Liposome having inner water phase containing sulfobutyl ether cyclodextrin salt

Abstract

A liposome comprising bilayer and inner water phase is disclosed. Said inner water phase may contain sulfobutyl ether cyclodextrin and an active compound.

Claims

1. A liposome comprising bilayer, inner water phase and metal cation ionophore in the outer phase, wherein the inner water phase comprises sulfobutyl ether cyclodextrin, one or more active compounds selected from the group consisting of doxorubicin, epirubicin, pirarubicin, idarubicin and mitoxantrone, and one or more of sodium ion, potassium ion and calcium ion, wherein the sulfobutyl ether cyclodextrin forms precipitate with the active compound in the inner water phase.

2. The liposome according to claim 1, wherein the metal cation ionophore is selected from the group consisting of nikkomycin, A23187 and ionomycin.

3. The liposome according to claim 1, wherein the sulfobutyl ether cyclodextrin is sulfobutyl ether-α-cyclodextrin, sulfobutyl ether-β-cyclodextrin or sulfobutyl ether-γ-cyclodextrin.

4. The liposome according to claim 1, wherein the sulfobutyl ether cyclodextrin has about 6.5 sulfo groups at average per molecule.

5. The liposome according to claim 1, wherein the bilayer comprises phospholipid, cholesterol and hydrophilic polymer-modified lipid.

6. A liposome comprising bilayer and inner water phase, wherein the inner water phase comprises sulfobutyl ether cyclodextrin, one or more active compounds selected from the group consisting of doxorubicin, epirubicin, pirarubicin, idarubicin and mitoxantrone, and one or more of ammonium hydroxide, triethylamine and triethanolamine, wherein the sulfobutyl ether cyclodextrin forms precipitate with the active compound in the inner water phase.

7. The liposome according to claim 6, wherein the sulfobutyl ether cyclodextrin is sulfobutyl ether-α-cyclodextrin, sulfobutyl ether-β-cyclodextrin or sulfobutyl ether-γ-cyclodextrin.

8. The liposome according to claim 6, wherein the sulfobutyl ether cyclodextrin has about 6.5 sulfo groups at average per molecule.

9. The liposome according to claim 6, wherein the bilayer comprises phospholipid, cholesterol and hydrophilic polymer-modified lipid.

10. A process for preparing the liposome according to claim 1, comprising: (1) hydrating lipid phase powders with a first aqueous solution of a sulfobutyl ether cyclodextrin salt that comprises one or more of sodium ion, potassium ion and calcium ion, to form a blank liposome comprising the first aqueous solution of the sulfobutyl ether cyclodextrin salt as inner water phase, (2) removing the salt of sulfobutyl ether cyclodextrin in the outer phase of the blank liposome, to form an anion gradient, (3) adding a metal cation ionophore to the outer phase of the blank liposome, to form a pH gradient, and (4) incubating the blank liposome with the active compound in a second aqueous solution, to encapsulate the active compound into the liposome.

11. A liposomal pharmaceutical preparation, comprising the liposome according to claim 1 and a pharmaceutically acceptable carrier and/or excipient.

12. A liposomal pharmaceutical preparation according to claim 11, wherein the carrier and/or excipient comprises osmotic regulator and/or antioxidant.

13. A method of treating a tumor patient, comprising administering a liposome according to claim 1 to the patient in need thereof.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 indicates the in vitro release results of doxorubicin liposomes comparing different inner phases;

(2) FIG. 2 indicates the in vitro release results of mitoxantrone liposomes comparing different inner phases;

(3) FIG. 3 indicates the in vitro release results of topotecan liposomes comparing different inner phases;

(4) FIG. 4 indicates the in vitro release results of irinotecan liposomes comparing different inner phases.

DETAILED DESCRIPTION OF THE INVENTION

(5) The present invention is illustrated by the following examples, which is only exemplary and should not be construed as a limitation to the scope of the present invention.

(6) As used herein, the drug/lipid ratio refers to weight ratio of drug to phospholipid, and “the content of DSPE-mPEG” Refers to its molar percentage in the total phospholipid components in liposomal bilayer.

