GnRH analogue formulations

Abstract

The present invention relates to compositions forming a low viscosity mixture of: a) at least one diacyl glycerol; b) at least one phosphatidyl choline; c) at least one oxygen containing organic solvent; d) at least one GnRH analog; Wherein the pre-formulation forms, or is capable of forming, at least one liquid crystalline phase structure upon contact with an aqueous fluid. The invention further relates to methods of treatment comprising administration of such compositions, pre-filled administration devices and kits containing the formulations.

Claims

1. A pre-formulation consisting essentially of a low viscosity mixture of: a) at least one diacyl glycerol comprising at least 50% glycerol dioleate (GDO); b) at least one phosphatidyl choline (PC); c) at least one oxygen containing organic solvent comprising ethanol; and d) at least one peptide GnRH analogue comprising 12 or fewer amino acids; wherein the ratio of a:b (w/w) is in the range of 40:60 to 70:30; wherein the pre-formulation forms at least one liquid crystalline phase structure upon contact with an aqueous fluid.

2. A pre-formulation as claimed in claim 1 wherein component a) comprises at least 80% glycerol dioleate (GDO).

3. A pre-formulation as claimed in claim 1 wherein component b) comprises soy PC.

4. A pre-formulation as claimed in any of claims 1 to 3 wherein said pre-formulation comprises at least one GnRH analogue selected from leuprolide and goserelin.

5. A pre-formulation comprising a low viscosity mixture of: a) 40-70 wt. % of at least one diacyl glycerol comprising at least 50% GDO; b) 30-60 wt. % of at least one phosphatidyl choline; c) at least one oxygen containing organic solvent comprising ethanol; d) 0.1-10 wt. % of at least one peptide GnRH analogue comprising 12 or fewer amino acids; wherein the ratio of a:b (w/w) is in the range of 40:60 to 70:30; wherein the pre-formulation forms at least one liquid crystalline phase structure upon contact with an aqueous fluid at physiological temperature.

6. A pre-formulation as claimed in claim 5 comprising: a) 43-60 wt. % of at least one diacyl glycerol comprising at least 50% GDO; b) 35-55 wt. % of at least one phosphatidyl choline; c) 0.1-10 wt. % of at least one oxygen containing organic solvent comprising ethanol; d) 0.1-10 wt. % of at least one peptide GnRH analogue comprising 12 or fewer amino acids; wherein the ratio of a:b (w/w) is in the range of 40:60 to 70:30; wherein the pre-formulation forms, or is capable of forming, at least one liquid crystalline phase structure upon contact with an aqueous fluid.

7. A pre-formulation as claimed in claim 5, wherein the ratio of a:b (w/w) is 45:55 to 60:40.

8. A pre-formulation as claimed in claim 5, wherein the ratio of a:b (w/w) is 48:52 to 55:45.

9. A pre-formulation as claimed in claim 5 wherein component b) comprises soy PC.

10. A pre-formulation as claimed in claim 5 wherein said pre-formulation comprises at least one GnRH analogue selected from leuprolide and goserelin.

11. A pre-formulation comprising a low viscosity mixture of: a) 32.5-70 wt. % of at least one diacyl glycerol comprising at least 50% GDO; b) 30-60 wt. % of at least one phosphatidyl choline; c) at least one oxygen containing organic solvent comprising ethanol; d) 0.1-10 wt. % of at least one peptide GnRH analogue comprising 12 or fewer amino acids; wherein the ratio of a:b (w/w) is in the range of 40:60 to 70:30; wherein the pre-formulation forms at least one liquid crystalline phase structure upon contact with an aqueous fluid at physiological temperature.

12. A pre-formulation as claimed in claim 11 comprising: a) 32.5-60 wt. % of at least one diacyl glycerol comprising at least 50% GDO; b) 35-55 wt. % of at least one phosphatidyl choline; c) 0.1-10 wt. % of at least one oxygen containing organic solvent comprising ethanol; d) 0.1-10 wt. % of at least one peptide GnRH analogue comprising 12 or fewer amino acids; wherein the ratio of a:b (w/w) is in the range of 40:60 to 70:30; wherein the pre-formulation forms, or is capable of forming, at least one liquid crystalline phase structure upon contact with an aqueous fluid.

13. A pre-formulation as claimed in claim 11, wherein the ratio of a:b (w/w) is in the range of 45:55 to 60:40.

14. A pre-formulation as claimed in claim 11, wherein the ratio of a:b (w/w) is in the range of 48:52 to 55:45.

15. A pre-formulation as claimed in claim 11 wherein component b) comprises soy PC.

16. A pre-formulation as claimed in claim 11 wherein said pre-formulation comprises at least one peptide GnRH analogue selected from leuprolide and goserelin.

17. A pre-formulation comprising a low viscosity mixture of: a) 32.5-70 wt. % of at least one diacyl glycerol comprising at least 50% GDO; b) 30-60 wt. % of at least one phosphatidyl choline; c) at least one oxygen containing organic solvent comprising ethanol; d) 0.1-10 wt. % of leuprolide; wherein the ratio of a:b (w/w) is in the range of 40:60 to 70:30; wherein the pre-formulation forms at least one liquid crystalline phase structure upon contact with an aqueous fluid at physiological temperature.

