WO2015154677A1 - 药物组合物及其应用 - Google Patents

药物组合物及其应用 Download PDF

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WO2015154677A1
WO2015154677A1 PCT/CN2015/076102 CN2015076102W WO2015154677A1 WO 2015154677 A1 WO2015154677 A1 WO 2015154677A1 CN 2015076102 W CN2015076102 W CN 2015076102W WO 2015154677 A1 WO2015154677 A1 WO 2015154677A1
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solution
paclitaxel
lapachone
lpc
pharmaceutical composition
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French (fr)
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钱锋
高金明
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Tsinghua University
University of Texas System
University of Texas at Austin
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Tsinghua University
University of Texas System
University of Texas at Austin
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers

Definitions

  • the present invention relates to the field of medicine, and in particular to pharmaceutical compositions and uses thereof, and more particularly to pharmaceutical compositions, uses of the pharmaceutical compositions in the preparation of medicaments, and methods of preparing micelles.
  • Beta-lapachone is a novel, promising anticancer drug that causes apoptosis in a variety of cancer cells, especially in cancer cells that overexpress quinone oxidoreductase (NQO1), such as breast cancer. Apoptosis is particularly evident in cells, prostate cancer cells, pancreatic cancer, lung cancer cells, and leukemia cells. --lapa is poorly soluble, with a solubility in water of only 0.038 mg/ml and a high effective dose (>500 mg).
  • ⁇ -lapachone and hydroxypropyl- ⁇ -cyclodextrin can greatly improve its solubility and bioavailability, but due to high dose of hydroxypropyl
  • the basal- ⁇ -cyclodextrin HP ⁇ -CD
  • ⁇ -lapach has a weak binding ability to albumin, and it cannot be effectively formed by forming an albumin complex like paclitaxel.
  • the present invention aims to solve at least one of the technical problems in the related art to some extent. To this end, it is an object of the present invention to provide a means for effectively treating or preventing cancer.
  • the present invention has been completed based on the following findings of the inventors: At present, many drugs have low bioavailability in the body, and side effects are large. One of the important reasons is that the water solubility of the drug is poor, the dosage is large, and there is no targeting effect. Can not effectively act on the lesion location, some drugs may also cause damage to normal human tissues; some poorly water-soluble drugs are also very easy to crystallize, unable to form a physically stable pharmaceutical preparation.
  • the use of polymer micelles as a drug carrier can effectively solubilize certain poorly water-soluble drugs. However, for a drug that is too fast in crystallization, the drug quickly forms crystals and escapes from the micelles, and a stable system cannot be formed, so that it cannot be effectively applied.
  • the invention provides a pharmaceutical composition.
  • the pharmaceutical composition comprises: an active ingredient; and a crystallization inhibitor.
  • the crystallization inhibitor can effectively inhibit the crystallization of the active ingredient, thereby significantly increasing the bioavailability of the active ingredient.
  • the active ingredient is a drug molecule having a fast crystallization rate.
  • the expression "the drug molecule with fast crystallization rate” as used herein refers to a drug having a crystallization ability of "Class I", that is, the molten drug is crystallized during the cooling process in DSC, and the literature is referred to as "JABaird”. , B. Van Eerdenbrugh, and LSTaylor. A classification system to assess the crystallization tendency of organic molecules from undercooled melts. Journal of Pharmaceutical Sciences. 99: 3787-3806 (2010).
  • the active ingredient is a drug molecule having a low solubility.
  • low solubility drug means that the dose of the drug cannot be dissolved by a clinically acceptable volume of aqueous solution.
  • the solubility is 0.38 mg/mL, but the dose is greater than 500 mg, and the volume of the aqueous solution preparation is too large to be clinically administered intravenously.
  • the crystallization inhibitor is a drug molecule which can inhibit the crystallization of the active ingredient and has a pharmacological synergistic effect with the active ingredient.
  • the expression "the drug molecule which can inhibit the crystallization of the active ingredient and has a pharmacological synergistic effect with the active ingredient” as used herein means that the crystallization inhibitor has a strong physical interaction with the active ingredient, and a substance having a higher glass transition temperature Tg (greater than 20 ° C), wherein the quantitative measurement of the physical interaction can be performed by the Flory-Huggins equation interaction parameter x, with a melting point reduction
  • the interaction parameter of the crystallization inhibitor and the active ingredient measured by the method is x ⁇ 0.
  • the active ingredient is at least one selected from the group consisting of ⁇ -lapachone, ⁇ -lapachone analogs, derivatives, or prodrugs.
  • Applicants believe that any known analogs, derivatives, or prodrugs of ⁇ -lapachone, ⁇ -lapachone, and analogs, derivatives, or prodrugs of ⁇ -lapachone may be used.
  • the types can be referred to the following documents: Xinpeng Ma, Xiumei Huang, et al, Prodrug Strategy to Achieve Lyophilizable, High Drug Loading Micelle Formulations Through Diester Derivatives of ⁇ -Lapachone, Adv. Healthcare Mater. 2014, DOI: 10.1002/adhm.201300590.
  • the crystallization inhibitor is at least one selected from the group consisting of paclitaxel and paclitaxel analogs/derivatives.
  • the paclitaxel analog/derivative may be docetaxel, cabazitaxel or the like.
  • the active ingredient is ⁇ -lapachone
  • the crystallization inhibitor is paclitaxel
  • the weight ratio of ⁇ -lapach and the paclitaxel is 1:0.1 ⁇ 3.
  • the weight ratio of the paclitaxel to the ⁇ -lapachone is 1:1 and 1:3.
  • the effect of paclitaxel on the inhibition of ⁇ -lapachone crystallization is remarkable, and the bioavailability of ⁇ -lapachone is significantly improved, and the synergistic therapeutic effect between the two is obvious.
  • the pharmaceutical composition is in various micelles, albumin nano preparations, capsules, pills In the form of a dose, a tablet, a granule, an oral liquid, an internal cream, an aerosol or a spray. Thereby, administration is easy.
  • the pharmaceutical composition is in the form of micelles and the micelles are formed from a polyethylene glycol polylactic acid block copolymer.
  • the encapsulation efficiency of the active ingredient in the micelle is from 11.7% to 100%.
  • the bioavailability of the drug is high, and the effect of treating or preventing cancer, especially lung cancer, is remarkable.
  • the invention provides the use of a pharmaceutical composition as hereinbefore described in the manufacture of a medicament for the treatment or prevention of cancer, preferably lung cancer, more preferably non-small cell lung cancer.
  • the invention also provides a method of preparing a micelle.
  • the method comprises: (1) mixing a paclitaxel solution, a ⁇ -lapachone solution, and an amphiphilic polymer solution, wherein the paclitaxel solution, ⁇ -lapachone solution, and amphipathicity
  • the polymer solution is an organic solution; (2) evaporating the mixture obtained in the step (1) to form a film, preferably evaporating to form a film within 5 minutes; (3) performing ultrasonic treatment after adding water to the film; (4)
  • the sonicated product obtained in the step (3) is filtered to obtain the micelle.
  • the method can be quickly and efficiently prepared by using the method of the invention, and the operation is simple, easy to control, and has no special requirements on the equipment, and is suitable for large-scale production.
  • paclitaxel can effectively inhibit ⁇ -lapachone crystallization, thereby significantly increasing the drug loading and bioavailability of ⁇ -lapach, and the formed micelles have good stability and can be effectively used for treating or preventing cancer, especially It is lung cancer.
  • the paclitaxel solution is a solution of paclitaxel in acetonitrile.
  • the concentration of the paclitaxel solution is 4 mg/ml.
  • the ⁇ -lapachone solution is a solution of ⁇ -lapachone in acetonitrile.
  • the concentration of the ⁇ -lapachone solution is 4 mg/ml.
  • the amphiphilic polymer is a polyethylene glycol polylactic acid block copolymer. Therefore, it is safe and non-toxic and can be effectively used for clinical treatment.
  • the amphiphilic polymer solution is a solution of an amphiphilic polymer in acetonitrile.
