Particle and pharmaceutical composition comprising an insoluble camptothecin compound with double core-shell structure and method for manufacturing the same
10980796 · 2021-04-20
Assignee
Inventors
Cpc classification
A61K9/19
HUMAN NECESSITIES
A61K9/5031
HUMAN NECESSITIES
A61K47/34
HUMAN NECESSITIES
Y10S977/906
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
Y10S977/773
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61K31/4745
HUMAN NECESSITIES
A61K9/1075
HUMAN NECESSITIES
International classification
A61K31/4745
HUMAN NECESSITIES
A61K9/50
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
A61K9/19
HUMAN NECESSITIES
Abstract
The present invention relates to a drug delivery system having a double core-shell structure and, specifically, to a double nano-drug delivery system having an inner core-shell containing a poorly soluble camptothecin compound and a water-soluble camptothecin compound inside and an amphiphilic polymer shell, and to a manufacturing method therefor. The double core-shell structured particles manufactured by the present invention form very stable particles and show a mono-distribution of particles before and after freeze-drying. The particles of the present invention show excellent results compared with existing monolayer micelles in animal efficacy tests and pharmacokinetic tests, and do not use a surfactant causing hypersensitivity, and thus the use of the particles of the present invention can provide a pharmaceutical composition or a drug delivery system platform, which are safe for the human body.
Claims
1. A composition of freeze-dried particles with particle size of less than 1 μm said freeze-dried particles comprising: (1) an inner core-shell particle comprising i) a hydrophobic camptothecin compound; and ii) a hydrophilic camptothecin compound, wherein the hydrophobic camptothecin compound and the hydrophilic camptothecin compound form the inner core; and (2) an outer shell surrounding the inner core, said outer shell being formed of an amphiphilic block copolymer, wherein the hydrophobic camptothecin compound is 7-ethyl-10-hydroxylcamptothecin (SN-38), wherein the hydrophilic camptothecin compound is irinotecan, topotecan, or SN-38 glucuronide, and wherein the amphiphilic block copolymer is selected from the group consisting of PEG-PBLA, mPEG-PLA, PEG-PCL, PEG-PLA, mPEG-PGA, mPEG-PLGA, PEG-p(Glu), PEG-PLA-PEG, and PEG-p(Asp), wherein a proportion of the freeze-dried particles with a size more than 200 nm in the composition is 11.8% or less as measured by dynamic light scattering (DLS) method, and wherein the composition does not contain a surfactant that causes hypersensitivity.
2. The composition of freeze-dried particles of claim 1, wherein a weight ratio of the hydrophobic camptothecin compound and the hydrophilic camptothecin compound is 1:10 to 10:1.
3. The composition of freeze-dried particles of claim 1, wherein a weight ratio of the sum of the hydrophobic camptothecin compound and the hydrophilic camptothecin compound to the amphiphilic block copolymer is 1:200 to 10:1.
4. The composition of freeze-dried particles of claim 1, wherein the number average particle size of the particle is 20-200 nm.
5. The composition of freeze-dried particles of claim 1, wherein the hydrophilic camptothecin compound is irinotecan, and the amphiphilic block copolymer is mPEG-PLA; and wherein a proportion of the particle size more than 200 nm is between 2.8% and 7.4% during first 6 months of storage of the freeze-dried particles under condition of 40° C. and 75% relative humidity.
6. The composition of freeze-dried particles of claim 1, further comprising a cryoprotectant.
7. The composition of freeze-dried particles of claim 6, wherein the cryoprotectant is mannitol or trehalose.
8. The composition of freeze-dried particles of claim 1, wherein the hydrophobic camptothecin compound, the hydrophilic camptothecin compound, and the amphiphilic block copolymer are respectively: i) SN-38, irinotecan, and an amphiphilic block copolymer selected from the group consisting of PEG-PBLA, mPEG-PLA, PEG-PCL, PEG-PLA, mPEG-PGA, mPEG-PLGA, PEG-p(Glu), PEG-PLA-PEG, and PEG-p(Asp); or ii) SN-38, topotecan, and mPEG-PLA.
9. The composition of freeze-dried particles of claim 1, wherein the proportion of the freeze-dried particles with a size more than 200 nm is 10% or less.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3)
(4)
(5)
DETAILED DESCRIPTION
(6) Hereinafter, the present invention will be described in detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.
EXAMPLES
(7) Throughout the present specification, the term “%” used to express the concentration of a specific material, unless otherwise particularly stated, refers to (wt/wt)% for solid/solid, (wt/vol)% for solid/liquid, and (vol/vol)% for liquid/liquid.