Example 1

General Process of Preparation of Liposomes with Sulfobutyl Ether Cyclodextrin (SBE-CD) as Inner Water Phase (with the Formulation of SBE-CD)

(7) HSPC, cholesterol and DSPE-mPEG2000 at a mass ratio of 3:1:1 were mixed and dissolved in 95% t-butyl alcohol. The organic solvent was removed by lyophilization to obtain a loose lipid powder. The powder was hydrated with aqueous solution of sulfobutyl ether β-cyclodextrin at 50-60° C. and incubated for 1 hour to obtain a heterogenous multivesicular liposome. The particle size of the liposome was reduced by a micro-jet apparatus. Anion in outer phase of the blank liposome was removed by an ultrafiltration apparatus to form a dynamic transmembrane gradient. An aqueous drug solution was added to the blank liposome at an appropriate drug/lipid ratio, and the drug loading was achieved by incubation at 60° C. for 1 hour.

Example 2

General Process of Preparation of Liposomes with Triethylamine Salt of Sulfobutyl Ether Cyclodextrin as Inner Water Phase (with the Formulation of SBE-CD/TA)

(8) HSPC, cholesterol and DSPE-mPEG2000 at a mass ratio of 3:1:1 were mixed and dissolved in 95% t-butyl alcohol. The organic solvent was removed by lyophilization to obtain a loose lipid powder. The powder was hydrated with aqueous solution of triethylamine salt of sulfobutyl ether cyclodextrin at 50-60° C. and incubated for 1 hour to obtain a heterogenous multivesicular liposome. The particle size of the liposome was reduced by a high pressure extrusion apparatus. Anion in outer phase of the blank liposome was removed by an ultrafiltration apparatus to form a dynamic transmembrane gradient. An aqueous drug solution was added to the blank liposome at an appropriate drug/lipid ratio, and the drug loading was achieved by incubation at 60° C. for 1 hour.

Example 3

General Process of Preparation of Liposomes with Sodium Salt of Sulfobutyl Ether Cyclodextrin as Inner Water Phase (with the Formulation of SBE-CD/Na)

(9) HSPC, cholesterol and DSPE-mPEG2000 at a mass ratio of 3:1:1 were mixed and dissolved in 95% t-butyl alcohol. The organic solvent was removed by lyophilization to obtain a loose lipid powder. The powder was hydrated with aqueous solution of sodium salt of sulfobutyl ether cyclodextrin at 50-60° C. and incubated for 1 hour to obtain a heterogenous multivesicular liposome. The particle size of the liposome was reduced by a high pressure extrusion apparatus. Anion in outer phase of the blank liposome was removed by column chromatography, and then ethanol solution of nikkomycin in an appropriate amount was added (20 ng nikkomycin/1 mg HSPC). The resulting mixture was incubated at 60° C. for ten minutes, so as to exchange hydrogen ion and sodium ion across the liposomal membrane, so as to form a pH gradient. An aqueous drug solution was added to the blank liposome at an appropriate drug/lipid ratio, and the drug loading was achieved by incubation at 60° C. for 1 hour.

Example 4

Comparison of Encapsulation Rate of Liposomes Containing Various Internal Water Phase

(10) The liposomes of various drugs with 3 respective inner water phases were prepared as described in Example 1, 2 and 3, at a drug/lipid ratio of 2:9.58 (see table 1).

(11) TABLE-US-00001 TABLE 1 Effect of intraliposomal trapping agent on drug loading Encapsulation rate of liposomes having different inner water phases (%) Drug SBE-CD SBE-CD/TA SBE-CD/Na Mitoxatrone hydrochloride 7.6 48.5 77.6 Topotecan hydrochloride 4.8 63.6 74.6 Irinotecan hydrochloride 5.3 64.1 96.1 Doxorubicin hydrochloride 11.3 63.5 91.8 Vinorelbine bitartrate 4.7 38.2 75.9 Vincristine sulfate 3.8 47.8 79.7

(12) Conclusion: as can be seen from encapsulation rate as disclosed, the liposome having SBE-CD as inner water phase has a poor encapsulation rate, while high encapsulation rates were achieved with SBE-CD/TA and SBE-CD/Na, which illustrates that a good encapsulation cannot be achieved unless a pH gradient is formed by ion transporting. The drug is firstly protonated after entering inner water phase of the liposome, and then associates with SBE-CD, while drug loading is hardly achieved depending exclusively on inclusion effect of SBE-CD.

