處方和粒徑減少提高難溶于水的抗瘧疾藥物的生物利用度

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（固體分散體的方法是：DQ為處方，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（吐溫）】, 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（處方粒徑平均200-4

4、00nm，在水相中至少穩(wěn)定3周）. Pharmacokinetic (PK) studies showed that compared to DQ microparticle suspension（相比起微粒懸?。? a nanoparticle formulation（納米處方） 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血藥濃度曲線下面積增加了14.17倍）and a 4.53-fold increase

5、 in AUC of DQ liver distribution（肝臟分配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（球蟲?。?in the d

7、igestive system of poultry for many years without any obvious adverse effects （DQ作為一種獸藥，可以抑制家禽消化系統(tǒng)球蟲病的發(fā)展而沒有明顯的副作用）1. This compound has been shown experimentally to have efficacy against diarrheal（腹瀉） disease caused by Cryptosporidium parvum（隱孢子蟲）（對隱孢子蟲引起的腹瀉有作用） 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（在某些嚙齒和靈長類瘧疾模型中，DQ在血液和肝臟階段對瘧原蟲都非常有效） 48. Unlike the classic antimalarial drugs such as chloroquine（氯喹/氯奎寧）, which enters the red blood cells, is selectively accumulated in the

9、 Plasmodium lysosome（瘧原蟲溶酶體）, 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在瘧原蟲肝臟階段體內(nèi)外均是強效的抑制劑） and acts by selectively and speci

10、fically inhibiting the parasites mitochondrial electron transport chain（DQ選擇并特異抑制瘧原蟲的線粒體電子傳遞鏈） 6. DQ exhibited minimal cross-resistance with its analog compound atovaquone（阿托伐醌）, an existing antimalarial drug（DQ和它的同類藥物-阿托伐醌顯示了最小的交叉抗藥性）. Although both DQ and atovaquone inhibit cytochrome bc1 complex（

11、雖然DQ和阿托伐醌都抑制細胞色素bc1復(fù)合體）, there is evidence that they have distinctly different modes of binding within the ubiquinol-binding site（泛醌結(jié)合的位點） of cytochrome b （但證據(jù)說明他們在細胞色素b泛醌結(jié)合位點是不同的）6. DQ has also shown to have potent activity against developing gametocytes（配子體）（DQ強力抑制配子體的形成-瘧原蟲的傳播形式）, 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在水的難溶性限制著其成為系統(tǒng)治療的藥物）. 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-羥基喹啉）, is highly lipophilic and its water solubility is exceptionally low（高親脂性，水溶性極低） 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（在體外有著強效作用的水難溶性藥物在提高其生物利用度后，也許可以轉(zhuǎn)化成有用的治療藥物）. Drug microparticles and nanoparticles have been shown to significantly improve drug permeability, bioavailability and enhance drug exposure for oral

15、and parenteral（胃腸外的） dosage forms（微粒和納米粒藥物顯著提高藥物穿透性，生物利用度和提高藥物在口服和胃腸外的暴露）. 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倍的表體比）10. This increase in su

16、rface area has a profound impact on the dissolution rate and absorption of the molecule（增加的表面積對分子的溶出度和吸收有著重要影響）. 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（高壓均質(zhì)法） (HPH) 12. Typically, drug microparticles and nanocrystals are generated in a liquid d

18、ispersion medium（藥物微粒和納米晶粒產(chǎn)生于液體分散介質(zhì)） (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（在這過程中可以獲得在液體穩(wěn)定的表面活性劑或聚合物的微小的藥物懸浮顆粒） 1315. In contrast to micronized powd

19、ers, drug nanocrystals can be administered using very different administration routes（納米藥物和微粉相比，其給藥途徑不同） 10. Solid dispersion is also an important approach for improvement of bioavailability of poor water-soluble drugs （固體分散體也是一種提高水不溶性藥物的重要方式）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（這種方式就是不容的藥物先分散在其他以固體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（鹵泛群）, was formulated into various solid dispersions, and the reformulated dispersions showed a five- to seven-fold increase in absolute bioavailability（鹵泛群在固體分散體中的絕對生物利用度增加了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 （其他藥物都因藥物粒徑減少而提高生物利用度）1821.In this study, we have made DQ in nanoparticle formulations by solid dispersion（使用固體分散體制備DQ納米粒）, 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處方目標是達到納米級別）. 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（胃腸內(nèi)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（羥乙基纖維素） (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%的羥乙纖維素w/v和0.2%的吐溫-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，使用超聲波液體處理器，15%振幅，7mm直徑探針）. 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%振幅超聲5min，停止2min，再重新用15%振幅超聲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均質(zhì)方法則用一個裝有30mm發(fā)電機的均質(zhì)機，以20000-22000轉(zhuǎn)每分鐘，均質(zhì)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（用同樣均質(zhì)的方法制備WR299666的懸浮液）. The particle size of the starting