(8) Materials
(9) Among the compounds used in the present invention, hydrophobic and hydrophilic camptothecin-based compounds, polysaccharides such as trehalose, cyclodextrin, polyethylene glycol, and the like were used from Sigma-Aldrich, AbCem, Toronto Research Chemical (Canada) or Tocris (USA), and polymers were used from Akina Inc (USA), Advanced Polymer Materials (Canada), Shanghai Liang Chemical Co., LTD (China), NanoSoft Polymer (USA), and Samyang BioPharm (Korea).
Example 1: Solubility in Different Solvents, when Hydrophobic Camptothecin Compound was Dissolved Alone or Hydrophobic Camptothecin Compound and Hydrophilic Camptothecin Compound were Dissolved in Mixture
(10) The solubility change according to solvent was measured when the hydrophobic camptothecin compound, camptothecin or 7-ethyl-10-hydroxycamptothecin (SN-38) was dissolved alone or together with the hydrophilic camptothecin-based compound, irinotecan (CPT-11), topotecan, or belotecan (CKD-602).
(11) 1) Solubility in Different Solvents when Hydrophobic Camptothecin Compound was Dissolved Alone
(12) A supersaturated solution was prepared by adding 20 mg of the hydrophobic camptothecin compound (camptothecin or SN-38) to 5 ml of ethanol, acetonitrile, acetone, ethyl acetate, chloroform dimethyl sulfoxide, or distilled water, followed by ultrasonic treatment for 30 minutes. The prepared supersaturated solution was filtered through a 0.45-μm filter, and a filtrate was properly diluted, followed by HPLC analysis.
(13) 2) Solubility in Different Solvents when Hydrophobic Camptothecin Compound and Hydrophilic Cam Ptothecin Compound were Dissolved in Mixture
(14) The present inventors prepared a supersaturated solution by adding 20 mg of the hydrophilic camptothecin compound (irinotecan hydrochloride, topotecan hydrochloride, or belotecan) to 5 ml of ethanol, acetonitrile, chloroform, ethyl acetate, dimethyl sulfoxide, or distilled water, and then adding 20 mg of the hydrophobic camptothecin compound (SN-38 or camptothecin), followed by ultrasonication for 30 minutes. The prepared supersaturated solution was filtered through a 0.45-μm filter, and a filtrate was properly diluted, followed by HPLC analysis.
(15) HPLC (Agilent 1200 Series, USA) conditions were as follows. The column was CapcellPak C8 (5 m, 4.6 mm×25 cm, Shiseido); the mobile phase was a mixed solvent of methanol:acetonitrile:buffer (2.8 g/L sodium dihydrogenphosphate, 1.8 g/L 1-octanesulfonate aqueous solution)=17:24:59 (v/v); the flow rate was 1.5 mL/min; the measurement wavelength was UV 255 nm; and the sample injection amount was 15 μL. The solubility test results are shown in table 1 below.
(16) TABLE-US-00001 TABLE 1 Solubility changes of hydrophobic camptothecin-based compounds in polar organic solvents (unit: mg/ml) Comparative Test Comparative Test Test Test Solvent type example example example example example example Camptothecin Camptothecin SN-38 SN-38 SN-38 SN-38 20 mg 20 mg 20 mg 20 mg 20 mg 20 mg — Irinotecan Irinotecan Topotecan Belotecan 20 mg 20 mg 20 mg 20 mg Ethanol 0.149 1.414 0.779 2.885 2.205 2.047 Acetonitrile 0.101 1.090 0.402 1.972 1.694 1.338 Chloroform 0.002 0.051 0.009 0.025 0.088 0.104 Ethyl 0.134 0.295 0.078 0.425 0.239 0.221 acetate Dimethyl 3.291 >4 2.263 3.816 2.949 2.685 sulfoxide Distilled 0.009 0.011 <0.001 <0.001 <0.001 <0.001 water
(17) As shown in Table 1 above, the poorly soluble compound camptothecin or SN-38 alone was hardly dissolved in most solvents including water, and were dissolved at 2.26-3.29 mg/mL in only dimethyl sulfoxide (DMSO) as a non-volatile solvent. However, camptothecin or SN-38, when dissolved together with the relatively hydrophilic drug, irinotecan hydrochloride, topotecan hydrochloride, or belotecan, had solubility increased up to 15-fold, which corresponds to an appropriate level of solubility required for the manufacture of drugs. It was therefore confirmed from the above results that the solubility of the hydrophobic camptothecin compounds was remarkably increased when dissolved in mixing with a hydrophilic camptothecin compound.