Example 5

In Vitro Release of Liposomal Vincristine Formulations Containing Different Inner Water Phase (SBE-CD/TA Vs Ammonium Sulfate)

(13) 1, Samples

(14) The vincristine liposomes were prepared at a drug/lipid ratio of 3:9.58, respectively as described in Example 2 for the liposome having SBE-CD/TA as inner water phase, as described in Example 3 for the liposome having SBE-CD/Na as inner water phase, and as described in Example 2, with the exception of the replacement of sulfobutyl ether-β-cyclodextrin triethylamine salt with ammonium sulfate, for the liposome having ammonium sulfate as inner water phase.

(15) 2, Release Condition

(16) Samples of liposomal vincristine formulations were diluted by 10 times in release buffer (5 mM NH.sub.4Cl/10 mM histidine/260 mM glucose, pH 7.0) and transferred into the dialysis bags. The dialysis was performed against a 200-fold volume of dialysis buffer in dissolution flask. Release test was performed at 37° C., 75 rpm. At various time points (1 h, 2 h, 4 h, 6 h, 8 h, 24 h), aliquots were withdrawn for analysis.

(17) 3, Results

(18) TABLE-US-00002 TABLE 2 Release of vincristine liposomes with different inner water phases Drug release rate at different time (%) Inner water phase 1 h 2 h 4 h 6 h 8 h 24 h t.sub.1/2(h) SBE-CD/TA 22 31 44 52 61 94 7.2 SBE-CD/Na 23 31 47 58 68 96 6.8 ammonium sulfate 26 62 91 97 98 99 1.1

(19) Conclusion: In comparison to the liposome having ammonium sulfate as inner water phase, the liposome having SBE-CD/TA as inner water phase and the liposome having SBE-CD/Na as inner water phase both significantly extended the retention of drug in inner water phase.

Example 6

In Vitro Release of Liposomal Vinorelbine Formulations Containing SBE-CD/NH.SUB.3 .and Ammonium Sulfate as a Mixed Inner Water Phase

(20) 1, Samples

(21) The vinorelbine liposomes were prepared at a drug/lipid ratio of 3:9.58, as described in Example 2 with the exception of the replacement of sulfobutyl ether-β-cyclodextrin triethylamine salt with the mixed solution of SBE-CD/NH.sub.3 and ammonium sulfate as described in A-F of table 3.

(22) TABLE-US-00003 TABLE 3 Formulations for Liposomal Vinorelbine having SBE-CD/NH.sub.3 and ammonium sulfate as a mixed inner water phase Concentration (mM) Number [H.sup.+] of SBE-CD Ammonium sulfate A 280.8 86.4 B 236.7 108.9 C 204.3 126.0 D 180.0 138.6 E 160.2 148.5 F 0 225.0
2, Release Condition

(23) Samples of liposomal formulations were diluted by 10 times in release buffer (2 mM NH.sub.4Cl/10 mM histidine/250 mM glucose, pH 7.5) and transferred into the dialysis bags. The dialysis was performed against a 200-fold volume of dialysis buffer in dissolution flask. Release test was performed at 37° C., 75 rpm. At various time points (1 h, 2 h, 4 h, 8 h), aliquots were withdrawn for analysis.

(24) 3, Results

(25) TABLE-US-00004 TABLE 4 In vitro release of liposomal vinorelbine formulations having different internal water phase Sampling Release rate for different inner water phase (%) time (h) A B C D E F 1 34.9 25.1 33.2 36.0 39.1 68.3 2 56.6 51.8 59.0 63.1 67.7 91.5 4 83.6 83.5 89.3 90.2 93.4 98.6 8 97.4 97.2 98.0 98.5 98.6 99.3

(26) Conclusion: The liposomes having high SBE-CD/NH.sub.3 proportion in the mixed inner water phase displayed relatively slow drug release, indicating that ammonium salt of SBE-CD could extend drug release.

