LIPID FORMULATIONS COMPRISING A THIOLATED ANTIOXIDANT

Abstract

The present invention provides a formulation comprising a lipid matrix; at least one thiolated antioxidant; at least one bioactive agent; and optionally at least one chelating agent. The bioactive agent may be a gonadotrophin-releasing hormone (GnRH) agonist; a gonadotrophin-releasing hormone (GnRH) antagonist; a luthenizing hormone releasing hormone (LHRH); and/or a luthenizing hormone releasing hormone (LHRH) related peptide.

Claims

1) A formulation comprising: i) a lipid matrix; ii) at least one thiolated antioxidant; iii) at least one of: <a gonadotrophin-releasing hormone (GnRH) agonist; a gonadotrophin-releasing hormone (GnRH) antagonist; a luthenizing hormone releasing hormone (LHRH); and/or a luthenizing hormone releasing hormone (LHRH) related peptide; and iv) optionally at least one chelating agent.

2) A formulation as claimed in claim 1, wherein said lipid matrix comprises: a) at least one diacyl glycerol and/or tocopherol; b) at least one phospholipid; c) at least one oxygenated organic solvent; and d) optionally at least one fragmentation agent.

3) A formulation as claimed in claim 2, wherein the lipid matrix is in the form of at least one non-lamellar phase, or generates at least one non-lamellar phase upon exposure to an aqueous fluid.

4) A formulation as claimed in claim 1, wherein the thiolated antioxidant is selected from the group consisting of a thiolated sugar, thiolated amino acid, a thiolated amino ester, and a thiolated polyol.

5) A formulation as claimed in claim 4, wherein said thiolated antioxidant is selected from the group consisting of mono-thioglycerol, cysteine, and N-acetyl cysteine.

6) A formulation as claimed in claim 1 additionally comprising a chelating agent.

7) A formulation as claimed in claim 6, wherein said chelating agent is EDTA.

8) A formulation as claimed in claim 1 additionally comprising a lipid soluble acid.

9) A formulation as claimed in claim 1, wherein said at least one gonadotrophin-releasing hormone (GnRH) agonist and/or GnRH antagonist is selected from the group consisting of buserelin, luprorelin, goserelin, triptorelin, avorelin, deslorelin, abarelix, and degarelix.

10) A method for reducing oxidative degradation in a formulation comprising a lipid matrix and at least one of: a gonadotrophin-releasing hormone (GnRH) agonist; a gonadotrophin-releasing hormone (GnRH) antagonist; luthenizing hormone releasing hormone (LHRH); and/or a luthenizing hormone releasing hormone (LHRH) related peptide; said method comprising adding at least one thiolated antioxidant and optionally at least one chelating agent to the formulation.

11) A method according to claim 10, wherein said at least one gonadotrophin-releasing hormone (GnRH) agonist and/or GnRH antagonist is selected from the group consisting of buserelin, luprorelin, goserelin, triptorelin, avorelin, deslorelin, abarelix, and degarelix.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0114] FIG. 1 Shows the effect of MTG concentration on the protection of buprenorphine (BUP) in the lipid formulation of the invention towards oxidation degradation during stress tests with hydrogen peroxide.

[0115] FIG. 2 Shows the recovery of BUP (HPLC) after 1 month at 70 C. (accelerated stability testing) as a function of MTG concentration in the formulation of the invention.

[0116] FIG. 3 Shows the assayed glucagon (GLU) content after storage at 25 C./60% RH for 7 days, plotted as the % of initial (time zero) GLU content, with different antioxidants included in the formulations.

[0117] FIG. 4 Shows the amount of detected degradation products (% Total Area) in GLU formulations after storage at 25 C./60% RH for 7 days, with different antioxidants included in the formulations.