  • the concentration of the amphiphilic polymer solution is 36 mg/ml.
  • the sonication is performed at 300 watts for 5 minutes. Thereby, the formation of micelles is facilitated, and the efficiency of preparing micelles is improved.
  • the filtration is carried out using a nylon filter having a pore size of 0.45 ⁇ m.
  • Figure 1a is a graph showing the results of DSC detection of LPC during cooling at a cooling rate of 10 ° C / min.
  • Figure 1b is a graph showing DSC results of sample powders 9/1 LPC/PTX, 7/3 LPC/PTX, 5/5 LPC/PTX, and LPC at a heating rate of 1 ° C/min;
  • FIG. 2 shows a chromatogram of a mixed solution of an LPC solution, a PTX solution, and LPC and PTX, according to an embodiment of the present invention, wherein
  • 2A is a chromatogram of a mixed solution of LPC solution (LPC) and LPC and PTX (5/5LPC/PTX) at a detection wavelength of 257 nm.
  • LPC LPC solution
  • PTX 5/5LPC/PTX
  • 2B is a chromatogram of a PTX solution (PTX) and a mixed solution of LPC and PTX (5/5LPC/PTX) at a detection wavelength of 227 nm;
  • FIG. 3 shows an LPC concentration-time curve of a supersaturated solution of an LPC supersaturated solution and a mixture of LPC and PTX, in accordance with one embodiment of the present invention
  • FIG. 4 shows a scanning electron micrograph of a sample precipitated from an LPC precipitated sample and a mixture of LPC and PTX, according to one embodiment of the present invention, wherein
  • Figure 4A is a scanning electron micrograph of a sample of LPC precipitated
  • 4B is a scanning electron micrograph of a precipitated sample of a mixture of LPC and PTX;
  • Figure 5 shows a powder X-ray diffraction spectrum of an LPC precipitated sample, a PTX precipitated sample, and a precipitated sample of LPC and PTX mixture (LPC/PTX), in accordance with one embodiment of the present invention
  • Figure 6 shows a photograph of different proportions of micelles in accordance with one embodiment of the present invention.
  • Figure 7 shows the results of DLS detection of different proportions of micelles according to an embodiment of the present invention, wherein
  • Figure 7A shows the results of DLS detection of LM micelles.
  • Figure 7B shows the results of DLS detection of 7L3PM micelles.
  • Figure 7C shows the results of DLS detection of 5L5PM micelles
  • Figure 8 shows a transmission electron micrograph of different proportions of micelles in accordance with one embodiment of the present invention.
  • Figure 9 shows a nuclear magnetic resonance spectrum of different proportions of micelles in accordance with one embodiment of the present invention.
  • Figure 10 shows an in vitro drug release profile of different proportions of micelles, in accordance with one embodiment of the present invention, wherein
  • Figure 10A is a drug release curve when phosphate buffer is used as a release medium
  • Fig. 10B is a drug release curve when salicylate is used as a release medium.
  • the invention provides a pharmaceutical composition.
  • the medicament comprises: an active ingredient; and a crystallization inhibitor.
  • the crystallization inhibitor can effectively inhibit the crystallization of the active ingredient, thereby significantly increasing the bioavailability of the active ingredient.
  • the active ingredient is a drug molecule having a fast crystallization rate.
  • the expression "the drug molecule with fast crystallization rate” as used herein refers to a drug having a crystallization ability of "Class I", that is, the molten drug is crystallized during the cooling process in DSC, and the literature is referred to as "JABaird”. , B. Van Eerdenbrugh, and LSTaylor. A classification system to assess the crystallization tendency of organic molecules from undercooled melts. Journal of Pharmaceutical Sciences. 99: 3787-3806 (2010).
  • the active ingredient is a drug molecule having a low solubility.
  • low solubility drug means that the dose of the drug cannot be dissolved by a clinically acceptable volume of aqueous solution.
  • the solubility is 0.38 mg/mL, but the dose is >500 mg, and the volume of the aqueous solution preparation is too large to be clinically realized.
  • the crystallization inhibitor is a drug molecule which can inhibit the crystallization of the active ingredient and has a pharmacological synergistic effect with the active ingredient.
  • the expression "the drug molecule which can inhibit the crystallization of the active ingredient and has a pharmacological synergistic effect with the active ingredient” as used herein means that the crystallization inhibitor has a strong physical interaction with the active ingredient, and It has its own higher glass transition temperature Tg (greater than 20 ° C), in which physical interaction can be qualitatively verified by spectroscopy, such as infrared, Raman, etc., quantitative measurement can use the Flory-Huggins formula (Flory-Huggins equation)
  • Tg glass transition temperature
  • the active ingredient is at least one selected from the group consisting of ⁇ -lapachone, ⁇ -lapachone analogs, derivatives, or prodrugs.
  • Applicants believe that any known analog, derivative, or prodrug of ⁇ -lapachone, ⁇ -lapachone, and analogs, derivatives, or prodrugs of ⁇ -lapachone may be used.
  • the types can be referred to the following documents: Xinpeng Ma, Xiumei Huang, et al, Prodrug Strategy to Achieve Lyophilizable, High Drug Loading Micelle Formulations Through Diester Derivatives of ⁇ -Lapachone, Adv. Healthcare Mater. 2014, DOI: 10.1002/adhm.201300590.
  • the crystallization inhibitor is at least one selected from the group consisting of paclitaxel and paclitaxel analogs/derivatives.
  • the paclitaxel analog/derivative may be docetaxel or cabazitaxel or the like.
  • ⁇ -Lappa is poorly soluble and has a solubility in water of only 0.038 mg/ml.
  • HP ⁇ -CD hydroxypropyl- ⁇ -cyclodextrin
  • ⁇ -lapach has a weak binding ability to albumin, and it cannot be effectively formed by forming an albumin complex like paclitaxel.
  • ⁇ -lapachone has a very high crystallization rate and is easily detached from the carrier when it is delivered to the body by a drug carrier, and the bioavailability is low.
  • the active ingredient is ⁇ -lapachone
  • the crystallization inhibitor is paclitaxel
  • the weight ratio of ⁇ -lapachone to the paclitaxel is 1:0.1. ⁇ 3.
  • the crystallization inhibitor paclitaxel can effectively inhibit the ⁇ -lapachone crystallization of the active ingredient, improve the stability of the system, the bioavailability of the active ingredient is remarkably improved, and ⁇ -lapach and paclitaxel have synergistic effects. To effectively treat or prevent cancer, especially The treatment effect on lung cancer is remarkable.
  • the weight ratio of the paclitaxel to the ⁇ -lapachone is 1:1 and 1:3.
  • the effect of paclitaxel on the inhibition of ⁇ -lapachone crystallization is remarkable, and the bioavailability of ⁇ -lapachone is significantly improved, and the synergistic therapeutic effect between the two is obvious.
  • the dosage form of the pharmaceutical composition is not particularly limited, and those skilled in the art can flexibly select according to actual conditions.
  • the pharmaceutical composition is in the form of a micelle, capsule, pill, tablet, granule, oral liquid, internal cream, aerosol or spray. Thereby, administration is easy.
  • Polymer micelles are attracting more and more attention as drug carriers.
  • the polymer micelles form a unique core-shell structure, the outer shell is formed by hydrophilic segments, and the inner core is formed by hydrophobic segments, which encapsulates the hydrophobic drug in the core of the micelle and protects the loaded drug.
  • the micelle size is generally 10-100 nm, which makes it possible to escape the phagocytosis of the human reticuloendothelial system (RES) and the environment of others, and at the same time increase the high permeability and retention effect (EPR) of solid tumors.
  • RES human reticuloendothelial system
  • EPR high permeability and retention effect
  • the pharmaceutical composition is in the form of micelles and the carrier of the micelles is a polyethylene glycol polylactic acid diblock copolymer (PEG-PLA).
  • PEG-PLA polyethylene glycol polylactic acid diblock copolymer
  • the active ingredient can be prevented from being excreted by the human reticuloendothelial system and degraded into non-toxic monomers, and the hydrophilic segment of PEG has the advantages of being easily soluble in water, easy to flow and low toxicity, and can be achieved. The effect of long loops.