18. A pre-formulation as claimed in claim 17 comprising: a) 32.5-60 wt. % of at least one diacyl glycerol comprising at least 50% GOO; b) 35-55 wt. % of at least one phosphatidyl choline; c) 0.1-10 wt. % of at least one oxygen containing organic solvent comprising ethanol; d) 0.1-10 wt. % of leuprolide; wherein the ratio of a:b (w/w) is in the range of 40:60 to 70:30; wherein the pre-formulation forms, or is capable of forming, at least one liquid crystalline phase structure upon contact with an aqueous fluid.

19. A pre-formulation as claimed in claim 17, wherein the ratio of a:b (w/w) is in the range of 45:55 to 60:40.

20. A pre-formulation as claimed in claim 17, wherein the ratio of a:b (w/w) is in the range of 48:52 to 55:45.

21. A pre-formulation as claimed in claim 17 wherein component b) comprises soy PC.

22. A pre-formulation as claimed in claim 14, wherein the at least one peptide GnRH analogue comprising 12 or fewer amino acids is a constrained peptide of 6 to 12 amino acids.

23. A pre-formulation as claimed in claim 22, wherein the at least one peptide GnRH analogue comprises Gly-NH.sub.2, N-Et-NH.sub.2 or AzaGly-NH.sub.2 at the N-terminus.

24. A pre-formulation as claimed in claim 22, wherein the at least one peptide GnRH analogue is selected from GnRH-I, GNRH-II, GnRH III, Fertirelin, Leuprorelin (Leuprolide), Buserelin, Histrelin, Deslorelin, Goserelin, Narafelin and Triptorelin.

Description

(1) The Invention will now be further illustrated by reference to the following non-limiting Examples and the attached Figures, in which;

(2) FIG. 1 shows leuprolide plasma levels in the rat model following administration of leuprolide formulation precursor (0.3 wt % in leuprolide).

(3) FIG. 2 shows testosterone plasma levels in the rat model following administration of testosterone undecanoate (TEU) and testosterone enanthate (TEE) formulation precursors (25 wt % in testosterone ester).

EXAMPLES

Example 1: Availability of Various Liquid Crystalline Phases in the Depot by Choice of Composition

(4) Injectable formulations containing different proportions of phosphatidyl choline (“PC”—Lipoid S100) and glycerol dioleate (GDO) and with EtOH as solvent were prepared to illustrate that various liquid crystalline phases can be accessed after equilibrating the depot precursor formulation with excess water.

(5) Appropriate amounts of PC, GDO and EtOH were weighed in glass vials and the mixture was placed on a shaker until the PC completely dissolved to form a clear liquid solution. GDO was then added to form an injectable homogenous solution.

(6) Each formulation was injected in a vial and equilibrated with excess water. The phase behaviour was evaluated visually and between crossed polarizes at 25° C. Results are presented in Table 1.

(7) TABLE-US-00001 TABLE 1 Formulation PC (wt %) GDO (wt %) EtOH (wt %) Phase in H.sub.2O A 22.5 67.5 10.0 L.sub.2 B 28.8 61.2 10.0 I.sub.2 C 45.0 45.0 10.0 I.sub.2/H.sub.II D 63.0 27.0 10.0 H.sub.II/L.sub.α L.sub.2 = reversed micellar phase I.sub.2 = reversed cubic liquid crystalline phase H.sub.II = reversed hexagonal liquid crystalline phase L.sub.α = lamellar phase

Example 2: Viscosity in PC/GDO Mixtures on Addition of Co-Solvent

(8) Mixtures of PC/GDO and co-solvent were prepared according to the methods of Example 1. The EtOH content was adjusted by first evaporating the EtOH from the PC/GDO-mixture on a rotary evaporator leaving a viscous liquid mixture of essentially only PC and GDO. Co-solvents were then added in the proportions indicated in Table 2 below.

(9) The samples were allowed to equilibrate for several days before viscosity measurements were performed using a Physica UDS 200 rheometer at 25° C.

(10) TABLE-US-00002 TABLE 2 PC/GDO EtOH/ Glycerol/ H.sub.2O/ Viscosity/ Sample (wt/wt) wt % wt % wt % mPas 1 50/50 3 — — 1900 2 50/50 5 — — 780 3 50/50 7 — — 430 4 50/50 8 — — 300 5 50/50 10 — — 210 6 50/50 15 — — 100 7 45/55 3 — — 1350 8 45/55 5 — — 540 9 45/55 7 — — 320 10 45/55 8 — — 250 11 45/55 10 — — 150 12 45/55 15 — — 85 13 40/60 3 — — 740 14 40/60 5 — — 400 15 40/60 7 — — 240 16 40/60 8 — — 200 17 40/60 10 — — 130 18 40/60 15 — — 57 19 40/60 — 10 — 8 * 10.sup.6 20 40/60 — — 3 2.5 * 10.sup.8   21 40/60 — — 5 4 * 10.sup.7

(11) This example illustrates the need for a solvent with viscosity lowering properties in order to obtain injectable formulations. The mixtures containing glycerol (sample 19) or water (samples 20 and 21) are too viscous to be injectable at solvent concentrations equivalent to the samples containing EtOH (compare with samples 13, 14 and 17).

Example 3: Preparation of Depot Compositions Containing the Peptide Leuprolide

(12) Leuprolide acetate is an acetate salt of a synthetic nonapeptide and an analogue of gonadotropin-releasing hormone (GnRH) (also known as luteinizing hormone-releasing hormone (LHRH)). As a result of leuprolide administration to a subject, there is an initial increase in follicle stimulating hormone (FSH) and luteinizing hormone (LH) secretion (so-called flare effect) which in turn stimulates the production of testosterone by the testes in men and estrogens by ovaries in women. After about 10 days, a profound hypogonadal effect, equivalent to surgical castration, is achieved through down-regulation. Generally this induced and reversible hypogonadism is the therapeutic goal.