Example 7

In Vitro Release of Liposomal Doxorubicin Formulations Containing SBE-CD/TA, SBE-CD/Mg and Ammonium Sulfate Respectively as Inner Water Phase

(27) 1, Samples

(28) The doxorubicin liposomes were prepared at a drug/lipid ratio of 2:9.58, respectively as described in Example 2 for the liposome having SBE-CD/TA as inner water phase, as described in Example 3 (with the exception of replacing SBE-CD/Na with SBE-CD/Mg and replacing nikkomycin with ionomycin) for the liposome having having SBE-CD/Mg as inner water phase, and as described in Example 2 (with the exception of the replacement of sulfobutyl ether-β-cyclodextrin triethylamine salt with ammonium sulfate) for the liposome having (NH.sub.4).sub.2SO.sub.4 as inner water phase.

(29) 2, Release Condition

(30) Samples were diluted by 200 times in release buffer (50 mM NH.sub.4Cl/20 mM histidine/200 mM glucose, pH 6.5) and placed in a water bath of 52° C.±1° C. for incubation. The excitation wavelength for fluorescence spectrophotometer was set at 480 nm, and the emission wavelength at 560 nm. The change of fluorescence was scanned, and the release rate was calculated.

(31) 3, Results

(32) TABLE-US-00005 TABLE 5 Result of encapsulation rate and particle size of doxorubicin liposomal comprising different inner phases Inner water phase encapsulation rate (%) particle size (nm) (NH.sub.4).sub.2SO.sub.4 100 94 SBE-CD/TA 99.5 91 SBE-CD/Mg 99.8 93

(33) Note: in this table, the concentration of ammonium ion in the inner water phase of SBE-CD/TA and the concentration of Mg ion in the inner water phase of SBE-CD/Mg were both 500 mM; the concentration of ammonium ion in the inner water phase of SBE-CD/TA in Example 4 is 300 mM; and the concentration of sodium ion in the inner water phase of SBE-CD/Na in Example 4 is 300 mM, so the entrapment rates are different. The same concentration in internal water phase in the formula, similar particle size, and entrapment rate can ensure the in vitro release data more comparable.

(34) Conclusion: In comparison to the liposome having ammonium sulfate as inner water phase, the liposome having SBE-CD/TA as inner water phase and the liposome having SBE-CD/Mg as inner water phase both significantly extended the retention of drug in inner water phase.

Example 8

In Vitro Release of Liposomal Mitoxantrone Formulations Containing SBE-CD/NH.SUB.3., SBE-CD/Ca and Ammonium Sulfate Respectively as Inner Water Phase

(35) 1, Samples

(36) The mitoxantrone liposomes were prepared at a drug/lipid ratio of 1:9.58, respectively as described in Example 2 (with the exception of the replacement of sulfobutyl ether-β-cyclodextrin triethylamine salt with ether-β-cyclodextrin arginine salt) for the liposome having SBE-CD/NH.sub.3 as inner water phase, as described in Example 3 (with the exception of replacing SBE-CD/Na with SBE-CD/Ca and replacing nikkomycin with calcimycin) for the liposome having having SBE-CD/Ca as inner water phase, and as described in Example 2 (with the exception of the replacement of sulfobutyl ether-β-cyclodextrin triethylamine salt with ammonium sulfate) for the liposome having (NH.sub.4).sub.2SO.sub.4 as inner water phase.

(37) 2, Release Condition

(38) Samples were diluted by 2 times in release buffer (2 M NH.sub.4Cl/200 mM histidine, pH 6.5) and placed in a water bath of 52° C.±1° C. for incubation. The samples were taken respectively at time of 0, 5, 15, 30, 60, 120 and 180 min, the encapsulation rates were determined, thereby the release rates can be calculated.

(39) 3, Results

(40) TABLE-US-00006 TABLE 6 Result of encapsulation rate and particle size of mitoxantrone liposomal comprising different inner phases Inner water phase encapsulation rate (%) particle size (nm) (NH.sub.4).sub.2SO.sub.4 99.0 72 SBE-CD/NH.sub.3 99.2 73 SBE-CD/Ca 99.4 72

(41) The results of FIG. 2 showed that compared with the liposome containing ammonium sulfate as inner water phase, the liposomes containing SBE-CD/NH.sub.3 or SBE-CD/Ca as inner water phase can significantly prolong the retention of the drug in the inner water phase of the liposome.