  • the micellar system can effectively increase the drug loading and bioavailability of the active ingredient, thereby better functioning in treating or preventing cancer.
  • the encapsulation efficiency of the active ingredient in the micelle is from 11.7% to 100%.
  • the bioavailability of the drug is high, and the effect of treating or preventing cancer, especially lung cancer, is remarkable.
  • the invention provides the use of a pharmaceutical composition as hereinbefore described in the manufacture of a medicament for the treatment or prevention of cancer, preferably lung cancer, more preferably non-small cell lung cancer.
  • the invention also provides a method of preparing a micelle. According to an embodiment of the invention, the method comprises the steps of:
  • the paclitaxel solution, the ⁇ -lapachone solution, and the amphiphilic polymer solution are mixed, wherein the paclitaxel solution, the ⁇ -lapachone solution, and the amphiphilic polymer solution are all organic solutions.
  • the paclitaxel solution is a solution of paclitaxel in acetonitrile.
  • the concentration of the paclitaxel solution is 4 mg/ml.
  • the ⁇ -lapachone solution is a solution of ⁇ -lapachone in acetonitrile.
  • the concentration of the ⁇ -lapachone solution is 4 mg/ml.
  • the amphiphilic polymer is a polyethylene glycol polylactic acid diblock copolymer. Therefore, it is safe and non-toxic and can be effectively used for clinical treatment.
  • the amphiphilic polymer solution is a solution of an amphiphilic polymer in acetonitrile.
  • the concentration of the amphiphilic polymer solution is 36 mg/ml.
  • the mixture obtained in the step (1) is evaporated to form a film. According to an embodiment of the present invention, the mixture obtained in the step (1) is evaporated to form a film within 5 minutes.
  • Ultrasonic treatment is carried out after adding water to the film.
  • the sonication is performed at 300 watts for 5 minutes. Thereby, it is advantageous for dispersion and improves the efficiency of preparing micelles.
  • the sonicated product obtained in the step (3) is filtered to obtain the micelle.
  • the filtration is carried out using a nylon filter having a pore size of 0.45 ⁇ m.
  • the method can be quickly and efficiently prepared by using the method of the invention, and the operation is simple, easy to control, and has no special requirements on the equipment, and is suitable for large-scale production.
  • paclitaxel can effectively inhibit ⁇ -lapachone crystallization, thereby significantly increasing the drug loading and bioavailability of ⁇ -lapach, and the formed micelles have good stability and can be effectively used for treating or preventing cancer, especially It is lung cancer.
  • DSC differential scanning calorimetry
  • the sample powder and LPC obtained above were respectively placed in an aluminum pan covered with a pinhole, and the aluminum pan was placed on a sample stage of a DSC Q2000 Differential Scanning Calorimeter (TA Instruments, New Castle, DE, USA), first The sample in the aluminum pan is preheated to 105 degrees Celsius to remove the remaining solvent and moisture, then the sample is heated to above the melting point of LPC at a temperature increase rate of 1 degree Celsius/minute or 10 degrees Celsius/minute, and then quenched to 0 degrees Celsius. Wherein, the temperature at which all crystals are completely melted is referred to as T end .
  • the partial test results are shown in Fig. 1.
  • Fig. 1a is a DSC test result of LPC during cooling at a cooling rate of 10 ° C / min.
  • the upper left side of Fig. 1a is a polarizing microscope photograph of LPC;
  • Fig. 1b is The DSC test results of the sample powders 9/1 LPC/PTX, 7/3 LPC/PTX, 5/5 LPC/PTX, and LPC at a heating rate of 1 degree Celsius/minute.
  • Fig. 2A is a chromatogram of a mixed solution of LPC solution (LPC) and LPC and PTX (5/5LPC/PTX) at a detection wavelength of 257 nm
  • Fig. 2B shows a detection wavelength of 227 nm. Chromatogram of PTX solution (PTX) and mixed solution of LPC and PTX (5/5LPC/PTX).
  • LPC 20 mg of LPC was dissolved in 4 ml of methanol to obtain a LPC solution having a concentration of 5 mg/ml, and then 20 mg of a mixture of LPC and PTX was dissolved in 4 ml of methanol to obtain a mixed solution having an LPC concentration of 5 mg/ml.
  • PBS phosphate buffer solution
  • the concentration of LPC in the two supersaturated solutions was measured by HLPC at 2 min, 5 min, 10 min, 15 min, 20 min and 25 min, respectively, and the LPC concentration-time curve was plotted (see Fig. 3).
  • the chromatographic detection parameters were the same as in Example 2, and each sample was subjected to three repeated experiments.
  • the precipitates in the above supersaturated solution were collected and dried, respectively, using a FEI Quanta 200 Scanning Electron Microscope (Czech) and a powder X-ray diffractometer (PANalytical X'pert Powder X-ray diffractometer, Almelo, The Netherlands). The surface morphology and crystallization of the precipitated samples were observed. Scanning electron microscopy (SEM) photographs of LPC precipitated samples are shown in Figure 4A.
  • the LPC precipitated sample showed intact crystals, and in the presence of PTX, the morphology of the LPC crystal was different from that before, and the LPC and PTX mixture precipitated samples could not form intact crystals, indicating that PTX can inhibit LPC crystallization. It can also be seen from the results of Fig. 5 that the LPC crystallinity is significantly lowered when PTX is present. In summary, it is shown that PTX can effectively inhibit LPC crystallization under the condition of aqueous solution.
  • ⁇ -lapachone 40 mg was dissolved in 10 ml of acetonitrile, sonicated for 2 minutes to obtain a ⁇ -lapachone solution; 40 mg of paclitaxel (PTX) was dissolved in 10 ml of acetonitrile, and sonicated for 2 minutes.
  • a paclitaxel solution was obtained; 36 mg/ml of a polyethylene glycol polylactic acid block copolymer (PEG-PLA) aqueous solution was frozen, then sonicated for 30 minutes, and then lyophilized to dissolve 360 mg of the freeze-dried PEG-PLA in 10 ml.
  • the acetonitrile was sonicated for 2 minutes to obtain a polyethylene glycol polylactic acid block copolymer solution.
  • the ⁇ -lapachone solution, the paclitaxel solution and the polyethylene glycol polylactic acid block copolymer solution obtained above were mixed according to the ratios shown in Table 1, and the obtained mixture was sonicated for 2 minutes, at 5 Rotate to form a film in a minute, then add 10 ml of pure water at a temperature of 60 ° C to the obtained film, and after sonicating for 5 minutes, remove the unencapsulated micelle by a nylon filter having a pore size of 0.45 ⁇ m.
  • Paclitaxel and ⁇ -lapaquinone obtained different ratios of micelles, respectively. Photographs of different ratios of micelle solutions are shown in Figure 6.
  • the weight of the PE tube was weighed, and 0.5 ml of the micelle solution was added to a known weight of PE tube. After lyophilization, the weight of the lyophilized product was accurately weighed to calculate the packing density of the micelle.
  • Encapsulation rate mass of drug loaded in micelle / mass of drug added to micellar system ⁇ 100%
  • the particle size of the micelle is 40-50 nm, and it has been reported that this particle size range can effectively enhance the EPR effect.
  • TEM Transmission electron microscopy
  • Fig. 10A From the results of Fig. 10A, it can be seen that the presence of PTX in the presence of phosphate buffer as a release medium has little effect on the release of LPC. In 3 hours, the cumulative release of LPC in both LM and 5L5PM micelles reaches 50. %, while in the same time range, PTX is not released from PM micelles, depending on PM physicochemical properties. Compared with PM micelles, LPC can promote the release of PTX to some extent in 5L5PM micelles. It can be seen from Fig.
