ORAL PHARMACEUTICAL COMPOSITIONS COMPRISING LIPID CONJUGATES

Abstract

The invention relates to oral pharmaceutical compositions of an active agent and a lipid conjugate of a cell penetrating peptide conjugated to a lipid molecule. The conjugated lipid acts as a permeation enhancer for the active agent in the composition. In other words, oral bioavailability of the active agent increases when co-administered together with the lipid conjugate described herein.

Claims

1. A pharmaceutical composition for oral administration comprising a conjugate comprising a cell penetrating peptide conjugated to a lipid, and an active agent, wherein the composition is essentially free of liposomes.

2. The pharmaceutical composition according to claim 1, wherein the cell penetrating peptide is selected from penetratin, TAT (transactivator of transcription), MAP (model amphiphatic peptide), polyarginines (including R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12), pVEC, transportan, MPG, and functional derivatives, peptide-mimetics and combinations thereof.

3. The pharmaceutical composition according to claim 1, wherein the cell penetrating peptide is a cyclic peptide.

4. The pharmaceutical composition according to claim 1, wherein oral bioavailability of the active agent is increased by at least 50% compared to oral bioavailability of the same composition without the conjugate.

5. The pharmaceutical composition according to claim 1, wherein absolute oral bioavailability of the active agent in the composition is at least 2.5%.

6. The pharmaceutical composition according to claim 1, wherein the active agent is a peptide, such as a cyclic peptide.

7. The pharmaceutical composition according to claim 1, wherein the composition is liquid, solid, or semi-solid.

8. The pharmaceutical composition according to claim 1, wherein the amount of conjugated lipid is from 0.1 to 100 mol % relative to the total amount of oily components in the composition, wherein the amount of conjugated lipid is from 5.0 to 100 mol % relative to the total amount of oily components in the composition, wherein the total amount of oily components in the composition is from 0.1 to 100 mol % relative to the total amount of lipid conjugate in the composition, and/or wherein the total amount of oily components in the composition is from 5.0 to 100 mol % relative to the total amount of lipid conjugate in the composition.

9. The pharmaceutical composition according to claim 1, wherein the active agent exhibits a degradation half-life in simulated gastric and/or intestinal fluid at 37° C. of at least 10 minutes.

10. The pharmaceutical composition according to claim 1, wherein the lipid is a phospholipid, optionally selected from phosphatidylcholines, phosphatidylethanolamines, phosphatidylinosites, phosphatidylserines, cephalines, phosphatidylglycerols, lysophospholipids, and combinations thereof, and/or the lipid is selected from the group consisting of steroids (including cholesterol and its derivatives), fatty acids, fatty alcohols, fatty amines, hydrocarbons with carbon chain lengths of at least eight carbon atoms, sphingolipids, ceramides, glycolipids, etherlipids, carotenoids, glycerides and combinations thereof.

11. The pharmaceutical composition according to claim 1, wherein the composition comprises an aqueous solution with particles therein, wherein the particles comprise the lipid conjugate.

12. The pharmaceutical composition according to claim 11, wherein the particles have a particle size of less than 100 nm, or less than 75 nm.

13. The pharmaceutical composition according to claim 11, wherein the composition is essentially free of liposomes.

14. The pharmaceutical composition according to claim 1, wherein the amount of lipid conjugate in the composition is from 0.1 to 1000 mg/g.

15. A pharmaceutical composition comprising at least one conjugated lipid comprising a cell penetrating peptide conjugated to a lipid, such as a phospholipid or fatty acid, and at least one active agent, wherein the amount of conjugated lipid is from 0.1 to 100 mol % relative to the total amount of oily components in the composition.

16. The pharmaceutical composition according to claim 15, wherein the active agent is selected from the group consisting of peptide, polypeptide and protein.

17. The pharmaceutical composition according to claim 16, wherein the composition does not contain cholesterol in amounts of more than 1.0 mol % relative to the total amount of the oily components.

18. The pharmaceutical composition according to claim 15, wherein the total amount of oily components in the composition comprises the cumulative amounts of steroids (including cholesterol and its derivatives), fatty acids, fatty alcohols, fatty amines, hydrocarbons with carbon chain lengths of at least eight carbon atoms, phospholipids, sphingolipids, ceramides, glycolipids, etherlipids, polyethers, carotenoids, and glycerides (mono-, di- and/or triglycerides) and combinations thereof, including modified mono-, di- or triglycerides and/or modified fatty acids.

19. The pharmaceutical composition according to claim 15, wherein the oily components are the components of the composition that are immiscible with water at 25° C.

20. The pharmaceutical composition according to claim 15, wherein the oily components are components having saturated or unsaturated carbon chain lengths of more than 6, more than 8 or more than 10 carbon atoms.