Example 2: Manufacturing of Primary Core-Shell (monolayer Micelles) and Secondary Core-Shell (Double Core-Shell Particles) from Hydrophobic Camptothecin-Based Compound and Hydrophilic Camptothecin-Based Compound Under Organic Solvent and Evaluation of Particle Size
(18) In the present example, the hydrophobic camptothecin-based compound camptothecin or SN-38, and the hydrophilic camptothecin-based compound irinotecan hydrochloride, topotecan hydrochloride, belotecan, or SN-38-glucuronide, or an amphiphilic polymer (PEG-PBLA) were dissolved in an organic solvent to manufacture primary core-shell particles (monolayer micelles), and an amphiphilic polymer was added thereto to manufacture secondary core-shell particles (bilayer micelles).
(19) 1) Manufacturing of Primary Core-Shell (monolayer Micelles) (Comparative Examples 1 to 6)
(20) The primary core-shell particles as monolayer micelles were manufactured by using, as a main ingredient, a hydrophobic camptothecin (camptothecin or SN-38), and water-soluble camptothecin (irinotecan hydrochloride, topotecan hydrochloride, or SN-38-glucuronide) as an amphiphilic compound for micelle formation or an amphiphilic polymer (poly(ethyleneglycol)-poly (β-butyrolactone-co-lactic acid); PEG-PBLA), as shown in table 2 below.
(21) Specifically, 20 mg of water-soluble camptothecin (irinotecan hydrochloride, topotecan hydrochloride, or SN-38 glucuronide) or PEG-PBLA was added to 20 mg of hydrophobic camptothecin, and 100 ml of an organic solvent (a 50:50 mixture solution of ethanol and acetonitrile) was added thereto to attain complete dissolution, followed by drying in a rotary vacuum evaporator. 20 ml of distilled water was added to the dried product, followed by ultrasonication at 20-30° C. for 20 minutes in an ultrasonic cleaner (UC-20, 20 Hz, 400W, Jeio Tech, Korea), thereby obtaining nano-sized particles (monolayer micelles) in a dispersed state in an aqueous solution. 600 mg of D-trehalose was added to the aqueous solution containing the nano-sized particles, thereby attaining complete dissolution, and then the solution was filtered through a 0.22 μm sterile filter, and the filtrate was freeze-dried. The freeze-drying was conducted for a total of 62 hours under a temperature cycle of −45° C..fwdarw.−20° C..fwdarw.0° C..fwdarw.20° C. at a vacuum pressure of 100 mTorr or lower, and a freeze-drier from Operon (Korea) was used. An aliquot of the prepared freeze-dried product was taken, and again dissolved in distilled water for injection, and the size of particles was measured by the dynamic light scattering (DLS) (Zetasizer™, Malvern, UK).
(22) 2) Manufacturing of Double Core-Shell (Bilayer Micelles) (Test Examples 1 to 4)
(23) In order to prepare a bilayer particle composition having a double core-shell structure, the present inventors added 20 mg of each of hydrophobic cam ptothecin and hydrophilic camptothecin into 100 ml of an organic solvent (a 50:50 mixture solution of ethanol and acetonitrile) to be dissolved therein, followed by drying using a rotary vacuum evaporator (Buchi). 200 ml of distilled water was added to the dried product, followed by ultrasonication at 20-30° C. for 20 minutes in an ultrasonic cleaner, thereby obtaining nano-sized core-shell particles (monolayer micelles) in a dispersed state in an aqueous solution. While the aqueous solution mixed with the core-shell particles was stirred, 90 mg of the amphiphilic block copolymer methoxy poly(ethylene glycol)-poly(lactide) (mPEG-PLA) (mPEG molecular weight:PLA molecular weight=2,000:1,500) previously dissolved in 10 ml of distilled water was slowly added, followed by stirring at 20-30° C. for 6 hours, thereby preparing double core-shell particles in a dispersed state in an aqueous solution. 600 mg of D-trehalose as a cryoprotectant was added to the aqueous solution containing double core-shell particles to be dissolved therein, and then the mixture was filtered through a 0.22-μm sterile filter, and then freeze-drying was conducted by the same method as in the manufacturing of primary core-shell particles, thereby obtaining a freeze-dried product as a white powder. A predetermined amount of the freeze-dried product was again dissolved in injection water to measure the size of the particles. In addition, the average particle sizes after/before freeze-drying were measured, and the proportion of particle with 200 nm or more and the distribution form (mono- or multi-modal distribution) were compared.