Example 9

In Vitro Release of Liposomal Topotecan Formulations and Liposomal Irinotecan Formulations Containing SBE-CD/Na, SBE-CD/Ca and Ammonium Sulfate Respectively as Inner Water Phase

(42) 1, Samples

(43) The topotecan liposomes and irinotecan liposomes were prepared at a drug/lipid ratio of 2:9.58, respectively as described in Example 3 for the liposome having SBE-CD/Na as inner water phase, as described in Example 3 (with the exception of replacing SBE-CD/Na with SBE-CD/Ca and replacing nikkomycin with calcimycin) for the liposome having SBE-CD/Ca as inner water phase, and as described in Example 2 (with the exception of the replacement of sulfobutyl ether-β-cyclodextrin triethylamine salt with ammonium sulfate) for the liposome having (NH.sub.4).sub.2SO.sub.4 as inner water phase.

(44) 2, Release Condition

(45) Samples were diluted by 100 times in release buffer (20 mM NH.sub.4Cl/10 mM histidine/250 mM glucose, pH 6.5) and placed in a water bath of 52° C.±1° C. for incubation. For topotecan, the excitation wavelength for fluorescence spectrophotometer was set at 380 nm, and the emission wavelength at 520 nm; for irinotecan, the excitation wavelength for fluorescence spectrophotometer was set at 370 nm, and the emission wavelength at 420 nm. The change of fluorescence was scanned, and the release rate was calculated.

(46) 3, Results

(47) TABLE-US-00007 TABLE 7 Results of encapsulation rate of topotecan liposome and irinotecan liposome comprising different inner phases encapsulation rate (%) Inner water phase topotecan irinotecan (NH.sub.4).sub.2SO.sub.4 97.8 98.5 SBE-CD/Na 98.2 98.7 SBE-CD/Ca 98.3 99.1

(48) The results of FIG. 3 and FIG. 4 showed that compared with the liposome containing ammonium sulfate as inner water phase, the SBE-CD/Na and SBE-CD/Ca inner water phases can both significantly prolong the retention of the drugs in the inner water phase of the liposomes.

Example 10

Pharmacokinetics for the Liposomes Having Ammonium Sulfate, Different Ammonium Salts of SBE-CD as Inner Water Phase

(49) 1, Samples

(50) Vinorelbine, vincristine and irinotecan liposomes were prepared at a drug/lipid ratio of 2:9.58, as described in Example 2 with exception of the replacement of SBE-β-CD/TA with (NH.sub.4).sub.2SO.sub.4 for (NH.sub.4).sub.2SO.sub.4 as inner water phase, as described in Example 2 for SBE-CD/TA as inner water phase, and as described in Example 2 with exception of the replacement of SBE-β-CD/TA with SBE-β-CD/NH.sub.3 for SBE-CD/NH.sub.3 as inner water phase.

(51) 2, Animals and Dosage

(52) This example was conducted in male DBA/2 mice, and the dosage was 10 mg/kg.

(53) 3, Results

(54) TABLE-US-00008 TABLE 5 Plasma pharmacokinetics of liposome formulations having different inner water phase Half-life for different drug liposome (h) Inner water phase Vinorelbine Vincristine Irinotecan SBE-CD/TA 4.4 67.3 8.6 SBE-CD/NH.sub.3 5.4 46.2 11.3 (NH.sub.4).sub.2SO.sub.4 3.1 27.6 4.1

(55) Conclusion: As shown in pharmacokinetic results, in comparison to the liposome having ammonium sulfate as inner water phase, the liposomes having SBE-CD/NH.sub.3 as inner water phase exhibit significantly extended half life.

Example 11

Efficacies of Vinorelbine Liposomes Having Different Inner Water Phase on LLC Tumor Model

(56) 1, Formulations

(57) Formulation 1: SBE-CD/TA as inner water phase, prepared as described in Example 2.

(58) Formulation 2: Ammonium sulfate as inner water phase, prepared as described in Example 2 with exception of the replacement of SBE-β-CD/TA with ammonium sulfate.

(59) In both formulations, drug/lipid ratio is 3:9.58, and the content of DSPE-mPEG2000 is 0.5%.

(60) 2, Experiments

(61) LLC lung cancer cells were collected, and diluted with DMEM medium. After dilution, the tumor cell number was modulated to 2.0×10.sup.6 cells/ml. 0.2 mL of the tumor cell suspension containing about 4×10.sup.5 tumor cells was inoculated into forward limb oxter subcutaneous tissue of female C57 mice under aseptic condition. Fourteen days after inoculation, mice were randomized by tumor volume into three groups and administered with a single i.v. injection at a dose of 10 mg/kg.

(62) The mice were bred normally after administration. Tumor diameters were measured to dynamically evaluate anti-tumor efficacies of different formulations. Tumor volume (TV) was calculated with the following formula:
TV=½×a×b.sup.2,
in which a and b represent length and width, respectively.

(63) The tumor volumes were calculated by using the measurement results. The experiment data were analyzed using SPSS 11.5 statistics software.

(64) 3, Results

(65) TABLE-US-00009 TABLE 6 anti-tumor efficacies of vinorelbine liposomes having different inner water phase on LLC tumor model (n = 10, x ± sd) Day after Tumor volume (mm.sup.3) administration SBE-CD/TA ammonium sulfate 5% glucose solution 0 785.0 ± 343.0  692.2 ± 259.3  780.8 ± 353.3 1 1214.5 ± 732.4   979.7 ± 507.3  1154.8 ± 618.0  2 1179.6 ± 730.0   940.7 ± 415.1  1378.2 ± 753.2  3 1420.5 ± 716.3   1116.8 ± 503.5   1964.3 ± 1004.2 4 1591.6 ± 1056.1  1091.6 ± 562.3**  2456.5 ± 1170.1 6 1665.2 ± 1121.3*  1353.7 ± 631.6**  3173.9 ± 1591.2 7 2034.7 ± 1233.8*  1846.7 ± 1051.5** 4117.7 ± 2022.8 9 1939.0 ± 1171.0** 2086.5 ± 1446.8** 4715.0 ± 2203.6 11 2605.2 ± 1683.3** 3142.4 ± 1643.0*  6307.6 ± 3194.9 12 2893.5 ± 1656.5** 3650.4 ± 1931.8** 7562.9 ± 3819.7 14 3793.5 ± 2671.7** 5106.1 ± 2465.1** 9464.8 ± 4151.7 **P < 0.01, *P < 0.05, in comparison with 5% glucose control

(66) In comparison with 5% glucose, the growth of tumor was significantly suppressed from day 4 for the liposomes having ammonium sulfate as inner water phase and from day 6 for the liposomes having SBE-CD as inner water phase.

(67) Relative tumor proliferation rate T/C (%) was calculated with the following formula: T/C %=TRTV/CRTV×100%, in which TRTV and CRTV represent relative tumor volume (RTV) of treatment group and of negative control group, respectively. RTV=Vt/Vo. Vo means tumor volume of day 0 (initial dosage), and Vt means tumor volume at each measuring day. Regarding relative tumor volume proliferation rate of SBE-CD group and ammonium sulfate group, the lowest T/C % were 51.8% and 31.1% respectively. That is, anti-tumor efficacy of SBE-CD group on LLC lung cancer was superior to that of ammonium sulfate group.

Example 12

Anti-Tumor Efficacies of Topotecan Liposomes Having Different Inner Water Phase on Prostate RM-1 Tumor Model

(68) 1, Formulations

(69) Formulation 1: SBE-CD/TA as inner water phase, prepared as described in Example 2.

(70) Formulation 2: Sucrose octasulfate as inner water phase, prepared as described in Example 2 with exception of the replacement of SBE-β-CD/TA with sucrose octasulfate.

(71) In both formulations, drug/lipid ratio is 3:9.58, and the content of DSPE-mPEG2000 is 0.5%.

(72) 2, Experiments

(73) RM-1 lung cancer cells were collected, and diluted with 1640 medium. After dilution, the tumor cell number was modulated to 2.0×10.sup.6 cells/ml. 0.2 mL of the tumor cell suspension containing about 4×10.sup.5 tumor cells was inoculated into forward limb oxter subcutaneous tissue of female C57 mice under aseptic condition. Twelve days after inoculation, mice were randomized by tumor volume into groups and administered with a single i.v. injection at a dose of 10 mg/kg.

(74) The mice were bred normally after administration. Tumor diameters were measured to dynamically evaluate anti-tumor efficacies of different formulations. Tumor volume (TV) was calculated with the following formula:
TV=½×a×b.sup.2,
in which a and b represent length and width, respectively.

(75) The tumor volumes were calculated by using the measurement results. The experiment data were analyzed using SPSS 11.5 statistics software.

(76) 3, Results

(77) TABLE-US-00010 TABLE 7 The antineoplastic effects of topotecan liposomes on RM-1 tumour model (n = 10, x ± sd) Tumor volume (mm.sup.3) Day after Sucrose Free 5% glucose administration SBE-CD/TA octasulfate topotecan control 0 220.1 ± 70.1  218.8 ± 67.3   223.0 ± 65.7  219.6 ± 60.2  2 339.2 ± 145.0* 336.8 ± 96.3*  484.0 ± 154.7 468.9 ± 137.7 4 397.3 ± 234.4* 347.0 ± 117.8** 606.0 ± 183.1 765.3 ± 415.2 6  483.1 ± 253.6** 500.3 ± 165.5** 1060.7 ± 393.0  1376.9 ± 689.3  8 690.2 ± 656.7* 640.7 ± 280.7** 1301.8 ± 563.7  2082.9 ± 1508.7 9 914.0 ± 691.4* 734.2 ± 343.6*  1628.5 ± 835.4  2598.7 ± 2148.2 13 1876.2 ± 1931.9* 1247.8 ± 858.7**  3592.9 ± 1523.5 4499.4 ± 2946.5 15 2833.9 ± 3016.7* 2571.1 ± 2844.9** 6639.3 ± 2388.2 7504.9 ± 4335.9 **P < 0.01, *P < 0.05, in comparison with 5% glucose control group

(78) In comparison with 5% glucose for injection as control, free topotecan did not significantly suppress the growth of tumor (p>0.05), while the tumor growth was significantly suppressed in the two groups of the liposomes having different inner water phase. Significant differences were observed in comparison with free topotecan groups with equal dosages, while no significant difference of the suppression on RM-1 tumor was observed between the two liposomal formulations.

Example 13

Toxicity of Different Liposomal Topotecan Formulations in KM Mice

(79) 1, Formulations

(80) Formulation 1: SBE-CD/TA as inner water phase, prepared as described in Example 2.

(81) Formulation 2: Sucrose octasulfate as inner water phase, prepared as described in Example 2 with exception of the replacement of SBE-β-CD/TA with sucrose octasulfate.

(82) In both formulations, drug/lipid ratio is 3:9.58, and the content of DSPE-mPEG2000 is 0.5%.

(83) 2, Experiments

(84) Regarding the three liposomal drugs and free drug, each dosage group has two female KM mice, beginning with a maximum dose of 40.6 mg/kg of topotecan and continuing with a descending dose factor of 1.25 (i.e. dosages: 40.6, 32.5, 26.0, 20.8, 16.6, 13.3 and 10.6 mg/kg). Mice was observed in terms of general health and weighed every day for a period of 14 days.

(85) TABLE-US-00011 TABLE 8 toxicity of liposomal topotecan formulations having different inner water phase Number of Animals number of dead animals with >15% weight loss Dosage Sucrose Sucrose level SBE- octa- Free SBE- octa- Free (mg/kg) CD/TA sulfate topotecan CD/TA sulfate topotecan 40.6 1 2 1 2 2 2 32.5 1 2 — 2 2 1 26.0 — 2 — 2 2 — 20.8 — 2 — 2 2 — 16.6 — 2 — 2 2 — 13.3 — 1 — 2 1 — 10.6 — 1 — 2 1 —

(86) As shown in Table 8, the order of toxicity was: free topotecan<liposome having SBE-CD/TA as inner water phase<liposome having sucrose octasulfate as inner water phase. The sucrose octasulfate liposome caused animal death in a relative low dosage.

(87) The present inventors further prepared the liposomes of vinorelbine, vincristine and irinotecan, and similarly evaluated their toxicities in KM mice. The same results as that of topotecan were obtained. The order of toxicity was: free drug<liposome having SBE-CD/TA as inner water phase<liposome having sucrose octasulfate as inner water phase. The sucrose octasulfate liposome caused animal death in a relative low dosage.