(13) Leuprolide was first mixed with PC, GDO and EtOH where EtOH was added in excess to dissolve the peptide and the lipids to give a homogenous and clear solution. Typically, the EtOH content at this stage was about 50-80 wt %. The excess EtOH was then removed by rotary evaporation or freeze-drying and the final EtOH content was thereafter adjusted as required. The final compositions of the samples are given in Table 3 below.

(14) TABLE-US-00003 TABLE 3 Formulation Leuprolide/wt % PC/wt % GDO/wt % EtOH/wt % A 0.30 47.35 47.35 5.0 B 0.66 47.17 47.17 5.0 C 2.0 46.5 46.5 5.0 D 4.5 45.25 45.25 5.0 E 6.0 44.5 44.5 5.0

(15) Injecting the formulation precursor into excess aqueous phase (syringe 23 G; 0.6 mm×30 mm) resulted in a monolithic liquid crystalline phase i.e. leuprolide did not change monolith formation and phase behaviour after exposure to an aqueous environment.

(16) The leuprolide depot precursor formulations in this Example were tested for stability against crystallization during storage. Each formulation was stable at 4-8° C. for at least two weeks.

Example 4: In Vivo Release Study from Depot Formulation Containing Leuprolide Subcutaneously Administered

(17) In an in vivo rat model the drug release of leuprolide was followed during 28 days. The formulation was administered subcutaneously between the scapulae by using a syringe (23 G, 0.6 mm×25 mm). The leuprolide concentration in the rat plasma was followed for a period of 28 days (see FIG. 1). The dose was 3 mg/kg and the dose volume 1 ml/kg corresponding to a drug load of 0.3 wt % leuprolide in the depot formulation precursor (Formulation A in Example 3).

(18) FIG. 1 shows leuprolide plasma levels in the rat model following administration of leuprolide formulation precursor (0.3 wt % in leuprolide). It appears that the investigated formulation gives a release profile with a minimal initial release (low “burst”) and a sustained release duration of at least 28 days.

Example 5: Preparation of Depot Compositions Containing the Peptide Goserelin

(19) Goserelin is a potent synthetic decapeptide analogue of luteinizing hormone-releasing hormone (LHRH), also known as a GnRH agonist analogue. Goserelin binds to the gonadotropin releasing hormone (GnRH) receptor and, after prolonged administration, inhibits endogenous secretion of gonadotropin, resulting in suppression of sex hormone production in the ovary and testes. This agent reduces testosterone production to castration levels and may inhibit androgen receptor-positive tumor progression.

(20) Goserelin was first mixed with PC, GDO and EtOH where EtOH was added in excess to dissolve the peptide and the lipids to give a homogenous and clear solution. The EtOH content at this stage was about 50-80 wt %. The excess EtOH was then removed by rotary evaporation or freeze-drying and the final EtOH content was thereafter adjusted as required. The final compositions of the samples are given in Table 4 below.

(21) TABLE-US-00004 TABLE 4 Formulation Goserelin/wt % PC/wt % GDO/wt % EtOH/wt % A 1.08 46.96 46.96 5.00 B 2.16 46.42 46.42 5.00 C 4.32 45.34 45.34 5.00

(22) Injecting the formulation precursor into excess aqueous phase (syringe 23 G; 0.6 mm×30 mm) resulted in a monolithic liquid crystalline phase i.e. goserelin did not change monolith formation and phase behaviour after exposure to an aqueous environment.

(23) The goserelin depot precursor formulations in this Example were tested for stability against crystallization during storage. Each formulation was stable at 4-8° C. for at least two weeks.

Example 6: Preparation of Depot Compositions Containing the Peptide Triptorelin

(24) Triptorelin is a potent synthetic decapeptide analogue of luteinizing hormone-releasing hormone (LHRH), also known as a GnRH agonist analogue. Triptorelin binds to the gonadotropin releasing hormone (GnRH) receptor and, after prolonged administration, inhibits endogenous secretion of gonadotropin, resulting in suppression of sex hormone production in the ovary and testes. This agent reduces testosterone production to castration levels and may inhibit androgen receptor-positive tumor progression.

(25) Triptorelin was first mixed with PC, GDO and EtOH where EtOH was added in excess to dissolve the peptide and the lipids to give a homogenous and clear solution. The EtOH content at this stage was about 50-80 wt %. The excess EtOH was then removed by rotary evaporation or freeze-drying and the final EtOH content was thereafter adjusted as required. The final compositions of the samples are given in Table 5 below.

(26) TABLE-US-00005 TABLE 5 Formulation Triptorelin/wt % PC/wt % GDO/wt % EtOH/wt % A 0.75 47.125 47.125 5.00 B 1.5 46.75 46.75 5.00

(27) Injecting the formulation precursor into excess aqueous phase (syringe 23 G; 0.6 mm×30 mm) resulted in a monolithic liquid crystalline phase i.e. triptorelin did not change monolith formation and phase behaviour after exposure to an aqueous environment.

(28) The triptorelin depot precursor formulations in this Example were tested for stability against crystallization during storage. Each formulation was stable at 4-8° C. for at least two weeks.