EXAMPLES Abbreviations used in examples:

[0118]

TABLE-US-00001 Name Abbreviation Supplier Phosphatidylcholine, soy SPC Lipoid, Germany Glycerol dioleate GDO Danisco, Denmark Ethanol (99.5%) EtOH Kemetyl, Sweden Mono-thioglycerol MTG Fluka, Sweden N-acetyl cysteine N-AcCys Sigma-Aldrich, Sweden -tocopherol TOC DSM, Switzerland Propyl gallate PGall Sigma-Aldrich, Sweden Butyl hydroxytoluene BHT Fluka, Sweden

Example 1

Effects of Different Antioxidants in Lipid Formulations of Buprenorphine (BUP)

[0119] Lipid formulations of BUP also containing antioxidant were prepared in the following way: First, a liquid lipid stock solution of SPC/GDO/EtOH/antioxidant (or a reference stock solution without antioxidant) in the required proportions were prepared by weighing of all components into glass vials followed by mixing by end-over-end rotation for about 8 h or until completely homogenous liquids were obtained. Thereafter, BUP (powder) was added to achieve a nominal concentration of 7.9 wt % in all cases. The final nominal compositions of the respective formulations are given in Table 1. The antioxidants were added in an amount corresponding to common previous use in pharmaceutical products.

TABLE-US-00002 TABLE 1 Nominal compositions (wt %) of formulations studied Formulation BUP SPC GDO EtOH MTG PGall TOC BHT A 7.9 41.1 41.1 10.0 B 7.9 40.6 40.6 9.9 1.1 C 7.9 41.0 41.0 10.0 0.03 D 7.9 40.9 40.9 10.0 0.3 E 7.9 41.0 41.0 10.0 0.13

[0120] The following stress test was performed to evaluate the effect of the four different antioxidants (Table 1): Oxidative degradation was induced by adding 20 L H.sub.2O.sub.2 (30%)/mL formulation followed by equilibration of the formulations for 48 hours at RT (dark). Analysis of BUP and any oxidation degradation product (DP) in the formulations was performed by HPLC (reversed phase column) with UV detection at 288 nm. The relative retention time (RR) of BUP was set to 1 and the main oxidation DP had an RR of 1.053.

[0121] As shown in Table 2, all of the antioxidants had some effect compared with the reference formulation, however; MTG was clearly superior in protecting BUP from oxidation degradation in the lipid formulation.

TABLE-US-00003 TABLE 2 Effect of different antioxidants on BUP oxidation in stressed formulations (H.sub.2O.sub.2 treated formulations) Relative area % of main Formulation Antioxidant oxidation DP (RR = 1.053) A None (reference formulation) 1.36 B MTG 0.12 C PGall 0.70 D TOC 0.72 E BHT 0.74

Example 2

Effect of Different MTG Concentrations

[0122] The protective effect of different MTG concentrations was assessed by performing a stress test according to Example 1. Briefly, to a lipid formulation of BUP with the nominal composition BUP/SPC/GDO/EtOH =7.9/41.1/41.1/10.0 wt % was added MTG at concentrations between 0-1.0 wt %. To the resulting formulations was added 20 L H.sub.2O.sub.2 (30%)/mL formulation followed by equilibration for 48 hours at RT (dark). The formulations were thereafter analysed by HPLC as described in Example 1 and the relative area % of the main oxidation degradation product (DP) peak (relative retention RR=1.053) was determined. As shown in FIG. 1, already low concentrations of MTG had a significant protective effect.

Example 3

Effect of Different MTG Concentrations on BUP Stability During Accelerated Stability Studies

[0123] Lipid formulations of BUP (nominal concentration 7.9 wt %) comprising different concentrations of MTG (0-1.0 wt %) were prepared as described in Examples 1 and 2. The formulations were filled in glass vials, the headspace flushed with nitrogen and the vials were capped with Teflon-coated rubber stoppers and tear-off aluminium caps. The vials were thereafter transferred to a heating cabinet held at 70 C. and stored for 1 month before HPLC analysis as described in Examples 1 and 2. As shown in FIG. 2, the addition of low concentrations of MTG increased the recovery of BUP from the stored samples.