21. The pharmaceutical composition according to claim 15, wherein the amount of conjugated lipid is from 0.1 to 100 mol %, or from 5.0 to 100 mol %, of the total lipid content in the composition, wherein the total lipid content is the sum of the proportions of conjugated lipid and oily component in the composition.

Description

BRIEF DESCRIPTION OF FIGURES

[0110] FIG. 1A Chemical structure of activated lipid Tfp-PEG.sub.13-DSPE.

[0111] FIG. 1B Chemical structure of activated lipid Mal-PEG.sub.12-DSPE.

[0112] FIG. 2 Diagram of particle size and PDI of lipid conjugates and octreotide formed in citrate buffer, and changes of size and PDI over time

[0113] FIG. 3 Diagram of zeta potential measured at 10 minutes, 3 hours and 24 hours after preparation of particles

[0114] FIG. 4 Diagram of absolute oral bioavailability of octreotide in compositions with lipid conjugate, without lipid conjugate and in CPP-lipid liposomes

[0115] FIG. 5 Plasma concentration curves and resulting diagram of absolute oral bioavailability of octreotide in compositions with lipid conjugate and without lipid conjugate

[0116] FIG. 6 Plasma concentration curves and resulting diagram of absolute oral bioavailability of pasireotide in compositions with lipid conjugate and without lipid conjugate

[0117] FIG. 7 Plasma concentration curves of pasireotide after administration of active agent with (black symbols) and without lipid conjugates (white symbols)

[0118] FIG. 8 Synthesis of exemplary conjugates

[0119] FIG. 9 Synthesis of exemplary conjugates

[0120] FIG. 10 Structure of a modified phospholipid used to make a conjugate.

[0121] FIG. 11 Example conjugate and modified phospholipid

[0122] FIG. 12 Further example conjugate and modified phospholipid

[0123] FIG. 13 Comparison of zetapotentials of both octreotide and exenatide containing SEDDS

[0124] FIG. 14 Comparison of particles sizes of both octreotide and exenatide containing SEDDS

[0125] FIG. 15 Comparison of PDI values of both octreotide and exenatide containing SEDDS

[0126] FIG. 16 Example of a conjugated lipid

[0127] FIG. 17 Diagrams of size, PDI and zetapotential of micelles containing a conjugated lipid

[0128] FIG. 18 Diagram of absolute oral bioavailability of octreotide in compositions with lipid conjugate SEDDS and without lipid conjugate

[0129] FIG. 19 Diagram of absolute oral bioavailability of exenatide in compositions with lipid conjugate SEDDS, lipid conjugate micelles and without lipid conjugate.

EXAMPLES

[0130] Procedures

[0131] Zetasizer Measurements

[0132] The particle size, PDI and zeta potential were determined at room temperature using a zetasizer Nano ZS from Malvern™ (Malvern Instruments Ltd., Worcestershire, United Kingdom). Size and PDI were measured after dilution to a lipid concentration of 0.07 mg/ml with a 10 mM phosphate buffer with a pH of 7.4 using the automatic mode. The zeta potential was determined after dilution to a lipid concentration of 0.14 mg/ml by a 50 mM phosphate buffer with a pH of 7.4. The default settings of the automatic mode of the zetasizer Nano ZS from Malvern™ (Malvern Instruments Ltd., Worcestershire, United Kingdom) were the following: number of measurements=3; run duration=10 s; number of runs=10; equilibration time=60 s; refractive index solvent 1.330; refractive index polystyrene cuvette 1.590; viscosity=0.8872 mPa s; temperature=25° C.; dielectric constant=78.5 F/m; backscattering mode (173°); automatic voltage selection; Smoluchowski equation.

[0133] 1. Conjugate Synthesis

[0134] The synthesis of the lipid conjugate consists of the solid-phase peptide synthesis of lysinyl-nona-arginine utilizing the Fmoc/tBu strategy. Purity is controlled after loading of lysine on a solid phase resin, after coupling of five arginines, and after completion of the solid-phase synthesis. The peptide is cleaved from the resin with side-chain protection groups intact using HFIP/DCM and purified via HPLC. The head-to-tail cyclization of the side-chain protected peptide is performed in solution (ACN/DCM) using HATU/DI EA, which impedes racemization, followed by deprotection with TFA/water/anisole and precipitation with MTBE to obtain the cyclo(Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Lys) as intermediate. Purification of the peptide intermediate is performed by HPLC on a Phenyl/Hexyl column.

[0135] The peptide intermediate is conjugated in solution (DMF/water) with 1.2 equivalents of the second intermediate (2R)-3-((((4,46-dioxo-46-(2,3,5,6-tetrafluorophenoxy)-7,10,13,16,19,22,25,28,31,34,37,40,43-tridecaoxa-3-azahexatetracontyl)oxy)(hydroxy)-phosphoryl)oxy)propane-1,2-diyldistearate and purified via HPLC on a Phenyl/Hexyl column with concurrent ion exchange to acetic acid.