(24) TABLE-US-00002 TABLE 2 Proportions in manufacturing of monolayer micelles and bilayer particles (unit: mg) Poorly soluble camptothecin Water-soluble camptothecin PEG- mPEG- Micelle SN- Irinotecan Topotecan SN-38 PBLA PLA type Camptothecin 38 hydrochloride hydrochloride glucuronide (5k:6.5k) (2k:1.5k) Comparative Mono- 20 — 20 — — — — example 1 Comparative Mono- 20 — — — 20 — — example 2 Test Bi- 20 — 20 — — — 90 example 1 Comparative Mono- — 20 20 — — — — example 3 Comparative Mono- — 20 — 20 — — — example 4 Comparative Mono- — 20 — — 20 — — example 5 Comparative Mono- — 20 — — — 20 — example 6 Test Bi- — 20 20 — — — 90 example 2 Test Bi- — 20 — 20 — — 90 example 3 Test Bi- — 20 — — 20 — 90 example 4 *PEG-PBLA: poly(ethylene glycol)-b-poly(β-benzyl-L-aspartic acid) *mPEG-PLA: methoxypoly(ethylene glycol)-b-poly(lactic acid)
(25) TABLE-US-00003 TABLE 3 Comparision results of particle size before/after freeze- drying in monolayer micelles and bilayer particles Particle size before freeze- Particle size after freeze- drying (n = 3) drying (n = 3) Average >200 nm Average >200 nm Micelle particle size Propotion particle size proportion Micelle type (nm) (%) (nm) (%) >1 μm distribution Comparative Mono- 123.9 5.9 156.1 40.1 ◯ Multi- example 1 Comparative Mono- 133.6 7.1 152.8 38.6 ◯ Multi- example 2 Test example 1 Bi- 135.8 3.6 148.9 4.8 ND Mono- Comparative Mono- 76.2 0.0 108.4 16.4 ◯ Multi- example 3 Comparative Mono- 78.5 0.1 115.4 28.5 ◯ Multi- example 4 Comparative Mono- 92.1 2.7 131.2 30.4 ◯ Multi- example 5 Comparative Mono- 102.8 5.9 138.4 34.2 ◯ Multi- example 6 Test example 2 Bi- 83.3 0.0 93.8 2.4 ND Mono- Test example 3 Bi- 88.2 0.1 99.8 2.8 ND Mono- Test example 4 Bi- 93.3 0.2 105.4 3.1 ND Mono- *ND: Not detected
(26) As shown in Table 3 above, as for the average particle size after freeze-drying, the primary core-shell particles (monolayer micelles) composed of poorly soluble camptothecin and water-soluble camptothecin or the primary core-shell particles composed of poorly soluble camptothecin and an amphiphilic polymer were increased to about 1.2- to 1.5-fold, but the double-core-shell particles (bilayer micelles) only increased about 1.1-fold.
(27) With respect to the primary core-shell particles (monolayer micelles), after the freeze-drying, the particles of 200 nm or more were produced in large amounts, about 16-40%, and especially, the particles of several micrometers (μm) or more, capable of influencing safety when administered to the human body, were produced (comparative examples 1 to 6 on table 3 and
(28) In the particular distribution, the primary core-shell particles (monolayer micelles) showed a distribution with multiple peaks (
Example 3: Evaluation of Stability of Primary Core-Shell Particles (Monolayer Micelles) and Double Core-Shell Particles (Bilayer Micelles)
(29) In the present example, monolayer micelles (comparative examples 1, 3, and 6) and double core-shell particles (test examples 1 and 2) were compared for the change in particle size, wherein sample products manufactured according to the drug stability test standards were stored for six months in accelerated test conditions (40° C., 75% relative humidity). The results are shown in Table 4.