Example 7: Preparation of Depot Compositions Containing the Peptide Buserelin

(29) Buserelin is a highly potent synthetic nonapeptide analogue of luteinizing hormone-releasing hormone (LHRH), also known as a GNRH agonist analogue. Buserelin binds to the gonadotropin releasing hormone (GnRH) receptor and, after prolonged administration, inhibits endogenous secretion of gonadotropin, resulting in suppression of sex hormone production in the ovary and testes. This agent reduces testosterone production to castration levels and may inhibit androgen receptor-positive tumor progression.

(30) Buserelin was first mixed with PC, GDO and EtOH where EtOH was added in excess to dissolve the peptide and the lipids to give a homogenous and clear solution. The EtOH content at this stage was about 50-80 wt %. The excess EtOH was then removed by rotary evaporation or freeze-drying and the final EtOH content was thereafter adjusted as required. The final compositions of the samples are given in Table 6 below.

(31) TABLE-US-00006 TABLE 6 Formulation Buserelin/wt % PC/wt % GDO/wt % EtOH/wt % A 0.66 47.17 47.17 5.00 B 1.32 46.84 46.84 5.00 C 1.98 46.51 46.51 5.00

(32) Injecting the formulation precursor into excess aqueous phase (syringe 23 G; 0.6 mm×30 mm) resulted in a monolithic liquid crystalline phase i.e. buserelin did not change monolith formation and phase behaviour after exposure to an aqueous environment.

(33) The buserelin depot precursor formulations in this Example were tested for stability against crystallization during storage. Each formulation was stable at 4-8° C. for at least two weeks.

Example 8: Degradation of Depot Formulation in the Rat

(34) Various volumes (1, 2, 6 ml/kg) of the depot precursor (36% wt PC, 54% wt GDO, and 10% wt EtOH) were injected in the rat and were removed again after a period of 14 days. It was found that substantial amounts of the formulations were still present subcutaneously in the rat after this time, see Table 7.

(35) TABLE-US-00007 TABLE 7 Dose (ml/kg) Mean diameter day 3 (mm) Mean diameter day 14 (mm) 1 (n = 3) 15.8 12.5 2 (n = 3) 18.5 15.3 6 (n = 3) 23.3 19.3

Example 8B: Further Compositions Containing GnRH Agonist Analogues

(36) Formulations were prepared as described in Examples 3, 5, 6 and 7 by mixing the peptide active with a mixture of GDO (at one of several purity levels), PC, ethanol and optionally dioleoyl PG in the proportions (by weight) indicated in Table 8.

(37) TABLE-US-00008 TABLE 8 Formu- lation Peptide EtOH PC GDO1 GDO2 GDO3 DOPG 1 2.25 10 35.1 — — 52.65 — (LEU) 2 2.25 10 35.1 52.65 — — — (LEU) 3 2.25 10 35.1 — 52.65 — — (LEU) 4 2.25 7 36.3 — — 54.45 — (LEU) 5 2.16 10 35.14 — — 52.70 — (GOS) 6 2.16 7 36.34 — — 54.50 — (GOS) 7 2.16 5 37.14 — — 55.70 — (GOS) 8 1.50 10 35.4 — — 53.1 — (TRI) 9 1.50 7 36.6 — — 54.9 — (TRI) 10 1.50 5 37.4 — — 56.1 — (TRI) 11 1.32 10 35.47 — — 53.21 — (BUS) 12 1.32 7 36.67 — — 55.01 — (BUS) 13 1.32 5 37.47 — — 56.21 — (BUS) 14 2.25 10 39.49 — — 48.26 — (LEU) 15 2.25 5 45.375 — — 45.375 2 (LEU) 16 2.25 5 44.375 — — 44.375 4 (LEU) Abbreviations: LEU = Leuprolide; GOS = Goserelin; TRI = Triptorelin; BUS = Buserelin where EtOH is ethanol, PC is LIPOID S100 soybean phosphatidylcholine, GDO is glycerol dioleate and DOPG is dioleoyl phosphatidylglycerol

(38) TABLE-US-00009 GDO quality (according to AC) Monoglycerides Diglycerides Triglycerides GDO1 10.9% 87.5% 1.4% GDO2 4.2% 92.1% 3.5% GDO3 0.5% 95.3% 4.0%

Example 8C: Preparation of Depot Compositions of Glucagon-Like Peptide 1 (GLP-1)

(39) Depot precursors of GLP-1 were prepared in two different ways: 1) GLP-1 was first mixed with PC, GDO and EtOH where EtOH was added in excess to facilitate mixing. Typically, the EtOH content at this stage was about 50-80 wt %. The excess EtOH was then removed by rotary evaporation or freeze-drying and the final EtOH content was thereafter adjusted as required. 2) GLP-1 was first dissolved in a small amount of sterile water. A pre-made liquid mixture of PC, GDO and EtOH, where the EtOH content was about 5-10% by weight, was then added to the GLP-1/water solution. The resulting mixture was mixed by vortex mixing for 1 min.

(40) The final compositions of the samples are given in Table 9 below. Several purity levels of GDO and both soy and egg phosphatidylcholine (PC) were used.