[0136] The resulting lipid conjugate is shown in FIG. 11.

[0137] 2. Synthesis of the Lipid Conjugates

[0138] 3 equivalents of CPP (cyclic R9-peptides) and 10 equivalents of DIPEA were added to maleimido-PEG(12)-distearoylphosphatidylethanolamine (Iris Biotech) (shown in FIG. 1B) or PEG(13)-distearoylphosphatidylethanolamine-tetrafluorophenyl ester (Iris Biotech) (shown in FIG. 1A) dissolved in DMF in a concentration of 5 mg/ml. The reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with a 1:2 mixture of ACN/H.sub.2O and purification was performed via HPLC using a Chromolithe® Performance RP-C18e column (100×3 mm). Water and acetonitrile containing 0.05% TFA were used as eluents with a flow rate of 2 ml/min.

[0139] FIG. 12 shows the resulting lipid conjugate structure.

[0140] 3. Administration of Octreotide to Beagle Dogs

[0141] The lipid conjugate produced according to example 2 was used in lyophilized form. The lyophilized lipid conjugate was blended with lyophilized octreotide and suspended in citrate buffer. Upon suspension, the lipid conjugate formed particles. The particles had sizes of about 10 to 20 nm with a PDI in the range of from 0.17 to 0.20 (FIG. 2). Particles were remarkably stable over a period of 24 hours. FIG. 3 shows that the zeta potential of the particles was stable over 24 hours. The zeta potential was in a range of about 3.5 mV to about 7.0 mV.

[0142] A bioavailability study was performed at LPT, Hamburg, Germany (Study No 36229) by analyzing the systemic absolute bioavailability of octreotide. The pharmaceutical composition comprising 5 mg lipid conjugate and 1.5 mg octreotide, prepared in citrate buffer (100 mM, pH 5.5) was administered to four beagle dogs by gavage. The amount of active agent in plasma was measured and compared to administration of active agent alone in a dose of 1.5 mg, and with active agent in CPP-lipid-conjugate liposomes prepared in citrate buffer (100 mM, pH 5.5) according to WO 2018/178395 A1.

[0143] The results are shown in FIGS. 4 and 5. FIG. 5 illustrates the plasma concentration curves and the resulting absolute bioavailabilities. The absolute bioavailability of oral octreotide increased more than 4-fold due to the lipid-conjugate in the composition. The absolute bioavailability, i.e. compared to intravenous bolus administration of 0.1 mg/dog, was essentially the same as bioavailability of the same active agent in the liposome.

[0144] 4. Administration of Pasireotide to Beagle Dogs

[0145] The experiment of example 3 was repeated with 1.5 mg pasireotide as active agent, comparing the active agent in citrate buffer (100 mM, pH 5.5) with and without 5 mg of lipid conjugate.

[0146] The result is shown in FIG. 6. The bioavailability was more than doubled due to the lipid conjugate in the composition.

[0147] FIG. 7 shows the pharmacokinetics of pasireotide in plasma of beagle dogs after administration of free active agent (white symbols) and active agent with lipid conjugate (black symbols).

[0148] 5. Pharmacokinetic Evaluation of Octreotide Lipid Conjugate

[0149] The effect of lipid conjugate as absorption enhancer for octreotide was investigated in a study in beagle dogs at Charles River, Evreux, France (Study No 47656 PAC), by analyzing the systemic absolute bioavailability of octreotide. Animals were treated with a combination of octreotide/lipid conjugate or octreotide alone, prepared in citrate buffer (100 mM, pH 5.5), by single oral administration via gavage (table). For determination of the absolute bioavailability plasma concentrations after intravenous bolus injection were determined. Between the cycles, a washout phase of one week was performed.

[0150] The following observations were included in the overall study design:

[0151] Body weight, food consumption, and clinical observations:

[0152] Morbidity and mortality: each animal was checked for mortality and morbidity at least once a day during the study, including weekends and public holidays. Clinical signs: each animal was observed at least once a day, during pre-treatment and on the day of treatment, for the recording of clinical signs.

[0153] No signs of toxicity were observed throughout the study.

[0154] First, animals received an intravenous bolus injection of 0.01 mg/kg octreotide. The consecutive administrations were given orally by gavage. After a wash-out of one week animals #1 and #2 received 0.12 mg/kg free octreotide and animals #3 and #4 received 0.12 mg/kg octreotide with 0.3 mg/kg lipid-conjugate. After a wash-out of one week animals #1 and #2 received 0.12 mg/kg free octreotide with 1 mg/kg lipid-conjugate and animals #3 and #4 received 0.12 mg/kg octreotide with 3 mg/kg lipid-conjugate. After a wash-out of one week animals #1 and #2 received 0.12 mg/kg free octreotide with 10 mg/kg lipid-conjugate and animals #3 and #4 received 0.12 mg/kg octreotide with 30 mg/kg lipid-conjugate. After a wash-out of one week animals #1 and #2 received 0.012 mg/kg free octreotide with 1 mg/kg lipid-conjugate and animals #3 and #4 received 1.2 mg/kg octreotide with 1 mg/kg lipid-conjugate.