(30) TABLE-US-00004 TABLE 4 Test results of stability of primary core-shell particles (monolayer micelles) and double core-shell particles (bilayer micelles) 0 month 3 months 6 months Average >200 nm Average >200 nm Average >200 nm Micelle particle size Propotion particle size Propotion particle size Propotion type (nm) (%) (nm) (%) (nm) (%) Comparative Mono- 156.1 40.1 172.1 47.6 196.6 58.1 example 1 Test example 1 Bi- 148.9 4.8 155.0 5.6 168.3 7.4 Comparative Mono- 108.4 16.4 126.7 28.1 151.9 31.6 example 3 Comparative Mono- 138.4 34.2 149.9 44.6 174.2 48.4 example 6 Test example 2 Bi- 93.8 2.4 99.5 3.5 112.9 3.8
(31) The primary core-shell particles (monolayer micelles), containing camptothecin or SN-38 as a main ingredient, and double core-shell particles (bilayer micelles) were subjected to stability tests. As a result, in the case of the monolayer micelles, the average particle size was increased by about 50% or more and the proportion of the particles of 200 nm or more was rapidly increased to 58% in accelerated test conditions. In the case of the bilayer particles, the average particle size was increased by about 20%, and the proportion of the particles of 200 nm or more was restricted within 3.8% for SN-38 and 7.4% for camptothecin. Therefore, it can be seen that the structure of the bilayer particles of the present invention was significantly improved in view of stability, compared with the monolayer micelles.
Example 5: Manufacturing of Double Core-Shell Particles According to Type of Amphiphilic Polymer and Molecular Weight of Amphiphilic Polymer
(32) In the present example, double core-shell particles were manufactured using various amphiphilic polymers (block copolymers). As shown in Table 5, 20 mg of each of SN-38 and irinotecan hydrochloride as hydrophobic and hydrophilic cam ptothecin compounds, 500 mg of trehalose as a cryoprotectant, and 90 mg of each of amphiphilic polymers were used. The manufacturing method was carried out in the same manner as in the core-shell particle manufacturing method in example 2 above, and the materials were again dissolved in injection water after freeze-drying to compare particular sizes.
(33) TABLE-US-00005 TABLE 5 Particle size comparision of bilayer particles after freeze-drying according to amphiphilic polymer type and average molecular weight thereof Polymer type Particle >200 nm >1 μm Particle (Average molecular size Proportion presence distribution No weight) (nm) (%) or absence (Mono-/Multi-) PDI Test example 5 PEG-PCL (5k:2.5k) 133.8 5.0 ND Mono- 0.271 Test example 6 PEG-PCL(2k:1.5k) 102.0 3.1 ND Mono- 0.209 Test example 7 PEG-PLA(2.5k:1k) 99.2 2.9 ND Mono- 0.225 Test example 8 mPEG-PGA(2k:1.5k) 99.7 2.6 ND Mono- 0.213 Test example 9 mPEG-PLGA(1k:1k) 101.5 3.4 ND Mono- 0.247 Test example 10 PEG-PBLA(5k:6.5k) 137.9 9.8 ND Mono- 0.287 Test example 11 PEG-p(Glu)(5k:2.5k) 122.1 6.5 ND Mono- 0.245 Test example 12 mPEG-p(Asp)(5k:2.5k) 128.2 7.6 ND Mono- 0.231 Test example 13 PEG-PLA-PEG 156.4 11.8 ND Mono- 0.298 (2.5k-1k-2.5k) *PEG-PCL: poly(ethylene glycol)-b-poly(caprolactone) *PEG-PLA: poly(ethylene glycol)-b-poly(lactic acid) *mPEG-PGA: monomethoxy poly(ethylene glycol)-b-poly(glycolic acid) *mPEG-PLGA: monomethoxy poly(ethylene glycol)-b-poly(lactide-co-glycolide) *PEG-PBLA: poly(ethylene glycol)-b-poly(β-benzyl-L-aspartic acid) *PEG-p(Glu): poly(ethylene glycol)-b-poly(glutamic acid) *PEG-p(Asp): poly(ethylene glycol)-b-poly(aspartic acid) *PEG-PLA-PEG: poly(ethylene glycol)-b-poly(lactic acid)-b-poly(ethylene glycol) *PDI: Polydiversity index
(34) As shown in Table 5 above, it can be seen that the bilayer particles of the present invention can be manufactured by using various amphiphilic polymers (block copolymers) and amphiphilic polymers having various average molecular weights, and the stability of the particles was excellent.
Example 6: Evaluation of Particle Size According to Type of Cryoprotectant
(35) In the present example, the effects of a cryoprotectant in the manufacturing of primary core-shell particles (monolayer micelles) and double core-shell particles were observed. Here, 10-hydroxycamptothecin and SN-48 were selected as hydrophobic camptothecin compounds; irinotecan hydrochloride was selected as a hydrophilic camptothecin; and mPEG-PLA (2 k:1.5 k) was used as an amphiphilic polymer. As a cryoprotectant, 500 mg of each of D-trehalose, D-mannitol, PEG2000, and hydroxypropyl-β-cyclodextrin (HP-b-CD) was used. The double core-shell particles were manufactured using the compositions shown in Table 6, and the manufacturing method was carried out in the same manner as in example 2.