  • the toxicity test of the paclitaxel-resistant A549 cell line of the micelles prepared in Example 5 was carried out according to the following procedure:
  • Paclitaxel-resistant A549 cells were plated in 96-well black matrix (2000 cells/well). After 24 hours of culture, the old medium in 96-well plates was discarded and 200 ⁇ L of medium containing different drug micelle concentrations was added. The cells were cultured for 4 hours, and then cultured for 7 days after replacing 200 ⁇ L of fresh medium. After 7 days, the old medium was discarded, the cells were washed with 200 ⁇ L of PBS, and all PBS was discarded.
  • the 96-well plate was placed in a -80 ° C refrigerator for 2 hours, and after the cells were completely thawed, 200 ⁇ L of 1:1000 Hoechst 33258 and TNE (1 mM EDTA, 2 M NaCl) were added per well.
  • the dye solution of 10 mM Tris, pH 7.5) was stored at room temperature in the dark for 2 hours, and the plate was read by a Perkin Elmer instrument.
  • the excitation light was 356 nm and the emission light was 458 nm.
  • Example 5 The therapeutic effect of the micelles prepared in Example 5 on a paclitaxel-resistant tumor animal model was carried out according to the following procedure:
  • mice Female athymic nude mice (6-8weeks, 18-22g) were injected subcutaneously right hind leg 5 ⁇ 10 6-derived paclitaxel resistant human A549 NSCLC. When the tumor grew to approximately 200 mm 3 , the animals were re-randomized into groups of 5 animals each. The animals were administered once a week in the tail vein, and both the paclitaxel micelles and the composite micelles were administered in an amount of PTX contained therein, and PTX 30 mg/kg was administered three times in total. The blank control group was given an equal volume of blank micelles. After administration, the size of the tumor and the body weight of the animal were measured and weighed once every two days. The length (L) and width (W) of the tumor were measured using a vernier caliper, and the volume of the tumor was equal to L ⁇ W 2 /2.
  • the relative tumor inhibition rate of the PM group (PTX 30 mg/kg) against paclitaxel-resistant A549 human lung cancer xenograft nude mice model was 30%, and the equivalent dose of PTX was converted into 1L3PM.
  • the tumor inhibition rate was 95%.
  • the body weight changes of the PM group and the 1L3PM group did not fluctuate significantly.
  • the 1L3PM group was significantly better than the PM group in the paclitaxel-resistant A549 human lung cancer xenograft model, and no significant side effects were observed.
  • LPC and PTX composite nanomicelles have significant synergistic therapeutic effects on paclitaxel-resistant animal models.
  • the pharmaceutical composition of the present invention wherein the crystallization inhibitor is effective for inhibiting crystallization of the active ingredient, so that the bioavailability of the active ingredient in the pharmaceutical composition is very high.

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Abstract

提供了药物组合物及其应用，该药物组合物包含：活性成分；以及结晶抑制剂。

Description

药物组合物及其应用
优先权信息
本申请请求2014年4月8日向中国国家知识产权局提交的、专利申请号为201410138770.2的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本发明涉及医药领域，具体地，涉及药物组合物及其应用，更具体地，涉及药物组合物、药物组合物在制备药物中的用途和制备胶束的方法。
背景技术
β-拉帕醌(LPC)是一种新型的，有潜力的抗癌药物，能够引起多种癌细胞的凋亡，特别是对过表达醌氧化还原酶(NQO1)的癌细胞，例如乳腺癌细胞、前列腺癌细胞、胰腺癌、肺癌细胞及白血病细胞等，细胞凋亡效果尤其明显。β-拉帕醌水溶性较差，在水中的溶解度只有0.038mg/ml，而其有效剂量较高(>500mg)。有研究发现，β-拉帕醌与羟丙基-β-环糊精(HPβ-CD)之间的络合作用可以很大程度上提高其溶解度和生物利用度，但由于高剂量的羟丙基-β-环糊精(HPβ-CD)存在溶血副作用而无法在临床上满足β-拉帕醌的剂量需求。另外，β-拉帕醌与白蛋白的结合能力较弱，也无法像紫杉醇一样形成白蛋白络合物而有效应用。