(41) TABLE-US-00010 TABLE 9 Compositions containing GLP-1 Formu- GLP-1/ PC/ GDO1/ GDO2/ GDO3/ EtOH/ H.sub.2O/ lation wt % wt % wt % wt % wt % wt % wt % A 0.5 44.75 44.75 — — 10 — B 0.5 44.75 — 44.75 — 10 — C 0.5 44.75 — — 44.75 10 — D 1.0 44.5 — — 44.5 10 — E 1.0 46 — — 46 7 — F 1.0 47 — — 47 5 — G 2.0 44 — — 44 10 — H 2.0 45.5 — — 45.5 7 — I 2.0 46.5 — — 46.5 5 — J 3.0 46 — — 46 5 — K 0.5 35.775 — — 43.725 10 10 L 1.0 35.55 — — 43.45 10 10 M 2.0 37.35 — — 45.65 5 10 N 2.0 32.85 — — 40.15 10 15 O 2.0 30.4 — — 45.6 10 12 P 3.0 30 — — 45 10 12 Q 3.0 31.875 — — 43.125 10 12 R 3.0 32.4 — — 39.6 10 15 S 2.0* 46.5 — — 46.5 5 — T 2.0* 32.85 — — 40.15 10 15 U 2.0* 30.4 — — 45.6 10 12 where EtOH is ethanol, PC is LIPOID S100 soybean phosphatidylcholine or LIPOID E 80 egg phosphatidylcholine (marked with *) and GDO is glycerol dioleate

(42) TABLE-US-00011 TABLE 10 GDO qualities used GDO quality (according to AC) Monoglycerides Diglycerides Triglycerides GDO1 10.9% 87.5% 1.4% GDO2 4.2% 92.1% 3.5% GDO3 0.5% 95.3% 4.0%

Example 9: Preparation of Depot Compositions of Paclitaxel

(43) Depot precursors of paclitaxel were prepared by mixing paclitaxel, PC, GDO and EtOH where EtOH was added in excess to facilitate mixing. Typically, the EtOH content at this stage was about 50-80 wt %. The excess EtOH was then removed by rotary evaporation or freeze-drying and the final EtOH content was thereafter adjusted as required.

(44) The final compositions of the samples are given in Table 11 below. Both soy and egg phosphatidylcholine (PC) were used in the compositions.

(45) TABLE-US-00012 TABLE 11 Compositions containing paclitaxel Formulation Paclitaxel/wt % PC/wt % GDO3/wt % EtOH/wt % A 0.5 44.75 44.75 10 B 0.5 46.25 46.25 7 C 0.5 47.25 47.25 5 D 0.5 37.8 56.7 5 E  0.5* 47.25 47.25 5 F 1.0 44.5 44.5 10 G 1.0 46 46 7 H 1.0 47 47 5 I 1.0 37.6 56.4 5 J  1.0* 47 47 5 K 2.0 44 44 10 L 2.0 45.5 45.5 7 M 2.0 46.5 46.5 5 N 2.0 37.2 55.8 5 O  2.0* 46.5 46.5 5 P 5   42.5 42.5 10 Q 5   44 44 7 R 5   45 45 5 S 5   36 54 5 T 5*  45 45 5 U 10   40 40 10 V 10   41.5 41.5 7 W 10   42.5 42.5 5 X 10   34 51 5 Y 10*   42.5 42.5 5 where EtOH is ethanol, PC is LIPOID S100 soybean phosphatidylcholine or LIPOID E 80 egg phosphatidylcholine (marked with *) and GDO is glycerol dioleate (see below)

(46) TABLE-US-00013 GDO quality (according to AC) Monoglycerides Diglycerides Triglycerides GDO3 0.5% 95.3% 4.0%

Example 10: Preparation of Depot Compositions of Interferon Beta 1A

(47) Depot precursors of interferon beta 1A were prepared in two different ways: 1) Interferon beta 1A was first mixed with PC, GDO and EtOH where EtOH was added in excess to facilitate mixing. Typically, the EtOH content at this stage was about 50-80 wt %. The excess EtOH was then removed by rotary evaporation or freeze-drying and the final EtOH content was thereafter adjusted as required. 2) Interferon beta 1A was first dissolved in a small amount of sterile water. A pre-made liquid mixture of PC, GDO and EtOH, where the EtOH content was about 5-10% by weight, was then added to the Interferon beta 1A/water solution. The resulting mixture was mixed by vortex mixing for 1 min.

(48) The final compositions of the samples are given in Table 12 below. Both soy and egg phosphatidylcholine (PC) were used.

(49) TABLE-US-00014 TABLE 12 Compositions containing interferon beta 1A Interferon beta 1A/ PC/ GDO3/ EtOH/ H.sub.2O/ Formulation wt % wt % wt % wt % wt % A 0.03 44.985 44.985 10 — B 0.03 46.485 46.485 7 — C 0.03 47.485 47.485 5 — D 0.05 44.975 44.975 10 — E 0.05 46.475 46.475 7 — F 0.05 47.475 47.475 5 — G 0.1 37.96 56.94 5 — H 0.1 47.45 47.45 5 — I 0.1* 37.96 56.94 5 — J 0.1* 47.45 47.45 5 — K 0.05 35.98 43.97 10 10 L 0.05 37.98 45.97 10 10 M 0.05 31.98 47.97 10 10 N 0.1 35.96 43.94 10 10 O 0.1 31.96 47.94 10 10 P 0.1* 35.96 43.94 10 10 Q 0.1* 31.96 47.94 10 10 R 0.2 33.9 45.9 10 10 S 0.2 35.9 43.9 10 10 where EtOH is ethanol, PC is LIPOID S100 soybean phosphatidylcholine or LIPOID E 80 egg phosphatidylcholine (marked with *) and GDO is glycerol dioleate (see below)