(36) TABLE-US-00006 TABLE 6 Size of double core-shell particles according to cryoprotectant type 10- Cryo- Particle >200 nm >1 μm OHCamptothecin SN-38 Irinotecan mPEG- protectant size Proporiton presence or (mg) (mg) (mg) PLA (500 mg) (nm) (%) absence Test example 13 20 — 20 90 Mannitol 121.8 4.4 ND Test example 14 — 20 20 90 Mannitol 101.1 2.9 ND Test example 15 20 — 20 90 Trehalose 118.6 5.8 ND Test example 16 — 20 20 90 Trehalose 99.6 3.1 ND Test example 17 — 20 20 90 PEG2000 133.2 19.6 ◯ Test example 18 — 20 20 — PEG2000 164.9 32.8 ◯ Test example 19 — 20 20 90 HP-b-CD 126.8 9.3 ◯ Test example 20 — 20 20 — HP-b-CD 151.7 26.2 ◯ *PEG2000: Polyethyleneglycol 2000 *HP-b-CD: hydroxypropyl-β-cyclodextrin *ND: Not detected
(37) As shown in Table 6, favorable results were observed in view of the particle size when the polysaccharides mannitol and trehalose were used as cryoprotectants. Whereas, the particle sizes were somewhat large, for example, particles with a size of 1 μm or more were detected, in PEG2000 and HP-b-CD. In test examples 18 and 20 for monolayer micelles, relatively large particle of 200 nm or more and macroparticles of 1 μm or more were observed when polyethylene glycol and cyclodextrin were used as cryoprotectants.
Example 7: Manufacturing of Primary Core-Shell Particles and Double Core-Shell Particles in Water-Soluble Solvent Conditions
(38) The primary core-shell particles (monolayer micelles) can be manufactured in an aqueous solution as well as an organic solvent as in example 2. As shown in Table 7 below, 10 mg of SN-38 was completely dissolved in 0.1 ml of a 0.5 M sodium hydroxide aqueous solution, and the resultant solution was dropped and neutralized in an aqueous solution of irinotecan hydrochloride (1 mg/ml) previously dissolved in 15 ml of a 0.5 mM hydrochloride aqueous solution, and a hydrochloride aqueous solution was further added to control the pH to about 5, followed by ultrasonication, thereby obtaining primary core-shell particles (monolayer micelles). 40 mg of the amphiphilic polymer mPEG-PLA was added thereto, followed by stirring at room temperature for 6 hours, and 300 mg of D-trehalose was further added to be dissolved. The mixture solution was filtered through a 0.22-μm sterile filter, freeze-dried, and again dissolved in injection water, and then the particle size was measured (test example 21). SN-38 and irinotecan were dissolved using the organic alkali ethanol amine as a basic aqueous solution or the organic acid citric acid as an acidic aqueous solution, and bilayer particles were manufactured by the same method, thereby measuring the particle sizes, respectively (test examples 22 and 23).
(39) TABLE-US-00007 TABLE 7 Manufacturing of bilayer particles under basic and acidic aqueous solutions Type of acid and basic solvents and particle size (nm) SN-38 Irinotecan mPEG-PLA NaOH/ NaOH/ (mg) (mg) Trehalose (2k:1.5k) HCl Ethanolamine/HCl Citric acid Test example 21 10 10 300 40 120.1 — — Test example 22 10 10 300 40 — 122. 3 — Test example 23 10 10 300 40 — — 119.5
(40) As shown in Table 7, the double core-shell particles manufactured by dissolving hydrophobic camptothecin and hydrophilic camptothecin in basic and acidic aqueous solutions showed a particle size of about 120 nm, and thus the double core-shell particles were successfully manufactured.