然而，关于β-拉帕醌的临床研究仍有待深入，这就需要一种可用于临床新型药物制剂。
发明内容
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。为此，本发明的一个目的在于提出一种能够有效治疗或预防癌症的手段。
本发明是基于发明人的下列发现而完成的：目前，许多药物在体内的生物利用度不高，副作用大，其中一个重要原因是药物的水溶性差，药用剂量大，没有靶向性作用，不能有效作用于病变位置，一些药物还可能对人体正常组织造成伤害；有一些水溶性差的药物也非常易于结晶，无法形成物理稳定的药物制剂。采用聚合物胶束作为药物载体，能够有效增溶某些水溶性较差的药物。但对于结晶速度过快的药物，药物会很快形成结晶而从胶束中逸出，无法形成稳定的系统，进而仍无法得到有效地应用。经过大量的实验和艰苦的努力，发明人惊喜地发现，向易于结晶的药物中添加合适的、本身具备肿瘤治疗效果的结晶抑制剂，能够有效抑制药物的结晶，使药物形成稳定的药物制剂，提高药物的利用率，使药物在靶点位置缓慢释放，进而能够更好的发挥药效。
因而，在本发明的一个方面，本发明提供了一种药物组合物。根据本发明的实施例，该药物组合物包含：活性成分；以及结晶抑制剂。发明人发现，在本发明的药物组合物 中，结晶抑制剂能够有效抑制活性成分的结晶，从而显著提高活性成分的生物利用度。
根据本发明实施例的药物组合物，还可以具有如下附加技术特征：
根据本发明的实施例，所述活性成分为结晶速度快的药物分子。需要说明的是，在本文中所使用的表达方式“结晶速度快的药物分子”是指结晶能力为“I类”的药物，即在DSC中熔融药物在降温过程中结晶，文献参见“J.A.Baird,B.Van Eerdenbrugh,and L.S.Taylor.A classification system to assess the crystallization tendency of organic molecules from undercooled melts.Journal of Pharmaceutical Sciences.99:3787-3806(2010).”。
根据本发明的实施例，所述活性成分为溶解度低的药物分子。需要说明的是，在本文中所使用的表达方式“溶解度低的药物”是指药物的使用剂量不能被临床可接受体积的水溶液所溶解。比如，对于β-拉帕醌，其溶解度是0.38mg/mL，但是剂量大于500mg，水溶液制剂的体积太大，不能临床实现静脉注射。
根据本发明的实施例，所述结晶抑制剂为可以抑制活性成分结晶，并且和活性成分有药理学协同作用的药物分子。需要说明的是，在本文中所使用的表达方式“可以抑制活性成分结晶，并且和活性成分有药理学协同作用的药物分子”是指结晶抑制剂为和活性成分有较强物理相互作用，并且自身有较高玻璃化转变温度Tg(大于20℃)的物质，其中，物理相互作用的定量衡量可以用弗洛里-哈金斯公式(Flory-Huggins equation)相互作用参数x，用熔点降低的方法测得的结晶抑制剂和活性成分的相互作用参数x<0。
根据本发明的实施例，所述活性成分为选自β-拉帕醌、β-拉帕醌的类似物、衍生物、或者前药的至少一种。申请人认为可以采用现有的任何已知的β-拉帕醌、β-拉帕醌的类似物、衍生物、或者前药，关于β-拉帕醌的类似物、衍生物、或者前药的种类可参照以下文献：Xinpeng Ma,Xiumei Huang,et al,Prodrug Strategy to Achieve Lyophilizable,High Drug Loading Micelle Formulations Through Diester Derivatives ofβ-Lapachone,Adv.Healthcare Mater.2014,DOI:10.1002/adhm.201300590。
根据本发明的实施例，所述结晶抑制剂为选自紫杉醇、紫杉醇类似物/衍生物的至少一种。根据本发明的实施例，所述紫杉醇类似物/衍生物可以为多烯紫杉醇、卡巴他赛等。
根据本发明的实施例，所述活性成分为β-拉帕醌，所述结晶抑制剂为紫杉醇，并且所述β-拉帕醌与所述紫杉醇的重量比为的重量比为1：0.1～3。由此，结晶抑制剂能够有效抑制活性成分结晶，提高系统的稳定性，所述活性成分的生物利用度显著提高，并且β-拉帕醌和紫杉醇具有协同增效的作用，从而能够有效治疗或预防癌症，特别是对肺癌的治疗效果显著。
根据本发明的实施例，所述紫杉醇与所述β-拉帕醌的重量比为1：1和1:3。由此，紫杉醇抑制β-拉帕醌结晶的效果显著，β-拉帕醌的生物利用度明显提高，两者之间的协同治疗作用明显。
根据本发明的实施例，所述药物组合物呈各种胶束、白蛋白纳米制剂、胶囊剂、丸 剂、片剂、颗粒剂、口服液体、内服膏剂、气雾剂或喷雾剂的形式。由此，易于进行给药。
根据本发明的实施例，所述药物组合物呈胶束的形式，并且所述胶束是由聚乙二醇聚乳酸嵌段共聚物形成的。由此，不仅能够使活性成分免受人体网状内皮系统的吞噬及其他人体内环境的影响，在病变部位缓慢的进行释放，还能够有效提高活性成分的载药量及生物利用度，从而更好地发挥治疗或预防癌症的功效。
根据本发明的实施例，所述胶束中活性成分的包封率为11.7％～100％。由此，药物的生物利用较高，治疗或预防癌症，尤其是肺癌的效果显著。
在本发明的另一方面，本发明提供了前面所述的药物组合物在制备药物中的用途，所述药物用于治疗或者预防癌症，优选肺癌，更优选非小细胞肺癌。
在本发明的再一方面，本发明还提供了一种制备胶束的方法。根据本发明的实施例，该方法包括：(1)将紫杉醇溶液、β-拉帕醌溶液和两亲性聚合物溶液混合，其中，所述紫杉醇溶液、β-拉帕醌溶液和两亲性聚合物溶液均为有机溶液；(2)将步骤(1)中所得到的混合物蒸发至形成薄膜，优选5分钟内蒸发至形成薄膜；(3)向所述薄膜中加入水之后进行超声处理；(4)将步骤(3)中所得到的超声处理产物进行过滤，以便获得所述胶束。发明人发现，利用本发明的该方法能够快速有效地制备获得所述胶束，且操作简单、易于控制，对设备没有特殊要求，适合规模化生产。另外，紫杉醇能够有效抑制β-拉帕醌结晶，从而显著提高β-拉帕醌的载药量和生物利用度，且形成的胶束稳定性较好，能够有效用于治疗或预防癌症，特别是肺癌。
根据本发明的实施例，所述紫杉醇溶液为紫杉醇在乙腈中的溶液。根据本发明的实施例，所述紫杉醇溶液的浓度为4mg/ml。由此，有利于胶束的形成，提高制备胶束的效率。
根据本发明的实施例，所述β-拉帕醌溶液为β-拉帕醌在乙腈中的溶液。根据本发明的实施例，所述β-拉帕醌溶液的浓度为4mg/ml。由此，有利于胶束的形成，提高制备胶束的效率。
根据本发明的实施例，所述两亲性聚合物为聚乙二醇聚乳酸嵌段共聚物。由此，安全无毒，能够有效用于临床治疗。
根据本发明的实施例，所述两亲性聚合物溶液为两亲性聚合物在乙腈中的溶液。根据本发明的实施例，所述两亲性聚合物溶液的浓度为36mg/ml。由此，有利于胶束的形成，提高制备胶束的效率。
根据本发明的实施例，所述超声处理是于300瓦下，处理5分钟。由此，有利于胶束的形成，提高制备胶束的效率。
根据本发明的实施例，所述过滤是利用孔径为0.45μm的尼龙滤器进行的。由此，能够有效去除溶液中未被包封的，沉淀聚集的紫杉醇和β-拉帕醌。
附图说明
图1显示了根据本发明的一个实施例，不同配比LPC和PTX的混合物的DSC检测结果，其中，
图1a为冷却速度为10摄氏度/分钟时，冷却过程中LPC的DSC检测结果图，
图1b为升温速度为1摄氏度/分钟时，样品粉末9/1LPC/PTX、7/3LPC/PTX、5/5LPC/PTX以及LPC的DSC检测结果图；
图2显示了根据本发明的一个实施例，LPC溶液、PTX溶液和LPC和PTX的混合溶液的色谱图，其中，
图2A为检测波长为257nm条件下，LPC溶液(LPC)以及LPC和PTX的混合溶液(5/5LPC/PTX)的色谱图，
图2B为检测波长为227nm条件下，PTX溶液(PTX)以及LPC和PTX的混合溶液(5/5LPC/PTX)的色谱图；
图3显示了根据本发明的一个实施例，LPC过饱和溶液以及LPC和PTX混合物的过饱和溶液的LPC浓度-时间曲线；
图4显示了根据本发明的一个实施例，LPC沉淀样品以及LPC和PTX的混合物沉淀样品的扫描电子显微镜照片，其中，