Example 4

Lipid Formulation Comprising N-Acetyl Cysteine (N-AcCys)

[0124] A liquid lipid formulation comprising SPC/GDO/EtOH (42.5/42.5/15 wt %) was prepared by weighing all components in a glass vial followed by end-over-end mixing for 4 hours at RT. To the transparent and homogenous lipid formulation was added N-AcCys at a concentration of 0.25 wt % followed by further mixing for 12 hours. The resulting formulation was transparent and homogenous.

Example 5

Antioxidant Tests

[0125] To prevent breakdown of glucagon resulting from oxidation of the methionine residue (giving Met(O)27 glucagon), an antioxidant may be included in the compositions of the present invention. To establish the most suitable antioxidant, various common and less common antioxidants were tested in the peptide/lipid system of the present invention.

[0126] The nominal composition of the samples used for the exploratory stability study is given in Table 3. Note that the antioxidant content in the respective formulations, except for the reference formulations (#229 and #234), was 0.3 wt % for tocopherol (-TOC), acsorbyl palmitate (AscPalm) and mono-thioglycerol (MTG) and 0.1 wt % for butyl hydroxytoluene (BHT). Samples containing MTG were #233 and 235.

TABLE-US-00004 TABLE 3 Sample composition of formulations used for the exploratory stability study Formulation Number Composition (wt %) (antioxidant additive in bold) 229 GLU/SPC/GDO/P80/EtOH/m-Cres/PG/BzCOOH = 0.3/31.2/31.2/3.9/11.8/0.5/20.1/1.0 230 GLU/SPC/GDO/P80/EtOH/m-Cres/PG/BzCOOH/-TOC = 0.3/31.1/31.1/3.9/11.7/0.5/20.1/1.0/0.3 231 GLU/SPC/GDO/P80/EtOH/m-Cres/PG/BzCOOH/ AscPalm = 0.3/31.1/31.1/3.9/11.7/0.5/20.1/1.0/0.3 232 GLU/SPC/GDO/P80/EtOH/m-Cres/PG/BzCOOH/BHT = 0.3/31.15/31.15/3.85/11.75/0.5/20.1/1.0/0.1 233 GLU/SPC/GDO/P80/EtOH/m-Cres/PG/BzCOOH/MTG = 0.3/31.1/31.1/3.9/11.7/0.5/20.1/1.0/0.3 234 GLU/DOPC/GDO/P80/EtOH/m-Cres/PG/BzCOOH = 0.3/31.2/31.2/3.9/11.8/0.5/20.1/1.0 235 GLU/DOPC/GDO/P80/EtOH/m-Cres/PG/BzCOOH/MTG = 0.3/31.1/31.1/3.9/11.7/0.5/20.1/1.0/0.3 GLU = glucagon; SPC = Soy phosphatidylcholine; GDO = Glyceroldioleate; P80 = Polysorbate 80; EtOH = Ethanol; PG = Propylene glycol; m-Cres = meta-Cresol; BzCOOH = Benzoic acid

[0127] The stability data using a number of different antioxidants above have shown that MTG is more efficient than other lipid soluble antioxidants in suppressing the oxidative degradation of glucagon. This effect is outlined in the above Table and discussed by reference to the Figures below.

[0128] In FIG. 3, the assayed GLU content after storage at 25 C./60% RH for 7 days is plotted as the % of initial (time zero) GLU content. It can be seen that MTG gave best oxidation protection, with only this and BHT being better than the control (antioxidant-free) composition. a-tocopherol and AscPalm were degrading of glucagon in these tests.

[0129] FIG. 4 displays the amount of detected degradation products (% Total Area) with a relative retention RR<1 and RR>1, respectively, as compared with the peak corresponding to GLU. Because the assay is based on a normal phase (NP) HPLC column, degradation products with RR<1 are more hydrophobic compared with GLU whereas more hydrophilic degradation products give RR>1.

[0130] The major part of the detected degradation products with RR>1 is constituted by oxidized glucagon (Met(O)27 glucagon). It is clearly seen that MTG is most efficient in preventing the formation of Met(O)27 glucagon.