(50) TABLE-US-00015 GDO quality (according to AC) Monoglycerides Diglycerides Triglycerides GDO3 0.5% 95.3% 4.0%

Example 11: Preparation of Depot Compositions of Human Growth Hormone (hGH)

(51) Depot precursors of hGH were prepared in two different ways: 1) hGH was first mixed with PC, GDO and EtOH where EtOH was added in excess to facilitate mixing. Typically, the EtOH content at this stage was about 50-80 wt %. The excess EtOH was then removed by rotary evaporation or freeze-drying and the final EtOH content was thereafter adjusted as required. 2) hGH was first mixed in a small amount of sterile water. A pre-made liquid mixture of PC, GDO and EtOH, where the EtOH content was about 5-10% by weight, was then added to the hGH/water mixture. The resulting mixture was mixed by vortex mixing for 1 min.

(52) The final compositions of the samples are given in Table 13 below. Both soy and egg phosphatidylcholine (PC) were used.

(53) TABLE-US-00016 TABLE 13 Compositions containing hGH EtOH/ Formulation hGH/wt % PC/wt % GDO3/wt % wt % H.sub.2O/wt % A 0.5 44.75 44.75 10 — B 0.5 46.25 46.25 7 — C 0.5 47.25 47.25 5 — D 0.5 33.53 40.97 10 15 E 0.5 35.78 43.72 10 10 F 0.5 37.13 45.37 7 10 G 1 47 47 5 — H 1 31.6 47.4 10 10 I 1 34.65 42.35 10 12 J 1 33.75 41.25 10 14 K 1 33.3 40.7 10 15 L 1* 34.65 42.35 10 12 M 1.2 34.68 39.12 10 15 N 1.2 33.21 40.59 10 15 O 1.2* 34.68 39.12 10 15 P 1.2* 33.21 40.59 10 15 where EtOH is ethanol, PC is LIPOID S100 soybean phosphatidylcholine or LIPOID E 80 egg phosphatidylcholine (marked with *) and GDO is glycerol dioleate (see below)

(54) TABLE-US-00017 GDO quality (according to AC) Monoglycerides Diglycerides Triglycerides GDO3 0.5% 95.3% 4.0%

Example 12: Preparation of Depot Compositions of Naltrexone

(55) Depot precursors of naltrexone were prepared by mixing naltrexone, PC, GDO and EtOH where EtOH was added in excess to facilitate mixing. Typically, the EtOH content at this stage was about 50-80 wt %. The excess EtOH was then removed by rotary evaporation or freeze-drying and the final EtOH content was thereafter adjusted as required.

(56) The final compositions of the samples are given in Table 14 below.

(57) TABLE-US-00018 TABLE 14 Compositions containing naltrexone Naltrexone/ Formulation wt % PC/wt % GDO3/wt % EtOH/wt % A  5 42.5 42.5 10 B  5 44 44 7 C  5 45 45 5 D  5 36 54 5 E  5* 45 45 5 F 10 40 40 10 G 10 41.5 41.5 7 H 10 42.5 42.5 5 I 10 34 51 5 J 10* 42.5 42.5 5 K 15 37.5 37.5 10 L 15 39 39 7 M 15 40 40 5 N 15 32 48 5 O 15* 40 40 5 P 20 35 35 10 Q 20 36.5 36.5 7 R 20 37.5 37.5 5 S 20 30 45 5 T 20* 37.5 37.5 5 where EtOH is ethanol, PC is LIPOID S100 soybean phosphatidylcholine or LIPOID E 80 egg phosphatidylcholine (marked with *) and GDO is glycerol dioleate (see below)

(58) TABLE-US-00019 GDO quality (according to AC) Monoglycerides Diglycerides Triglycerides GDO3 0.5% 95.3% 4.0%

Example 13: Preparation of Depot Compositions of Bupivacaine and Levobupivacaine

(59) Depot precursors of bupivacaine (or levobupivacaine) were prepared by mixing bupivacaine or levobupivacaine, PC, GDO and EtOH where EtOH was added in excess to facilitate mixing. Typically, the EtOH content at this stage was about 50-80 wt %. The excess EtOH was then removed by rotary evaporation or freeze-drying and the final EtOH content was thereafter adjusted as required.

(60) The final compositions of the samples are given in Table 15 below. Both soy and egg phosphatidylcholine (PC) were used in the compositions.

(61) TABLE-US-00020 TABLE 15 Compositions containing bupivacaine or levobupivacaine Bupivacaine or levobupivacaine/ PC/ GDO3/ EtOH/ Formulation wt % wt % wt % wt % A  5 42.5 42.5 10 B  5 44 44 7 C  5 45 45 5 D  5 36 54 5 E  5* 45 45 5 F 10 40 40 10 G 10 41.5 41.5 7 H 10 42.5 42.5 5 I 10 34 51 5 J  10* 42.5 42.5 5 K 15 37.5 37.5 10 L 15 39 39 7 M 15 40 40 5 N 15 32 48 5 O  15* 40 40 5 where EtOH is ethanol, PC is LIPOID S100 soybean phosphatidylcholine or LIPOID E 80 egg phosphatidylcholine (marked with *) and GDO is glycerol dioleate (see below)

(62) TABLE-US-00021 GDO quality (according to AC) Monoglycerides Diglycerides Triglycerides GDO3 0.5% 95.3% 4.0%

Example 14: Preparation of Depot Compositions of Pramipexole

(63) Depot precursors of pramipexole were prepared by mixing pramipexole, PC, GDO and EtOH where EtOH was added in excess to facilitate mixing. Typically, the EtOH content at this stage was about 50-80 wt %. The excess EtOH was then removed by rotary evaporation or freeze-drying and the final EtOH content was thereafter adjusted as required.

