated for 7 days (quarantine). Mice were 7 weeks of age upon the initiation of dosing. The animals were housed singly in a cage maintained in a room wi

29、th a temperature range of 1826C, 3468% relative humidity and a 12-hour light/dark cycles. Food and water were provided ad libitum during quarantine and throughout the study. The animals were fed a standard rodent maintenance diet. Research was conducted in compliance with the AnimalWelfare Act and o

30、ther federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, NRC publication, 2011 edition2.2. Drug Suspension Preparation. The suspension of DQ in HECT（0.5%的羟乙纤维素w/v和0.2%的吐温-80）(

31、0.5% hydroxyethyl cellulose (w/v) and 0.2% Tween 80 (v/v) in distilled water) was predispersed for 5minutes using a Misonix Ultrasonic Liquid Processor Q 500 (Q Sonica, LLC., Newtown,CT) at 15% amplitude with a 7mm diameter probe（DQ在HECT分散5min，使用超声波液体处理器，15%振幅，7mm直径探针）. The unit was then programmed

32、to sonicate at 25% amplitude for another 5 minutes, shut off for 2 minutes, and then restarted at 15% amplitude for 2 minutes（改用25%振幅超声5min，停止2min，再重新用15%振幅超声2min）. A comparison of DQ homogenization techniques was also conducted utilizing a homogenizer equipped with a 30mm open slotted generator run

33、ning at 2000022000 rpm for 5minutes（DQ均质方法则用一个装有30mm发电机的均质机，以20000-22000转每分钟，均质5min）. Without homogenization or sonication, DQ particles were not suspended evenly in water or HECT, and the particle size of the raw material could not be measured accurately. The information on the particle size was no

34、t available.The suspension of WR299666 was also prepared in HECT in distilled water, using a homogenizer (PROScientific Inc. Monroe, CT) with a 30mm open slotted generator to homogenize the drug powder mixture at 2000022000 rpm for 5 minutes（用同样均质的方法制备WR299666的悬浮液）. The particle size of the starting

35、 raw material of WR 299666 was above 70 m in average without treatment（未经处理的WR299666粒径是70微米）. Homogenization was repeated for one minute intervals in an ice bath until a stable mean particle size value was obtained for each sample（冰浴均质间隔1min，直至每个样品测出稳定的粒径为止）. A sonication instrument (Sonics Vibra Ce

36、ll Sonicator) was subsequently utilized in a cycle of 5-second pulses and 5-second standbys for a total of 20 minutes in an ice bath to further reduce the particle size of the drug in the suspension（进一步减少粒径，药物在冰浴中使用超声法，以5s脉冲5s静止的循环，持续20min）.The particle size was measured using a Horiba LA-950 partic

37、le size distribution analyzer (Kyoto, Japan), based on laser scattering（测量粒径，我们使用一台基于激光散射的粒径分散仪）. This particular instrument has a measurement period of less than 2 minutes from introduction of sample to data presentation with a sample requirement for testing of 25mg per measurement in a 150mL flow

38、cell, depending on the sample type. All DQ and WR2996666 preparations were measured at a range of transmittance between 8090%（DQ和WR299666的透射比范围是80-90%）. Various volumes ranging from 100L to 800 L of samples （100-800微升样品体积）with about 510mg solid （5-10mg固体重量）from each vial were tested in this instrume

39、nt to determine the mean particle size, which was the particle size parameter referred in the results.2.3. DQ Nanoparticle Preparation. DQ nanoparticles were prepared by solid dispersion of DQ with polymers and surfactants followed by particle size reduction using sonication and high pressure homoge

40、nization (HPH)（用表面活性剂和聚合物的固体分散体的方法制备DQ纳米粒，用超声和高压均质进一步减少粒径）. Prior to solid dispersion, DQ, poly vinyl pyrrolidone (PVP 10 or povidone), and surfactants of either PC or Tween 80 or both, was individually dissolved in a mixture of ethanol and nbutyl chloride (5 : 3)（未分散前，DQ，PVP10，吐温-80分别溶于乙醇：1-氯丁烷=5:3

41、的溶剂）. Each component was then added to the already dissolved DQ solution according to the formulation design（DQ溶解后，每种按处方设计加入）. The solvents were removed by rotary evaporation or dried under vacuum（溶剂通过旋蒸或真空干燥移去）. The dried materials were hydrated with water and particle size reduction was performed（

42、干物料与水化合，并进行降低粒径操作）.Such formulated DQ suspended in water was sonicated (Elma S 40H, Singen, Germany) for about 530 minutes or until the particle size reduced to less than 10 m（上述得到的DQ超声5-30min，直到粒径小于10m）. The suspension was then homogenized by HPH in high pressure homogenizer (Nano DeBEE, BEE intern

43、ational, Inc. South Easton, MA) at a drop of 15002500 bars and a reflux temperature of about 30C（在1500-2500bars压力下进行高压均质，回流温度约为30）. Continuous cold water flow was adopted for cooling the samples（冷水回流冷却样品）. The particle size was measured periodically and the HPH process continued until the particles

44、could not be further reduced（粒径定期检测，HPH也不断进行，直到粒径不能再小为止）. The piston pump stroke frequency utilized was 30 minutes, followed by a pause of 30 minutes, and the entire process was run for a total of 36 hours, which is equivalent to a total of 70190 passes or homogenization cycles.The resulting samples