Example 8: Mixing Manufacturing of Double Core-Shell Particles
(41) The present example showed a method for manufacturing double core-shell particles by mixing all the hydrophobic camptothecin, hydrophilic camptothecin, and amphiphilic block copolymer in one step. Hydrophobic camptothecin compounds (10-hydroxycamptothecin and SN-38) were placed together with 20 mg of irinotecan hydrochloride in 100 ml of an organic solvent (50:50 mixture solution of ethanol:acetonitrile), and completely dissolved with stirring, and 90 mg of mPEG-PLA (2 k:1.5 k) dissolved in 10 ml of an organic solvent (50:50 mixture solution of ethanol:acetonitrile) was added thereto with stirring. The mixture solution was dried by a rotary vacuum evaporator, and 200 ml of distilled water was added to residues, followed by ultrasonication for 10 minutes in an a ultrasonic cleaner, thereby obtaining double core-shell particles of the present invention. 400 mg of D-mannitol as a cryoprotectant was added to be dissolved, and this solution was filtered through a 0.22-μm sterile filter, freeze-dried. A proper amount of the freeze-dried product was again dissolved in injection water to measure the particle size. The results are shown in Table 8.
(42) TABLE-US-00008 TABLE 8 Particle size of double core-shell particles (bilayer micelles) produced by mixing manufacturing SN- 10-OH mPEG- D- Particle >200 nm >1 μm 38 Camptothecin Irinotecan PLA mannitol size Proportion Presence or (mg) (mg) (mg) (mg) (mg) (nm) (%) absence Test 20 — 20 90 400 125.4 4.2 ND example 24 Test — 20 20 90 400 129.3 4.8 ND example 25 *ND: Not detected
(43) As shown in Table 8, the double core-shell particles manufactured by simultaneously mixing and dissolving hydrophobic camptothecin, hydrophilic camptothecin, and amphiphilic polymer in an organic solvent showed a mono-distribution of particle sizes of about 120-130 nm, wherein particles of 200 nm or more were detected in small amounts, 5% or less, but particles of 1 μm or more were not detected, and thus the particles were confirmed to have overall favorable stability.
Example 9: Tumor Inhibitory Effect Comparison Test of Primary Core-Shell Particles (Monolayer Micelles) and Double Core-Shell Particles (Bilayer Micelles) in Tumor Mouse Models (Colorectal Cancer)
(44) In colorectal mouse models, monolayer micelle compositions (comparative examples 3 and 6) and bilayer particle composition (test example 2) were measured for anticancer effect by the following method.
(45) The previously cultured colorectal cancer cell line (HT-29) was injected at 5×10.sup.6 cells/0.2 mL into the right flank of Balb/c nude mice, and after about 7 days, only tumors with a size of 150-200 mm.sup.3 were selected. Nine animals were assigned to each group, and were intravenously administered with a sham drug (non-treatment group), comparative example (monolayer micelles), comparative example 6 (monolayer micelles), and test example 2 (bilayer particles), once every three days, three times in total. The dose was 10 mg/kg on the basis of SN-38. The volume of tumor measured every three days after the administration of the test composition was used as a measurement index of the anticancer effect, and was observed for a total of 18 days. The results are shown in
(46) As a result of measurement of tumor inhibitory effect, the monolayer micelle compositions (comparative examples 3 and 6) showed a tumor inhibitory effect of about 50-60% compared with a negative control group, and the bilayer particle composition (test example 2) showed a tumor inhibitory effect of about 80% or more compared with a negative control group, indicating very excellent effects. These results were due to the fact that the stabilized micelle structure and the micelle structure having a size as small as 200 nm or less of the present invention were efficiently transferred to cancer tissues while stably staying in the body.
Example 10: Tumor Inhibitory Effect Comparison Test of Monolayer Micelle and Bilayer Particles in Pancreatic Cancer Mouse Models (AsPc-1)
(47) The tumor inhibitory effect of the monolayer micelle composition (comparative example 3) was compared with that of the bilayer particle composition (test example 2) in pancreatic cancer mouse models. The previously cultured pancreatic cancer cell line (AsPc-1) was injected at 5×10.sup.6 cells/0.2 mL into the right flank of male BALB/c-nu/nu mice, and after about 10 days, only tumors with a size of 100-150 mm.sup.3 were selected. Ten animals were assigned to each group, and were administered with a sham drug (non-treatment group), comparative example 3, and test example 2, once every seven days, three times in total. The dose was 10 mg/kg on the basis of SN-38. The volume of tumor measured every three days after the administration of the test composition was used as a measurement index of the anticancer effect, and was observed for a total of 24 days. The results are shown
(48) As a result of measurement of tumor inhibitory effect, the monolayer micelle composition (comparative example 3) showed a tumor inhibitory effect of about 27% compared with a negative control group, and the bilayer particle composition (test example 2) showed a tumor inhibitory effect of about 47% or more compared with a negative control group, indicating very excellent effects. The results were overall similar to those in the colorectal cancer models (Example 9), and it was confirmed that the bilayer particle composition of the present invention were very excellent in tumor inhibitory effects compared with monolayer micelles.