35、 raw material of WR 299666 was above 70 m in average without treatment（未經(jīng)處理的WR299666粒徑是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（冰浴均質(zhì)間隔1min，直至每個樣品測出穩(wěn)定的粒徑為止）. 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（進一步減少粒徑，藥物在冰浴中使用超聲法，以5s脈沖5s靜止的循環(huán)，持續(xù)20min）.The particle size was measured using a Horiba LA-950 partic

37、le size distribution analyzer (Kyoto, Japan), based on laser scattering（測量粒徑，我們使用一臺基于激光散射的粒徑分散儀）. 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的透射比范圍是80-90%）. Various volumes ranging from 100L to 800 L of samples （100-800微升樣品體積）with about 510mg solid （5-10mg固體重量）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)（用表面活性劑和聚合物的固體分散體的方法制備DQ納米粒，用超聲和高壓均質(zhì)進一步減少粒徑）. 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，吐溫-80分別溶于乙醇：1-氯丁烷=5:3

41、的溶劑）. Each component was then added to the already dissolved DQ solution according to the formulation design（DQ溶解后，每種按處方設(shè)計加入）. The solvents were removed by rotary evaporation or dried under vacuum（溶劑通過旋蒸或真空干燥移去）. The dried materials were hydrated with water and particle size reduction was performed（

42、干物料與水化合，并進行降低粒徑操作）.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超聲5-30min，直到粒徑小于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壓力下進行高壓均質(zhì)，回流溫度約為30）. Continuous cold water flow was adopted for cooling the samples（冷水回流冷卻樣品）. The particle size was measured periodically and the HPH process continued until the particles

44、could not be further reduced（粒徑定期檢測，HPH也不斷進行，直到粒徑不能再小為止）. 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（凍干） to powder or thin film under high vacuum at low temperature (91C) by using a lyophilizer （得到的納米粒在干冰上快速冷凍干燥，用丙酮浸潤，然后在高壓和低溫（-91攝氏度）用凍干機把樣品凍干成粉末）(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（凍干得到的東西4度保存待用或用水和生理鹽水溶解待用）.2.4. WR299666 Nanoparticle Preparation. A suspension of WR299666 was predispersed utilizing an Ultrasonic Liquid Processor Q 500（WR299666懸液使用超聲機進行超聲）. 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微米的顆粒在30-40ml的HECT懸浮，攪拌10min，獲得滿意的分散效果）. 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（懸浮液使用均質(zhì)機進行均質(zhì)，每次2000bar，30攝氏度回流溫度）. 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（均質(zhì)循環(huán)10-

49、15次，每次活塞泵工作20min，休息10min。然后運行1小時）.2.5. Evaluation of Drug Preparations. The rehydrated DQ nanoparticle formulations were evaluated by particle size measurement（再次水化的DQ納米粒進行粒徑測試）, 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全懸液和小于0.2微米的懸液測定藥物濃度，動物體內(nèi)進行藥動學(xué)研究）. The particle size was monitored throughout the preparation procedure and before and after animal studies（粒徑的監(jiān)測貫穿制備過程和動物實驗的前后）. 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.)（為保證藥物成分在處方過程中沒有發(fā)生改變，使用安捷倫液相儀進行藥物濃度監(jiān)測和藥物分

52、子鑒定，流動相是等度的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（水相的藥物使用甲醇稀釋，并強烈渦旋，HPLC的分析則使用乙腈）. This was done to obtain complete extraction of the compound prior to loading the samples to HPLC（先獲得化合物

53、的完整抽提物再進行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（制備10mg/ml的DQ水懸液，37攝氏度孵育2小時，過0.2微米的微孔濾膜，然后進行DQ的含量測定）. The filtrates were used for

54、HPLC analysis following the same dilution steps as above（HPLC檢測前也同樣進行過膜）. 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（在進行藥動學(xué)研究時，為了得到真實的藥物

55、劑量，不進行過膜步驟，直接用HPLC檢測）.2.6. PK Studies. PK studies were performed using single oral administration（單次口服給藥進行藥動學(xué)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周齡雄性ICR小鼠，體重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（平均粒徑1.5微米的WR299666的用量是50mg/Kg，平均粒徑0.39微米的DQ則是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（因為微米粒比納米粒的吸收要差得多，要獲取真實的微米DQ懸液濃度（粒徑36.88微米），給藥劑量要增至400mg/kg）. Both drug suspensions were dosed at 100 L/20 g（兩種藥物懸液是100微升每20g）. After dosing, plasma and liver samples were collected at each time point（給藥后，在每個時間點收集血漿和肝臟樣品）. The whole blood was collected by cardiac puncture（全血通過心臟穿刺收集）. Blood samples were collected in lithium heparin（肝素鋰） tubes with