图4A为LPC沉淀样品的扫描电子显微镜照片，
图4B为LPC和PTX的混合物沉淀样品的扫描电子显微镜照片；
图5显示了根据本发明的一个实施例，LPC沉淀样品、PTX沉淀样品以及LPC和PTX混合物沉淀样品(LPC/PTX)的粉末X射线衍射谱图；
图6显示了根据本发明的一个实施例，不同配比胶束的照片；
图7显示了根据本发明的一个实施例，不同配比胶束的DLS检测结果，其中，
图7A为LM胶束的DLS检测结果，
图7B为7L3PM胶束的DLS检测结果，
图7C为5L5PM胶束的DLS检测结果；
图8显示了根据本发明的一个实施例，不同配比胶束的透射电子显微镜照片；
图9显示了根据本发明的一个实施例，不同配比胶束的核磁共振谱图；
图10显示了根据本发明的一个实施例，不同配比胶束的体外药物释放曲线，其中，
图10A为以磷酸盐缓冲液作为释放介质时的药物释放曲线，
图10B为以水杨酸盐作为释放介质时的药物释放曲线。
发明详细描述
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
在本发明的一个方面，本发明提供了一种药物组合物。根据本发明的实施例，该药物 组合物包含：活性成分；以及结晶抑制剂。发明人发现，在本发明的药物组合物中，结晶抑制剂能够有效抑制活性成分的结晶，从而显著提高活性成分的生物利用度。
根据本发明实施例的药物组合物，还可以具有如下附加技术特征：
根据本发明的实施例，所述活性成分为结晶速度快的药物分子。需要说明的是，在本文中所使用的表达方式“结晶速度快的药物分子”是指结晶能力为“I类”的药物，即在DSC中熔融药物在降温过程中结晶，文献参见“J.A.Baird,B.Van Eerdenbrugh,and L.S.Taylor.A classification system to assess the crystallization tendency of organic molecules from undercooled melts.Journal of Pharmaceutical Sciences.99:3787-3806(2010).”。
根据本发明的实施例，所述活性成分为溶解度低的药物分子。需要说明的是，在本文中所使用的表达方式“溶解度低的药物”是指药物的使用剂量不能被临床可接受体积的水溶液所溶解。比如，对于β-拉帕醌，其溶解度是0.38mg/mL，但是剂量>500mg，水溶液制剂的体积太大，不能临床实现。
根据本发明的实施例，所述结晶抑制剂为可以抑制活性成分结晶，并且和活性成分有药理学协同作用的药物分子。需要说明的是，在本文中所使用的表达方式“可以抑制活性成分结晶，并且和活性成分有药理学协同作用的药物分子”是指结晶抑制剂为和活性成分有较强物理相互作用，并且自身有较高玻璃化转变温度Tg(大于20℃)的物质，其中，物理相互作用可以用光谱法，比如红外，拉曼等来定性验证，其定量衡量可以用弗洛里-哈金斯公式(Flory-Huggins equation)相互作用参数x，用熔点降低的方法测得的结晶抑制剂和活性成分的相互作用参数x<0或者为不远大于0的正值。
根据本发明的实施例，所述活性成分为选自β-拉帕醌，β-拉帕醌类似物、衍生物、或者前药的至少一种。申请人认为可以采用现有的任何已知的β-拉帕醌，β-拉帕醌的类似物、衍生物、或者前药，关于β-拉帕醌的类似物、衍生物、或者前药的种类可参照以下文献：Xinpeng Ma,Xiumei Huang,et al,Prodrug Strategy to Achieve Lyophilizable,High Drug Loading Micelle Formulations Through Diester Derivatives ofβ-Lapachone,Adv.Healthcare Mater.2014,DOI:10.1002/adhm.201300590。
根据本发明的实施例，所述结晶抑制剂为选自紫杉醇、紫杉醇类似物/衍生物的至少一种。根据本发明的实施例，所述紫杉醇类似物/衍生物可以为多西紫杉醇或者卡巴他赛等。
β-拉帕醌水溶性较差，在水中的溶解度只有0.038mg/ml，有研究发现，β-拉帕醌与羟丙基-β-环糊精(HPβ-CD)之间的络合作用可以很大程度上提高其溶解度和生物利用度，但由于存在溶血副作用而无法临床应用。并且，β-拉帕醌与白蛋白的结合能力较弱，也无法像紫杉醇一样形成白蛋白络合物而有效应用。此外，β-拉帕醌的结晶速度非常快，利用药物载体输送至体内时，很容易从载体上脱离，生物利用度较低。
在本发明的实施例中，所述活性成分为β-拉帕醌，所述结晶抑制剂为紫杉醇，并且所述β-拉帕醌与所述紫杉醇的重量比为的重量比为1：0.1～3。由此，结晶抑制剂紫杉醇能够有效抑制活性成分β-拉帕醌结晶，提高系统的稳定性，所述活性成分的生物利用度显著提高，并且β-拉帕醌和紫杉醇具有协同增效的作用，从而能够有效治疗或预防癌症，特别是 对肺癌的治疗效果显著。
根据本发明的实施例，所述紫杉醇与所述β-拉帕醌的重量比为1：1和1:3。由此，紫杉醇抑制β-拉帕醌结晶的效果显著，β-拉帕醌的生物利用度明显提高，两者之间的协同治疗作用明显。
根据本发明的实施例，所述药物组合物的剂型不受特别限制，本领域技术人员可以根据实际情况灵活选择。根据本发明的实施例，所述药物组合物呈胶束、胶囊剂、丸剂、片剂、颗粒剂、口服液体、内服膏剂、气雾剂或喷雾剂的形式。由此，易于进行给药。
聚合物胶束作为药物载体，越来越受到瞩目。聚合物胶束能够形成独特的核壳结构，外壳由亲水链段形成，内核由疏水链段形成，这样可以将疏水药物包裹在胶束的内核，并对所载入药物进行保护。胶束粒径一般为10-100纳米，使其可以逃离人体网状内皮系统(RES)的吞噬及其他人体内环境的影响，同时能够增加实体瘤的高通透性和滞留效应(EPR)。因而，根据本发明的实施例，所述药物组合物呈胶束的形式，并且所述胶束的载体是聚乙二醇聚乳酸二嵌段共聚物(PEG-PLA)。由此，能够阻止活性成分被人体网状内皮系统清除和降解成无毒的单体被排泄出体外，且亲水段的PEG具有易溶于水、链易流动和低毒的优点，可达到长循环的效果。此外，胶束体系还能够有效提高活性成分的载药量及生物利用度，从而更好地发挥治疗或预防癌症的功效。
根据本发明的实施例，所述胶束中活性成分的包封率为11.7％～100％。由此，药物的生物利用较高，治疗或预防癌症，尤其是肺癌的效果显著。
在本发明的另一方面，本发明提供了前面所述的药物组合物在制备药物中的用途，所述药物用于治疗或者预防癌症，优选肺癌，更优选非小细胞肺癌。
在本发明的再一方面，本发明还提供了一种制备胶束的方法。根据本发明的实施例，该方法包括以下步骤：
(1)将紫杉醇溶液、β-拉帕醌溶液和两亲性聚合物溶液混合，其中，所述紫杉醇溶液、β-拉帕醌溶液和两亲性聚合物溶液均为有机溶液。
根据本发明的实施例，所述紫杉醇溶液为紫杉醇在乙腈中的溶液。根据本发明的实施例，所述紫杉醇溶液的浓度为4mg/ml。由此，有利于胶束的形成，提高制备胶束的效率。
根据本发明的实施例，所述β-拉帕醌溶液为β-拉帕醌在乙腈中的溶液。根据本发明的实施例，所述β-拉帕醌溶液的浓度为4mg/ml。由此，有利于胶束的形成，提高制备胶束的效率。
根据本发明的实施例，所述两亲性聚合物为聚乙二醇聚乳酸二嵌段共聚物。由此，安全无毒，能够有效用于临床治疗。
根据本发明的实施例，所述两亲性聚合物溶液为两亲性聚合物在乙腈中的溶液。根据本发明的实施例，所述两亲性聚合物溶液的浓度为36mg/ml。由此，有利于胶束的形成，提高制备胶束的效率。
(2)将步骤(1)中所得到的混合物蒸发至形成薄膜。根据本发明的实施例，将步骤(1)中所得到的混合物5分钟内蒸发至形成薄膜。
(3)向所述薄膜中加入水之后进行超声处理。根据本发明的实施例，所述超声处理是于300瓦下，处理5分钟。由此，有利于分散，提高制备胶束的效率。
(4)将步骤(3)中所得到的超声处理产物进行过滤，以便获得所述胶束。根据本发明的实施例，所述过滤是利用孔径为0.45μm的尼龙滤器进行的。由此，能够有效去除溶液中未被包封的，沉淀聚集的紫杉醇和β-拉帕醌。