(64) The final compositions of the samples are given in Table 16 below. Both soy and egg phosphatidylcholine (PC) were used in the compositions.

(65) TABLE-US-00022 TABLE 16 Compositions containing pramipexole Pramipexole/ Formulation wt % PC/wt % GDO3/wt % EtOH/wt % A 1.0 44.5 44.5 10 B 1.0 46 46 7 C 1.0 47 47 5 D 1.0 37.6 56.4 5 E 1.0* 47 47 5 F 3 43.5 43.5 10 G 3 45 45 7 H 3 46 46 5 I 3 36.8 55.2 5 J 3* 46 46 5 K 5 42.5 42.5 10 L 5 44 44 7 M 5 45 45 5 N 5 36 54 5 O 5* 45 45 5 where EtOH is ethanol, PC is LIPOID S100 soybean phosphatidylcholine or LIPOID E 80 egg phosphatidylcholine (marked with *) and GDO is glycerol dioleate (see below)

(66) TABLE-US-00023 GDO quality (according to AC) Monoglycerides Diglycerides Triglycerides GDO3 0.5% 95.3% 4.0%

Example 15: Preparation of Depot Compositions of Clonidine

(67) Depot precursors of clonidine were prepared by mixing clonidine, PC, GDO and EtOH where EtOH was added in excess to facilitate mixing. Typically, the EtOH content at this stage was about 50-80 wt %. The excess EtOH was then removed by rotary evaporation or freeze-drying and the final EtOH content was thereafter adjusted as required.

(68) The final compositions of the samples are given in Table 17 below. Both soy and egg phosphatidylcholine (PC) were used in the compositions.

(69) TABLE-US-00024 TABLE 17 Compositions containing clonidine Clonidine/ Formulation wt % PC/wt % GDO3/wt % EtOH/wt % A 1.0 44.5 44.5 10 B 1.0 46 46 7 C 1.0 47 47 5 D 1.0 37.6 56.4 5 E 1.0* 47 47 5 F 3 43.5 43.5 10 G 3 45 45 7 H 3 46 46 5 I 3 36.8 55.2 5 J 3* 46 46 5 K 5 42.5 42.5 10 L 5 44 44 7 M 5 45 45 5 N 5 36 54 5 O 5* 45 45 5 where EtOH is ethanol, PC is LIPOID S100 soybean phosphatidylcholine or LIPOID E 80 egg phosphatidylcholine (marked with *) and GDO is glycerol dioleate (see below)

(70) TABLE-US-00025 GDO quality (according to AC) Monoglycerides Diglycerides Triglycerides GDO3 0.5% 95.3% 4.0%

Example 16: Preparation of Depot Compositions of Levothyroxine

(71) Depot precursors of levothyroxine were prepared by mixing levothyroxine, PC, GDO and EtOH where EtOH was added in excess to facilitate mixing. Typically, the EtOH content at this stage was about 50-80 wt %. The excess EtOH was then removed by rotary evaporation or freeze-drying and the final EtOH content was thereafter adjusted as required.

(72) The final compositions of the samples are given in Table 18 below. Both soy and egg phosphatidylcholine (PC) were used in the compositions.

(73) TABLE-US-00026 TABLE 18 Compositions containing levothyroxine Levothyroxine/ Formulation wt % PC/wt % GDO3/wt % EtOH/wt % A 0.5 44.75 44.75 10 B 0.5 46.25 46.25 7 C 0.5 47.25 47.25 5 D 0.5 37.8 56.7 5 E 0.5* 47.25 47.25 5 F 1.5 44.25 44.25 10 G 1.5 45.75 45.75 7 H 1.5 46.75 46.75 5 I 1.5 37.4 56.1 5 J 1.5* 46.75 46.75 5 K 3 43.5 43.5 10 L 3 45 45 7 M 3 46 46 5 N 3 36.8 55.2 5 O 3* 46 46 5 where EtOH is ethanol, PC is LIPOID S100 soybean phosphatidylcholine or LIPOID E 80 egg phosphatidylcholine (marked with *) and GDO is glycerol dioleate (see below)

(74) TABLE-US-00027 GDO quality (according to AC) Monoglycerides Diglycerides Triglycerides GDO3 0.5% 95.3% 4.0%

Example 17: Preparation of Depot Compositions of Buprenorphine

(75) Depot precursors of buprenorphine were prepared by mixing buprenorphine, PC, GDO and EtOH where EtOH was added in excess to facilitate mixing. Typically, the EtOH content at this stage was about 50-80 wt %. The excess EtOH was then removed by rotary evaporation or freeze-drying and the final EtOH content was thereafter adjusted as required.

(76) The final compositions of the samples are given in Table 19 below. Both soy and egg phosphatidylcholine (PC) were used in the compositions.