Example 11: Tumor Inhibitory Effect Comparison Test of Monolayer Micelle and Bilayer Particle Composition in Pancreatic Cancer Mouse Models (MiaPaca-2)
(49) The tumor inhibitory effect of the monolayer micelle composition (comparative example 3) was compared with that of the bilayer particle composition (test example 2) in pancreatic cancer mouse models (Orthotopic) on the basis of SN-38. After the left flank side of male BALB/c-nu/nu mice was incised at 0.7-1 cm, the entire pancreas and spleen were exposed to the outside, and then the pancreatic cancer cell line (MiaPaca-2 cell line) previously cultured using a syringe was injected at 1×10.sup.7 cells/0.1 mL. It was confirmed that the tumor cell suspension did not leak, and the organs exposed to the outside were relocated again, and the incision site was sutured by suture thread. About 10 days after the inoculation of the pancreatic cancer cell line, grouping was carried out by the body weight. Ten animals were assigned to each group, and were administered with a sham drug (non-treatment group), comparative example 3, and test example 2, once every seven days, three times in total. The dose was 20 mg/kg on the basis of SN-38. The animals were observed for a total of 28 days, and the tumor size and weight were measured by autopsy on day 28. The results are shown
(50) As a result of measurement of tumor inhibitory effect, the tumor weight of the non-treatment group (negative control group) was on average 0.46±0.17 g, the tumor weight of the monolayer micelle composition (comparative example 3) treatment group was 0.37±0.09 g, and the tumor weight of the bilayer particle composition (test example 2) treatment group was 0.21±0.05 g. Therefore, the bilayer particle composition showed a tumor inhibitory effect of 55% or more compared with the monolayer micelle, and thus a very excellent tumor inhibitory effect.
Example 12: Pharmacokinetic Test in Beagle Dogs
(51) The pharmacokinetic characteristics of the monolayer micelle composition (comparative example 3) were compared with those of the bilayer particle composition (test example 2) in beagle dogs. Male beagle dogs weighing 7-10 kg were divided into two groups, three dogs per each group, according to the body weight, and were intravenously administered with a monolayer micelle composition (comparative example 2) and a bilayer particle composition (test example 2) at 0.5 mg/kg on the basis of SN-38 for 10 minutes infusion. Blood samples were taken at 0.33, 0.67, 1, 1.5, 2, 4, 8, 12, 24, 36 hours after the end of the administration, and the plasma obtained by centrifuging the blood was pretreated by the following method to measure the drug concentration in plasma. For sample pretreatment, 20 μL of S-(+)-camptothecin (500 ng/mL, dissolved in acetonitrile) as an internal standard substance was first added to 100 μL of plasma, and 500 μL of acetonitrile was further added, followed by vortex-mixing for 30 seconds. After the mixture was centrifuged at 12,000 rpm for 3 minutes, the supernatant was taken, and was injected 2 μL into an LC-MS/MS system (API-5,000 model, AB Sciex). Separation was carried out while the column was Gemini C18 (3 μm, 2.0×50 mm, Phenomenex, USA), the mobile phase was a 50% acetonitrile solution containing 0.1% formic acid, and the flow rate was 0.25 mL/min. MS/MS detection conditions were positive ion mode, and SN-38 glucuronide was detected at m/z 569.3.fwdarw.393.2, and the internal standard was detected at m/z 349.2.fwdarw.305.2.
(52) The blood drug concentration over time after administration is shown in
(53) TABLE-US-00009 TABLE 9 Pharmacokinetic parameter comparison between monolayer micelle particle composition and bilayer particle composition Dose Cmax Tmax AUCt Relative No Micelle type (mg/kg) (ng/mL) (hr) (ng .Math. hr/mL) BA(%) Comparative Monolayer 0.5 25.30 ± 5.12 0.33 ± 0.00 131.84 ± 34.94 — example 3 micelles Test example 2 Double core- 0.5 66.25 ± 16.34 0.44 ± 0.20 361.29 ± 96.25 275.6 shell *mean ± SD (n = 3)
(54) In beagle dogs, SN-38 glucuronide produced directly from SN-38 solubilized in the particles of the present invention was analyzed. The results confirmed that the bilayer particles of the present invention showed an increase in bioavailability by about 2.75 times, compared with the monolayer micelles. It was determined from the above results that the bilayer particles of the present invention maximize the solubility of the poorly soluble drug SN-38 in vivo.
(55) Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention.