Sustained-release lipid pre-concentrate of GNRH analogues and pharmaceutical composition comprising the same

Abstract

Disclosed is a pharmaceutical composition, comprising: a) at least one sorbitan unsaturated fatty acid ester having a polar head with at least two or more OH (hydroxyl) groups; b) at least one phospholipid; c) at least one liquid crystal hardener which is free of an ionizable group and has a triacyl group with 15 to 40 carbon atoms or a carbon ring structure in a hydrophobic moiety; and d) at least one GnRH (gonadotropin-releasing hormone) analogue as a pharmacologically active substance, wherein said lipid pre-concentrate exists as a liquid phase in absence of aqueous fluid and forms into a liquid crystal in presence of aqueous fluid. The pharmaceutical composition is configured to enhance the sustained release of the pharmacologically active substance GnRH analogue.

Claims

1. A pharmaceutical composition, comprising: a) at least one sorbitan unsaturated fatty acid ester having a polar head with at least two or more OH (hydroxyl) groups; b) at least one phospholipid; c) at least one liquid crystal hardener, which is free of an ionizable group, having a hydrophobic moiety of 15 to 40 carbon atoms with a triacyl group or a carbon ring structure; and d) at least one GnRH (gonadotropin-releasing hormone) analogue as a pharmacologically active substance, wherein the pharmaceutical composition exists as a liquid phase in the absence of aqueous fluid and forms into a liquid crystal in the presence of aqueous fluid.

2. The pharmaceutical composition of claim 1, wherein the sorbitan unsaturated fatty acid ester is selected from the group consisting of sorbitan monooleate, sorbitan monolinoleate, sorbitan monopalmitoleate, sorbitan monomyristoleate, sorbitan sesquioleate, sorbitan sesquilinoleate, sorbitan sesquipalmitoleate, sorbitan sesquimyristoleate, sorbitan dioleate, sorbitan dilinoleate, sorbitan dipalmitoleate, sorbitan dimyristoleate, and a combination thereof.

3. The pharmaceutical composition of claim 1, wherein the sorbitan unsaturated fatty acid ester is selected from the group consisting of sorbitan monooleate, sorbitan monolinoleate, sorbitan monopalmitoleate, sorbitan monomyristoleate, sorbitan sesquioleate, and a combination thereof.

4. The pharmaceutical composition of claim 1, wherein the phospholipid contains a saturated or unsaturated alkyl ester group of 4 to 30 carbon atoms and is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerine, phosphatidylinositol, phosphatidic acid, sphingomyelin, and a combination thereof.

5. The pharmaceutical composition of claim 4, wherein the phospholipid is phosphatidylcholine.

6. The pharmaceutical composition of claim 1, wherein the liquid crystal hardener is selected from the group consisting of triglyceride, retinyl palmitate, tocopherol acetate, cholesterol, benzyl benzoate, ubiquinone, and a combination thereof.

7. The pharmaceutical composition of claim 1, wherein the liquid crystal hardener is tocopherol acetate, cholesterol, and a combination thereof.

8. The pharmaceutical composition of claim 1, wherein the GnRH analogue is a GnRH agonist or a GnRH antagonist.

9. The pharmaceutical composition of claim 8, wherein the GnRH agonist is selected from the group consisting of leuprolide, goserelin, triptorelin, nafarelin, buserelin, histrelin, deslorelin, meterelin, gonadorelin, a pharmaceutically acceptable salt thereof, and a combination thereof.

10. The pharmaceutical composition of claim 8, wherein the GnRH antagonist is selected from the group consisting of degarelix, abarelix, ganirelix, cetrorelix, a pharmaceutically acceptable salt thereof, and a combination thereof.

11. The pharmaceutical composition of claim 1, wherein the GnRH analogue is selected from the group consisting of leuprolide, goserelin, triptorelin, degarelix, abarelix, a pharmaceutically acceptable salt thereof, and a combination thereof.

12. The pharmaceutical composition of claim 1, wherein the GnRH analogue is leuprolide or a pharmaceutically acceptable salt thereof.

13. A method for treating a sex hormone-dependent disease, comprising administering a pharmaceutical composition of claim 1 to a subject in need thereof.

14. The method of claim 13, wherein the sex hormone-dependent disease is selected from the group consisting of prostate cancer, breast cancer, ovarian cancer, endometriosis, uterine fibroid, polycystic ovarian disease, precocious puberty, hypertrichosis, gonadotroph pituitary adenomas, sleep apnea syndrome, irritable bowel syndrome, premenstrual syndrome, benign prostatic hyperplasia, and infertility.

15. The pharmaceutical composition of claim 1, wherein a weight ratio of a) to b) ranges from 10:1 to 1:10.

16. The pharmaceutical composition of claim 1, wherein a weight ratio of a)+b) to c) ranges from 1,000:1 to 1:1.

17. The pharmaceutical composition of claim 1, wherein a weight ratio of a)+b)+c) to d) ranges from 10,000:1 to 1:1.

18. The pharmaceutical composition of claim 1, comprising: a) at least one sorbitan unsaturated fatty acid ester having a polar head with at least two or more OH (hydroxyl) groups in an amount of 9-90 weight %; b) at least one phospholipid in an amount of 9-90 weight %; c) at least one liquid crystal hardener which is free of an ionizable group and has a triacyl group with 15 to 40 carbon atoms or a carbon ring structure in a hydrophobic moiety in an amount of 0.1-50 weight %; and d) at least one GnRH (gonadotropin-releasing hormone) analogue in an amount of 0.01-50 weight %.

19. The pharmaceutical composition of claim 1, comprising: a) at least one sorbitan unsaturated fatty acid ester having a polar head with at least two or more OH (hydroxyl) groups in an amount of 9-64 weight %; b) at least one phospholipid in an amount of 18-76 weight %; c) at least one liquid crystal hardener which is free of an ionizable group and has a triacyl group with 15 to 40 carbon atoms or a carbon ring structure in a hydrophobic moiety in an amount of 1-36 weight %; and d) leuprolide or a pharmaceutically acceptable salt thereof in an amount of 0.1-50 weight %.

20. The pharmaceutical composition of claim 1, comprising: a) at least one sorbitan unsaturated fatty acid ester having a polar head with at least two or more OH (hydroxyl) groups in an amount of 9-64 weight %; b) at least one phospholipid in an amount of 18-76 weight %; c) at least one liquid crystal hardener which is free of an ionizable group and has a triacyl group with 15 to 40 carbon atoms or a carbon ring structure in a hydrophobic moiety in an amount of 1-36 weight %; and d) goserelin or a pharmaceutically acceptable salt thereof in an amount of 0.1-50 weight %.

21. The pharmaceutical composition of claim 1, comprising: a) at least one sorbitan unsaturated fatty acid ester having a polar head with at least two or more OH (hydroxyl) groups in an amount of 9-64 weight %; b) at least one phospholipid in an amount of 18-76 weight %; c) at least one liquid crystal hardener which is free of an ionizable group and has a triacyl group with 15 to 40 carbon atoms or a carbon ring structure in a hydrophobic moiety in an amount of 1-36 weight %; and d) degarelix or a pharmaceutically acceptable salt thereof in an amount of 2-50 weight %.

22. The pharmaceutical composition of claim 1, being in a formulation, said formulation being selected from the group consisting of an injection, an ointment, a gel, a lotion, a capsule, a tablet, a solution, a suspension, a spray, an inhalant, an eye drop, an adhesive, a plaster, and a pressure sensitive adhesive.

23. The pharmaceutical composition of claim 22, wherein the formulation is an injection.

24. A contraceptive, comprising the pharmaceutical composition of claim 1.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 illustrates phase change behaviors of compositions of Examples 2, 6, 9 and 12 upon exposure to aqueous fluid.

(2) FIG. 2 shows the liquid crystalline structures of the compositions of Examples 2 and 6, formed in aqueous fluid.

(3) FIG. 3 shows the in vivo drug release behaviors of the compositions of Example 2 and Comparative Example 1.

(4) FIG. 4 shows the in vivo drug release behaviors of the compositions of Example 6 and Comparative Example 2.

MODE FOR THE INVENTION

(5) The following non-limiting Examples serve to illustrate selected embodiments of the invention. It will be appreciated that variations in proportions and alternatives in elements of the components shown will be apparent to those skilled in the art and are within the scope of embodiments of the present invention.

(6) The additives and excipients used in the present invention satisfied the requirements of the Pharmacopoeia and were purchased from Aldrich, Lipoid, Croda, and Seppic.

[Examples 1 to 12] Preparation of Pharmaceutical Compositions

(7) Sorbitan unsaturated fatty acid esters, phospholipids, liquid crystal hardeners, and pharmacologically active substances were added, at the weight ratios given in Table 1, below.

(8) In Examples 1 to 12, the substances were homogeneously mixed in a water bath maintained at 2075 C. using a homogenizer (PowerGen model 125. Fisher) for 0.53 hrs at 10003000 rpm. The resulting lipid solutions were left at room temperature to come to thermal equilibrium at 25 C., followed by adding each of the pharmacologically active substances leuprolide acetate, goserelin acetate, and degarelix acetate thereto. Then, the substances were homogenized using a homogenizer for about 530 mins at 1,0003,000 rpm to prepare pharmaceutical compositions in a liquid phase.

(9) TABLE-US-00001 TABLE 1 Example (Unit: mg) 1 2 3 4 5 6 7 8 9 10 11 12 Leuprolide 3.75 3.75 3.75 3.75 11.25 11.25 22.5 22.5 acetate Goserelin 3.78 3.78 acetate Degarelix 80 80 acetate Sorbitan 32 35 75 150 36 51.0 25 monooleate Sorbitan 43.4 45.8 68 120 35.0 sesquioleate Phosphatidyl 40 45 95 101.3 156 202.6 40 50 35 choline Phosphatidyl 36.6 40.7 10 38.2 ethanolamine Tocopherol 6 9 15 45 30 65 60 15 10.0 5.8 12 acetate Cholesterol 10 4 13.5 15 11.3 22 22.6 4 4 Ubiquinone 4 5 DMSO 5 15 5 5 Ethanol 10 5 28.1 30 10 Form in Liquid Crystal aqueous phase

Comparative Examples 1 and 2

(10) For the formulation of Comparative Example 1, Leuplin DPS(CJ) containing leuprolide acetate as a pharmacologically active substance was used in an amount of 3.75 mg.

(11) As the formulation of Comparative Example 2, 11.25 mg of Leuplin DPS(CJ) containing the pharmacologically active substance leuprolide acetate was used.

[Experimental Example 1] Contents of Pharmacologically Active Substances in Pharmaceutical Compositions

(12) To examine whether the pharmaceutical compositions prepared in Examples contained pharmacologically active substances at a therapeutically effective concentration, the contents of leuprolide acetate were quantitated by HPLC, as follows.

(13) Each of the pharmaceutical compositions was dissolved in an amount corresponding to 2.5 mg of leuprolide acetate in a mobile phase (triethylamine buffer:acetonitrile:n-propyl alcohol=85:9:6), and centrifuged for 10 min at 1500 rpm, followed by filtering the supernatants of the test sample through a 0.2 m filter. For comparison, a standard sample with the same concentration as that of the test samples was prepared from a leuprolide acetate standard. The standard sample and the test samples were loaded in an injection volume of 20 L at a flow rate of 1.01.5 mL/min to 4.6100 mm, 3 m packing L1 column or like, and quantitatively analyzed at 220 nm using a UV spectrometer. Average contents of leuprolide acetate in the pharmaceutical compositions were obtained from three measurements (see Table 2).

(14) TABLE-US-00002 TABLE 2 Example (Unit: %) 1 2 3 4 5 6 7 8 Content 100.3 101.2 99.8 98.9 102.9 99.4 100.5 99.1

(15) As can be seen in Table 2, all of the pharmaceutical compositions prepared in Examples 1 to 8 ideally contained leuprolide acetate in amounts within standard content (100%)3%.

[Experimental Example 2] Formation of Liquid Crystals in Aqueous Fluid

(16) An examination was made to confirm whether the pharmaceutical compositions prepared in Examples form ideal liquid crystals in aqueous fluid. In this regard, the compositions of Examples 2, 6, 9, and 12 which were in liquid phase were loaded into syringes and then injected to 2 g of PBS (pH 7.4). The results are depicted in FIG. 1.

(17) The pharmaceutical compositions prepared in Examples 1 to 12 existed in liquid phase in the absence of aqueous fluid. When injected into an aqueous fluid (PBS), the pharmaceutical compositions in liquid phase forms into spherical liquid crystals, indicating that the pharmacologically active substance GnRH analogue has no influence on the formation of the pharmaceutical compositions into liquid crystals.

[Experimental Example 3] Structural Determination of Liquid Crystals in Aqueous Fluid

(18) The liquid crystals of the pharmaceutical compositions of Examples 2 and 6, formed in aqueous fluid, were observed for structure under a polarization microscope (Motic, BA 300 Pol) (FIG. 2).