(77) TABLE-US-00028 TABLE 19 Compositions containing buprenorphine Buprenorphine/ Formulation wt % PC/wt % GDO3/wt % EtOH/wt % A 1.0 44.5 44.5 10 B 1.0 46 46 7 C 1.0 47 47 5 D 1.0 37.6 56.4 5 E 1.0* 47 47 5 F 3 43.5 43.5 10 G 3 45 45 7 H 3 46 46 5 I 3 36.8 55.2 5 J 3* 46 46 5 K 5 42.5 42.5 10 L 5 44 44 7 M 5 45 45 5 N 5 36 54 5 O 5* 45 45 5 where EtOH is ethanol, PC is LIPOID S100 soybean phosphatidylcholine or LIPOID E 80 egg phosphatidylcholine (marked with *) and GDO is glycerol dioleate (see below)

(78) TABLE-US-00029 GDO quality (according to AC) Monoglycerides Diglycerides Triglycerides GDO3 0.5% 95.3% 4.0%

Example 18: Preparation of Depot Compositions of Testosterone Esters

(79) Depot precursors of the undecanoate and enanthate esters of testosterone were prepared by mixing the testosterone esters, PC, GDO and EtOH, where EtOH was added in excess to facilitate mixing. Typically, the EtOH content at this stage was about 50-80 wt %. The excess EtOH was then removed by rotary evaporation or freeze-drying and the final EtOH content was thereafter adjusted as required.

(80) The final compositions of the samples are given in Table 20 below. Both soy and egg phosphatidylcholine (PC) were used in the compositions.

(81) TABLE-US-00030 TABLE 20 Compositions containing testosterone undecanoate or testosterone enanthate Testosterone undecanoate or PC/ GDO3/ EtOH/ Formulation testosterone enanthate/wt % wt % wt % wt % A 10 40 40 10 B 10 41.5 41.5 7 C 10 42.5 42.5 5 D 10 34 51 5 E  10* 42.5 42.5 5 F 15 37.5 37.5 10 G 15 39 39 7 H 15 40 40 5 I 15 32 48 5 J  15* 40 40 5 K 20 35 35 10 L 20 36.5 36.5 7 M 20 37.5 37.5 5 N 20 30 45 5 O  20* 37.5 37.5 5 P 25 32.5 32.5 10 Q 25 34 34 7 R 25 35 35 5 S 25 28 42 5 T  25* 35 35 5 where EtOH is ethanol, PC is LIPOID S100 soybean phosphatidylcholine or LIPOID E 80 egg phosphatidylcholine (marked with *) and GDO is glycerol dioleate (see below)

(82) TABLE-US-00031 GDO quality (according to AC) Monoglycerides Diglycerides Triglycerides GDO3 0.5% 95.3% 4.0%

Example 19: In Vivo Release Study from Depot Formulations Containing Testosterone Esters Subcutaneously Administered

(83) In an in vivo rat model the drug release of testosterone undecanoate and testosterone enanthate was followed during 28 days. The formulations were administered subcutaneously between the scapulae by using a syringe (23 G, 0.6 mm×25 mm). The testosterone concentration in the rat plasma was followed for a period of 28 days (see FIG. 2). The dose was 125 mg/kg and the dose volume 0.5 ml/kg corresponding to a drug load of 25 wt % testosterone ester in the depot formulation precursor (Formulation R in Example 18).

(84) FIG. 2 shows testosterone plasma levels in the rat model following administration of testosterone undecanoate (TEU) and testosterone enanthate (TEE) formulation precursors (25 wt % in testosterone ester). It appears that the investigated formulations give release profiles with a sustained release duration of at least 28 days.

Example 20: Preparation of a Depot Precursor of hGH Using a 2-Part Mixing Device

(85) A depot precursor of hGH was prepared by first dissolving 7.5 mg of hGH in 0.15 g of sterile water. This solution was withdrawn into a 1 mL glass syringe. A liquid solution containing PC, GDO and EtOH (PC/GDO/EtOH=40.5/49.5/10 wt %) (0.84 g) was withdrawn into a second glass syringe.

(86) The two syringes containing hGH/water and the lipid mixture, respectively, were connected using a female to female Luer adapter and the two solutions were mixed by repeatedly pushing the content back and forth. After about 15 cycles of pushing back and forth, the depot precursor was collected in one of the syringes and injected into saline using a 23 gauge needle.

Example 21: Preparation of a Depot Precursor of Interferon Beta 1A Using a 2-Part Mixing Device

(87) A depot precursor of interferon beta 1A was prepared by first dissolving 1.0 mg of interferon beta 1A in 0.1 g of sterile water. This solution was withdrawn into a 1 mL glass syringe. A liquid solution containing PC, GDO and EtOH (PC/GDO/EtOH=40.5/49.5/10 wt %) (0.9 g) was withdrawn into a second glass syringe.

(88) The two syringes containing interferon beta 1A/water and the lipid mixture, respectively, were connected using a female to female Luer adapter and the two solutions were mixed by repeatedly pushing the content back and forth. After about 15 cycles of pushing back and forth, the depot precursor was collected in one of the syringes and injected into saline using a 23 gauge needle.

LEGEND TO FIGURES

(89) FIG. 1: Leuprolide plasma levels in the rat model following administration of leuprolide (3 mg/kg) formulation precursor (0.3% by weight leuprolide) according to Example 4.

(90) FIG. 2: Testosterone plasma levels in the rat model following administration of different testosterone derivatives according to Example 19 (TEU=testosterone undecanoate and TEE=testosterone enanthate).