发明人发现，利用本发明的该方法能够快速有效地制备获得所述胶束，且操作简单、易于控制，对设备没有特殊要求，适合规模化生产。另外，紫杉醇能够有效抑制β-拉帕醌结晶，从而显著提高β-拉帕醌的载药量和生物利用度，且形成的胶束稳定性较好，能够有效用于治疗或预防癌症，特别是肺癌。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品，例如可以采购自Illumina公司。
实施例1
按照以下步骤，对不同配比LPC和PTX的混合物以及LPC进行差示扫描热量法(DSC)检测，具体如下：
将0.1g质量比分别为9/1、7/3、5/5的LPC和PTX的混合物以及纯的LPC，分别溶于2ml二氯甲烷中，于100摄氏度下快速蒸发溶剂后，将蒸发产物置于真空条件下过夜，然后用研钵研碎，分别得到样品粉末9/1LPC/PTX、7/3LPC/PTX、5/5LPC/PTX以及LPC。
将上述得到的样品粉末及LPC分别装入盖有针孔的铝盘，将铝盘置于DSC Q2000差示扫描量热仪(TA Instruments,New Castle,DE,USA)的样品台上，先将铝盘中的样品预加热至105摄氏度，以除去剩余的溶剂和水分，然后，以1摄氏度/分钟或10摄氏度/分钟的升温速度，将样品加热至LPC的熔点以上，然后淬冷至0摄氏度，其中，所有晶体全部熔融时的温度记为Tend。部分检测结果如图1所示，其中，图1a为冷却速度为10摄氏度/分钟时，冷却过程中LPC的DSC检测结果图，图1a中左上侧的插图为LPC的偏光显微镜照片；图1b为升温速度为1摄氏度/分钟时，样品粉末9/1LPC/PTX、7/3LPC/PTX、5/5LPC/PTX以及LPC的DSC检测结果图。
由图1a可以看出，当冷却速度为10摄氏度/分钟时，LPC的DSC检测曲线中存在一个绳套曲线，当采用不同的降温速度时，冷却过程中，LPC的DSC检测曲线中仍存在一个绳套曲线(数据未示出)。这个现象说明LPC的结晶速度过快，以致于DSC检测仪来不及对放热过程进行反应。由图1b可以看出，将LPC和PTX以不同比例混合，LPC的熔点产生很大的变化，通过弗洛里-哈金斯公式(Flory-Huggins equation)计算得到的相互作用参数x为-0.01，而相互作用参数为负值说明LPC和PTX之间存在强烈的物理作用，同时表明在固体状态下，PTX能够显著的抑制LPC结晶。
实施例2
将10mg的LPC和10mg的PTX分别溶于10ml乙腈中，然后将得到的溶液稀释，分别得到浓度为5μg/ml的LPC溶液和PTX溶液，再将LPC溶液和PTX溶液混合，得到LPC和PTX在乙腈中的浓度均为5μg/ml的混合溶液。将上述得到的LPC溶液、PTX溶液以及LPC和PTX的混合溶液于室温下放置48小时后，利用高效液相色谱(SHIMADZU Prominence LC-20A,Japan)进行检测。其中，色谱柱为
Figure PCTCN2015076102-appb-000001
C18，检测温度为30摄氏度，流动相为体积比为1：1的乙腈和水的混合溶液，流速为1.0ml/min，进样量为10μl，每个样品进行三次重复实验。检测结果如图2所示，其中，图2A为检测波长为257nm条件下，LPC溶液(LPC)以及LPC和PTX的混合溶液(5/5LPC/PTX)的色谱图，图2B为检测波长为227nm条件下，PTX溶液(PTX)以及LPC和PTX的混合溶液(5/5LPC/PTX)的色谱图。
结果显示：在所有样品溶液中，LPC和PTX的保留时间均分别为6.7分钟和8.7分钟，与LPC溶液、PTX溶液相比，LPC和PTX的混合溶液没有新的色谱峰出现，而且与LPC和PTX相对应的色谱峰的面积也没有变化，这说明LPC和PTX之间是化学兼容的，有望开发同时负载LPC和PTX的胶束系统。
实施例3
将20mg的LPC溶于4ml的甲醇中，得到浓度为5mg/ml的LPC溶液，然后，将20mg的LPC和PTX的混合物溶于4ml的甲醇中，得到LPC浓度为5mg/ml的混合溶液，将1ml上述得到的LPC溶液和混合溶液分别加入9ml的磷酸盐缓冲溶液(PBS，PH＝7.4)中，得到过饱和溶液，接着，将所得到的两种过饱和溶液于37摄氏度下，以恒定速度搅拌，然后，利用HLPC于2min、5min、10min、15min、20min和25min时，分别测定两种过饱和溶液中LPC的浓度，绘制LPC浓度-时间曲线(见图3)。其中，色谱检测参数同实施例2，每个样品进行三次重复实验。
由图3的结果可知，与LPC过饱和溶液(LPC)相比，LPC和PTX混合物的过饱和溶液(LPC/PTX)中，LPC的含量明显增加，这说明PTX存在能够显著提高LPC的过饱和度。
实施例4
收集上述过饱和溶液中的沉淀，经干燥后，分别用范广达200扫描电子显微镜(FEI Quanta 200Scanning Electron Microscope，Czech)和粉末X射线衍射仪(PANalytical X’pert Powder X-ray diffractometer，Almelo，The Netherlands)观察沉淀样品的表面形态和结晶情况。LPC沉淀样品的扫描电子显微镜(SEM)照片见图4A，LPC和PTX的混合物沉淀样品的扫描电子显微镜(SEM)照片见图4B，LPC沉淀样品、PTX沉淀样品(获得方法同LPC沉淀样品)以及LPC和PTX混合物沉淀样品(LPC/PTX)的粉末X射线衍射(PXRD)结果见图5。
由图4可以看出，LPC沉淀样品呈完整的晶体，而有PTX存在时，LPC晶体的形态与之前不同，LPC和PTX混合物沉淀样品不能形成完整的晶体，表明PTX可以抑制LPC结晶。由图5的结果也可以看出，当存在PTX时，LPC结晶度显著下降。综上，表明在水溶液状态下，PTX能够有效的地抑制LPC结晶。
实施例5
将40mg的β-拉帕醌(LPC)溶于10ml的乙腈中，超声处理2分钟，得到β-拉帕醌溶液；将40mg的紫杉醇(PTX)溶于10ml的乙腈中，超声处理2分钟，得到紫杉醇溶液；将36mg/ml的聚乙二醇聚乳酸嵌段共聚物(PEG-PLA)水溶液冷冻，然后超声处理30分钟后，冷冻干燥，将360mg的经冷冻干燥的PEG-PLA溶于10ml的乙腈中，超声处理2分钟，得到聚乙二醇聚乳酸嵌段共聚物溶液。
将上述获得的β-拉帕醌溶液、紫杉醇溶液和聚乙二醇聚乳酸嵌段共聚物溶液，分别按照表1所示的比例进行混合，将所得到的混合物超声处理2分钟后，在5分钟内旋转蒸发至形成一层薄膜，然后向所得到的薄膜中加入10ml、温度为60摄氏度的纯水，超声处理5分钟后，利用孔径为0.45微米的尼龙滤器去除未包封进胶束的紫杉醇和β-拉帕醌，分别得到不同配比的胶束。不同配比的胶束溶液的照片见图6。
表1
Figure PCTCN2015076102-appb-000002
实施例6
1、按照以下步骤，分别检测实施例5中制备获得的不同配比的胶束中LPC或/和PTX的包封率、装填密度以及产率：
将0.5mL的胶束溶于9.5ml乙腈中，然后利用高效液相检测LPC(257nm)或PTX(227nm)，得到胶束溶液中LPC或PTX的含量(包括游离的和包封的LPC)。
称量PE管的重量，将0.5ml的胶束溶液加入已知重量的PE管中，冷冻干燥后，准确称量冻干产物的重量，以便于计算胶束的装填密度。
按照如下公式计算包封率、装填密度以及产率，结果见表2。
包封率＝胶束内装载的药物质量/加入胶束体系中的药物质量×100％
装填密度＝胶束内装载的药物质量/(胶束体系的质量-游离的药物质量)×100％
产率＝(胶束体系的质量-游离药物的质量)/理论胶束体系的质量×100％
表2
Figure PCTCN2015076102-appb-000003
然后，收集上述步骤中剩余的胶束溶液，于4摄氏度下肉眼观察，并拍摄照片，并利用动态光散射法(DLS)检测不同配比胶束的尺寸及多分散性。不同配比胶束的照片见图6，不同配比胶束的尺寸及多分散性结果见表2和图7，其中，图7A为LM胶束的DLS检测结果，图7B为7L3PM胶束的DLS检测结果，图7C为5L5PM胶束的DLS检测结果。