(19) A slide glass was very thinly coated with each of the pharmaceutical compositions of Examples 2 and 6, and left for 4 hrs in deionized water in a schale to form liquid crystals. After being covered with a cover glass to prevent the introduction of air, the test sample on slide glass was observed at 200 magnification using a polarization microscope (Motic, BA 300 Pol). As can be seen in FIG. 2, the pharmaceutical compositions of Examples 2 and 6 are formed into liquid crystals with typical hexagonal crystalline structures for excellent sustained release.

(20) When an account is taken of results from Experimental Examples 1 to 3, the pharmaceutical compositions of the present invention can form physiochemically stable, ideal liquid crystals in the presence of aqueous fluid even if they contain pharmacologically active substances which have large molecular weights and relatively high hydrophobicity.

[Experimental Example 4] In Vivo PK Profile of Pharmaceutical Compositions

(21) Drug release behaviors from the pharmaceutical compositions of the present invention were examined in vivo in the following test.

(22) Using a disposable syringe, each of the pharmaceutical compositions of Examples 2 and 6 was subcutaneously injected at a leuprolide acetate dose of 12.5 mg/kg (corresponding to a 28-day dose for humans) into the back of 6 SD rats (male), 9 weeks old, with an average body weight of 300 g. For comparison with PK profiles of PLGA microparticle formulations, the pharmaceutical compositions of Comparative Examples 1 and 2 were subcutaneously injected at a leuprolide acetate dose of 12.5 mg/kg (corresponding to a 28-day dose for humans) into the back of 6 SD rats (male), 9 weeks old, with an average body weight of 300 g.

(23) Leuprolide acetate concentrations in plasma samples taken from the SD rats were monitored for 28 days using LC-MS/MS (liquid chromatography-mass spectrometry) to draw PK profiles (pharmacokinetic profiles). The average of leuprolide acetate concentration taken from the 6 SD rats are plotted in graph of each of FIGS. 3 and 4, and expressed as calculated logarithm values in the lower graph of each of FIGS. 3 and 4 to examine a difference in drug concentration of rat plasma at the late phase.

(24) The PK profiles in SD rats of the pharmaceutical compositions of Comparative Example 1 and Example 2 are shown in FIG. 3. As a control (reference drug) for Example 2, Comparative Example 1 was of 3.75 mg of Leuplin DPS(CJ), which is widely used as a 1-month formulation of leuprolide acetate. Compared to the control (reference drug) of Comparative Example 1, the pharmaceutical composition of Example 2 exhibited ideal pK behavior and excellent sustained release. The pharmaceutical composition of Example 2 had an initial burst concentration of 81 ng/mL, which is about half reduced, compared to 155 ng/mL of Comparative Example 1, thus achieving an exceptional improvement in initial burst concentration, a typical problem with PLGA microparticle formulations. In contrast to the composition of Comparative Example 1 which became unstable in PK behavior from 5 days after administration, the pharmaceutical composition of Example 2 maintained very stable effective plasma concentration of leuprolide acetate.

(25) FIG. 4 shows the PK profiles in SD rats of the pharmaceutical compositions of Comparative Example 2 and Example 6. As a control (reference drug) for Example 6, Comparative Example 2 was of 11.25 mg of Leuplin DPS(CJ), which is widely used as a 3-month formulation of leuprolide acetate. Compared to the control (reference drug) of Comparative Example 2, the pharmaceutical composition of Example 6 exhibited ideal pK behavior required for sustained release formulations, and particularly excellent sustained release for a long-term. The composition of Comparative Example 2 showed an initial burst concentration about three times as high as that of Comparative Example 1, which was believed to be attributed to the difference of drug content therebetween. Although the pharmaceutical composition of Example 6 was 3-fold higher in drug content than that of Example 2, no observations were made of the rapid increase in initial burst concentration, unlike the composition of Comparative Example 2. The initial burst concentration was measured to be 114 ng/ml, which is about 4-fold smaller than 484 ng/ml, which was measured at the initial phase in the composition of Comparative Example 2. In addition, the composition of Comparative Example 2 had blood leuprolide acetate levels from 10 days after administration, which was significantly lower level compared to the composition of Comparative Example 1 in the mid- to later phase, drawing an unstable PK profile. On the other hand, the pharmaceutical composition of Example 6 allowed for a blood leuprolide acetate level curve which was similar to that of Example 2 in the mid- to late phase, demonstrating that the pharmaceutical composition of the present invention, even though 3-fold increasing in drug content, maintained excellent long-term sustained release.