45、 with nanoparticles were fast freeze dried in dry ice, soaked in acetone, and lyophilized（冻干） to powder or thin film under high vacuum at low temperature (91C) by using a lyophilizer （得到的纳米粒在干冰上快速冷冻干燥，用丙酮浸润，然后在高压和低温（-91摄氏度）用冻干机把样品冻干成粉末）(Lyo-Centre, Virtis, Gardiner,NY). The lyophilized materials wer

46、e stored at 4C for future use or reconstituted with water or normal saline (0.9% NaCl) for further study（冻干得到的东西4度保存待用或用水和生理盐水溶解待用）.2.4. WR299666 Nanoparticle Preparation. A suspension of WR299666 was predispersed utilizing an Ultrasonic Liquid Processor Q 500（WR299666悬液使用超声机进行超声）. The sample with p

47、articles less than 10 m was suspended in 3040mL of HECT and stirred for 10 minutes to achieve satisfactory dispersion（小于10微米的颗粒在30-40ml的HECT悬浮，搅拌10min，获得满意的分散效果）. The suspension was then homogenized with a high pressure homogenizer (HPH) set at a drop of 2000 bar for each pass (1 bar = approx 14.5ps

48、i) and a reflux temperature of about 30C（悬浮液使用均质机进行均质，每次2000bar，30摄氏度回流温度）. The piston pump stroke frequency duration was 20 minutes, followed by a pause of 10 minutes, and the process was then run for a total of 1 hour, which was equivalent to a total of 1015 passes or homogenization cycles（均质循环10-

49、15次，每次活塞泵工作20min，休息10min。然后运行1小时）.2.5. Evaluation of Drug Preparations. The rehydrated DQ nanoparticle formulations were evaluated by particle size measurement（再次水化的DQ纳米粒进行粒径测试）, determination of drug concentration in the whole suspension as well as in the fine particles less than 0.2 M, and PK stud

50、y in drug dosed animals（DQ全悬液和小于0.2微米的悬液测定药物浓度，动物体内进行药动学研究）. The particle size was monitored throughout the preparation procedure and before and after animal studies（粒径的监测贯穿制备过程和动物实验的前后）. To ensure that there was no change of the drug integrity in any of the formulation procedure, drug concentration

51、s and chromatography profiles of the drug molecule identity were examined by an Agilent HPLC (Agilent Technologies, Foster City, CA) with an isocratic mobile phase of 20% water and 80% acetonitrile (0.1% formic acid contained in water and acetonitrile, resp.)（为保证药物成分在处方过程中没有发生改变，使用安捷伦液相仪进行药物浓度监测和药物分

52、子鉴定，流动相是等度的20%水和80%乙腈，其中各含0.1%甲酸）. All drug preparations in aqueous system were diluted in methanol with vigorous vortex and then in acetonitrile for HPLC analysis（水相的药物使用甲醇稀释，并强烈涡旋，HPLC的分析则使用乙腈）. This was done to obtain complete extraction of the compound prior to loading the samples to HPLC（先获得化合物

53、的完整抽提物再进行HPLC的分析）. To evaluate the amount of DQ in the fine particles, formulation suspensions were prepared in the amount of 10 mg/mL of DQ in water, incubated at 37C for 2 hours and passed through a 0.2 M size filter（制备10mg/ml的DQ水悬液，37摄氏度孵育2小时，过0.2微米的微孔滤膜，然后进行DQ的含量测定）. The filtrates were used for

54、HPLC analysis following the same dilution steps as above（HPLC检测前也同样进行过膜）. In order to determine the actual dosage of the drug for animal studies, the concentration of the drug used for dosing animals was determined by HPLC without prior particle size fractionation with filtration（在进行药动学研究时，为了得到真实的药物

55、剂量，不进行过膜步骤，直接用HPLC检测）.2.6. PK Studies. PK studies were performed using single oral administration（单次口服给药进行药动学研究）. For each time point to be acquired, five 6-week old male ICR mice at 2832 g body weight were utilized（使用五只6周龄雄性ICR小鼠，体重28-32g）. The animals were dosed at 50mg/kg for WR299666 with mean p

56、article diameter of 1.5 M or 80mg/kg for DQ with nanoparticle size of 0.39 M（平均粒径1.5微米的WR299666的用量是50mg/Kg，平均粒径0.39微米的DQ则是80mg/kg）. In order to obtain valid values of drug concentrations for microsuspension of DQ with particle size of 36.88 M, the dosage was increased to 400mg/kg due to much less ef

57、ficient absorption of the drug in micronized particles than in nanoparticles（因为微米粒比纳米粒的吸收要差得多，要获取真实的微米DQ悬液浓度（粒径36.88微米），给药剂量要增至400mg/kg）. Both drug suspensions were dosed at 100 L/20 g（两种药物悬液是100微升每20g）. After dosing, plasma and liver samples were collected at each time point（给药后，在每个时间点收集血浆和肝脏样品）. The whole blood was collected by cardiac puncture（全血通过心脏穿刺收集）. Blood samples were collected in lithium heparin（肝素锂） tubes with