由表2的数据可以看出，随着PTX含量的增加，LPC在胶束中的含量明显提高，这点可以从图6中不同配比胶束的颜色中很直观的看出，特别是在LM胶束和5L5LM胶束中表现的尤其明显，LM胶束中，LPC的含量仅为1.3±0.4％，而在5L5PM胶束中，LPC的含量达到8.1±1.9％，LPC的包封率和在5L5PM胶束中的浓度均提高了7倍。同时，LPC和PTX的总负载率达到96％，这是非常高的。从表2和图7的结果可知，胶束的粒径为40-50nm，有研究报道，这个粒径范围能够有效加强EPR效应。另外，发明人发现在4摄氏度下保存，24小时内5L5PM胶束的粒径基本没有改变，经高效液相检测得到LPC的含量也没有明显变化，说明5L5PM能够形成装载LPC含量高、稳定性较好的聚合物胶束。
2、利用透射电子显微镜(TEM)观察不同配比的胶束，部分胶束的透射电子显微镜照片见图8。从图8中可以看出，胶束呈球形，具有核壳结构。
3、分别以CDCl3和D2O作为溶剂，对不同配比的胶束进行核磁共振检测，部分结果见图9。其中，a为以CDCl3作为溶剂，PM胶束的核磁共振谱图，b为以CDCl3作为溶剂，LM胶束的核磁共振谱图，c为以CDCl3作为溶剂，5L5PM胶束的核磁共振谱图，d为以D2O作为溶剂，5L5PM胶束的核磁共振谱图。
从图9可以看出，以CDCl3作为溶剂时，5L5PM胶束的核磁共振谱图出现多个共振峰，而当溶剂为D2O时，5L5PM胶束的核磁共振谱图中没有共振峰出现，这表明LPC分散于聚乳酸链段形成的胶束的内核部分。
实施例7
按照下述步骤，对实施例5中制备获得的不同配比的胶束进行体外药物释放实验：
将1ml的胶束样品加入透析装置(MWCO＝100,000Da,Spectrum Float-A-Lyzer)中，在37℃、缓慢搅拌条件下，于释放介质(80ml的磷酸盐缓冲液(PBS)或75ml 1M的水杨酸盐)中进行透析，透析过程中，分别于0.5h、1h、2h、3h,、5h、7h、12h、18h和24h时，取出0.5ml的释放介质，并利用HLPC检测释放介质的成分及各成分的含量，同时，向释放介质中加入等量的新鲜的释放介质。部分结果见图10。其中，图10A为以磷酸盐缓冲液作为释放介质时的药物释放曲线，图10B为以水杨酸盐作为释放介质时的药物释放曲线。
从图10A的结果可以看出，以磷酸盐缓冲液作为释放介质时，PTX是否存在，对LPC的释放几乎没有影响，3小时内，LM和5L5PM胶束中，LPC的累积释放量均达到50％，而在相同的时间范围内，PTX不从PM胶束中释放，这取决于PM物理化学性质。与PM胶束相比，在5L5PM胶束中，LPC能够在一定程度上促进PTX的释放。从图10B可以看出，以水杨酸盐作为释放介质时，在PM和5L5PM中，3小时内有50％的PTX被释放，而在LM和5L5PM中，1小时内LPC的累积释放量就达到100％。综上，在胶束中的PTX的释放速度总是慢于LPC的释放速度，这与LPC和PTX之间具有协同的药理学作用相符。
实施例8
按照下述步骤，对实施5中制备获得的胶束进行对紫杉醇耐药的A549细胞系的毒性实验：
将对紫杉醇耐药的A549细胞铺于96孔黑色底透板中(2000cells/well)，培养24小时后，弃去96孔板中旧的培养基，加入200μL含不同药物胶束浓度的培养基到细胞中，培养4小时，接着更换200μL新鲜的培养基后继续培养7天。7天后，弃去旧的培养基，用200μL的PBS清洗细胞后弃去所有PBS。接着，每孔加60μL的超纯水后，将96孔板置于-80℃冰箱中2小时，再待细胞完全解冻后，每孔加200μL的1:1000Hoechst 33258与TNE(1mM EDTA,2M NaCl,10mM Tris,PH7.5)的染料溶液，室温避光保存2小时后，Perkin Elmer仪器读板，激发光356nm，发射光458nm。
实验结果显示，1L3PM对紫杉醇耐药的A549细胞具有显著的细胞毒性。并且，经计算(Graph Pad Prism 5)可得PM的IC50值(对细胞生存抑制率为50％时的药物浓度)约为2μM，LM的IC50值约为6μM，而1L3PM的IC50值仅约为0.5μM。综上可得，LPC和PTX复合胶束对紫杉醇耐药的癌细胞具有显著的协同作用。
实施例9
按照下述步骤，对实施5中制备获得的胶束进行对紫杉醇耐药的肿瘤动物模型的疗效实验：
雌性的无胸腺裸鼠(6-8weeks,18-22g)右后腿皮下注射5×106对紫杉醇耐药的人源性A549非小细胞肺癌。待肿瘤长至约200mm3时，动物重新随机分组，每组5只。动物每周尾静脉给药一次，紫杉醇胶束和复合的胶束都以其中含有的PTX量给药，PTX 30mg/kg，共给药三次。空白对照组给予等体积的空白胶束。给药后，肿瘤的体积大小和动物的体重每两天相应地测量和称量一次。用游标卡尺测量肿瘤的长(L)和宽(W)，肿瘤的体积等于L×W2/2。
实验结果显示，给药治疗14天后，PM组(PTX 30mg/kg)对紫杉醇耐药的A549人肺癌异种移植裸鼠模型的相对肿瘤抑制率为30％，而1L3PM中折算为同等剂量的PTX给药时，肿瘤的抑制率为95％。治疗期间，PM组和1L3PM组动物的体重变化波动不明显。综合而言，1L3PM组对紫杉醇耐药的A549人肺癌异种移植裸鼠模型的疗效显著优于PM组，且未出现显著的毒副作用。LPC和PTX复合纳米胶束对紫杉醇耐药的动物模型具有显著的协同治疗作用。
工业实用性
本发明的药物组合物，其中的结晶抑制剂能够有效抑制活性成分的结晶，从而该药物组合物中活性成分的生物利用度非常高。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示意性实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。

Claims (10)

  1. 一种药物组合物，其特征在于，包含：
    活性成分；以及
    结晶抑制剂。
  2. 根据权利要求1所述的药物组合物，其特征在于，所述活性成分为结晶速度快的药物分子。
  3. 根据权利要求1所述的药物组合物，其特征在于，所述活性成分为溶解度低的药物分子。
  4. 根据权利要求1所述的药物组合物，其特征在于，所述结晶抑制剂为可以抑制所述活性成分结晶，并且与所述活性成分有药理学协同作用的药物分子。
  5. 根据权利要求1所述的药物组合物，其特征在于，所述活性成分为选自β-拉帕醌、β-拉帕醌的类似物、衍生物、或者前药的至少一种，
    任选地，所述结晶抑制剂为选自紫杉醇、紫杉醇类似物/衍生物的至少一种，其中，所述紫杉醇类似物/衍生物为多烯紫杉醇或者卡巴他赛。
  6. 根据权利要求5所述的药物组合物，其特征在于，所述活性成分为β-拉帕醌，所述结晶抑制剂为紫杉醇，并且所述β-拉帕醌与所述紫杉醇的重量比为1：0.1～3，
    优选地，所述紫杉醇与所述β-拉帕醌的重量比为1：1和1:3。
  7. 根据权利要求1所述的药物组合物，其特征在于，所述药物组合物呈胶束、胶囊剂、丸剂、片剂、颗粒剂、口服液体、内服膏剂、气雾剂或喷雾剂的形式，
    优选地，所述药物组合物呈胶束的形式，并且所述胶束是由聚乙二醇聚乳酸嵌段共聚物形成的，
    任选地，所述胶束中所述活性成分的包封率为11.7％～100％。
  8. 权利要求1～7任一项所述的药物组合物在制备药物中的用途，所述药物用于治疗或者预防癌症，优选肺癌，更优选非小细胞肺癌。
  9. 一种制备胶束的方法，其特征在于，包括：
    (1)将紫杉醇溶液、β-拉帕醌溶液和两亲性聚合物溶液混合，其中，所述紫杉醇溶液、β-拉帕醌溶液和两亲性聚合物溶液均为有机溶液；
    (2)将步骤(1)中所得到的混合物5分钟内蒸发至形成薄膜；
    (3)向所述薄膜中加入水之后进行超声处理；
    (4)将步骤(3)中所得到的超声处理产物进行过滤，以便获得所述胶束。
  10. 根据权利要求9所述的方法，其特征在于，所述紫杉醇溶液为紫杉醇在乙腈中的溶液，
    任选地，所述紫杉醇溶液的浓度为4mg/ml，
    任选地，所述β-拉帕醌溶液为β-拉帕醌在乙腈中的溶液，
    任选地，所述β-拉帕醌溶液的浓度为4mg/ml，
    任选地，所述两亲性聚合物为聚乙二醇聚乳酸嵌段共聚物，
    任选地，所述两亲性聚合物溶液为两亲性聚合物在乙腈中的溶液，
    任选地，所述两亲性聚合物溶液的浓度为36mg/ml，
    任选地，所述超声处理是于300瓦下，处理5分钟，
    任选地，所述过滤是利用孔径为0.45μm的尼龙滤器进行的。
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