[0155] The lipid conjugate increased bioavailability of oral octreotide up to more than 3-fold. With respect to the determined bioavailability of octreotide and to the efforts to use the minimal required amount of lipid conjugate for the desired formulation, a dose range of 0.3-1.0 mg/kg conjugate is considered to be the optimal dose. Effective octreotide plasma concentrations were reached with 0.12 mg/kg octreotide. The respective calculated absolute bioavailabilities are listed in the table below. Additionally, a 10-fold increase in octreotide dose (1.2 mg/kg) revealed approximate dose linearity when administered with 1 mg/kg lipid-conjugate.

TABLE-US-00001 conjugate dose [mg] 0 3 10 30 100 300 abs. bioavailability [%] 2.1 4.3 6.5 6.1 3.4 6.3

[0156] 6. Preparation of Conjugates

[0157] FIG. 8 illustrates the synthesis of exemplary conjugates. The linker used in this example was SM(PEG)8, a PEGylated, long-chain SMCC crosslinker, and succinimidyl([N-maleimidopropionamido]-ethyleneglycol)ester, respectively. R9-CPP stands for either the linear or cyclic nona-arginine peptide. In case of the cyclic it is cyclized via a lysine (R9K) and coupled at the side chain amino function of this lysine.

[0158] For coupling of cyclic CPP to the bifunctional PEG-linker, as a first step the cyclized CPP was coupled by an additional lysine to the linker (1.). For this reaction an excess of the CPP was used. In the second step this intermediate product was coupled to the thiol modified phospholipid (2.). The modified phospholipid was 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, sodium salt.

[0159] FIG. 9 illustrates the synthesis of exemplary conjugates.

[0160] Cysteine-modified penetratin was coupled to the headgroup-modified phospholipid which is shown in FIG. 10. Its chemical name is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000], ammonium salt.

[0161] FIG. 11 shows an exemplary conjugate and a modified phospholipid used to make the conjugate.

[0162] A further conjugate comprising cyclic R9C was prepared. The conjugate prepared and the modified lipid used to make it are shown in FIG. 12.

[0163] 7. Preparation of SEDDS

[0164] SEDDS containing either octreotide (c=0.21 mg/ml) or exenatide (c=0.14 mg/ml) with a lipid conjugate concentration of c=1.5 mg/ml were prepared as follows:

[0165] The oily component (corn oil; 310 mg, 10.33 mg/ml) was weighted in a vial. Subsequently, the required amounts of lipid-conjugate (as shown in FIG. 11) and the respective API were added. Afterwards, the SEDDS were prepared by the addition of the required amount of citrate buffer (pH=5.5). Upon suspension, the lipid-conjugate and the oily component formed SEDDS. The SEDDS had sizes of about 150 to 200 nm with a PDI in the range of 0.3 to 0.4 (FIGS. 14 and 15) and a strong positive zeta potential of about +8 mV (FIG. 13). The concentration of octreotide was 0.21 mg/ml, and exenatide concentration was 0.14 mg/ml. The number of measurements was n=3 for zeta potential and PDI, n=6 for particle size.

[0166] The encapsulation efficiency was 98.06% for octreotide and 67.55% for exenatide. The encapsulation efficiency of SEDDS containing either octreotide (c=0.21 mg/ml) or exenatide (c=0.14 mg/ml) with a lipid conjugate concentration of c=1.5 mg/ml was determined as follows:

[0167] After preparation of SEDDS, a volume of 500 μl of the respective formulation was loaded on a Sephadex G-25 gel filtration column (NAP-5 size exclusion column). Elution was performed with a volume of 1 ml of water. The amount of the respective API in this eluted fraction was compared with the API content of the unpurified fraction under consideration of potential dilution effects. Concentrations were determined by UPLC-MS/MS quantification.

[0168] 8. Bioavailability of Octreotide SEDDS Formulation

[0169] The administration of octreotide in SEDDS formulation was investigated in a study in beagle dogs at Charles River, Evreux, France (Study No 47656 PAC and 48482 PAC). Individual systemic absolute bioavailability of octreotide in SEDDS (dose: 1.5 mg/animal) was evaluated by comparison to intravenous administration of 0.1 mg/animal of free drug, and oral administration of free drug at a dose of 1.5 mg/animal. Animals were treated with octreotide in SEDDs formulation, or octreotide alone, prepared in citrate buffer (100 mM, pH 5.5), by single oral administration via gavage. For determination of the absolute bioavailability plasma concentrations after intravenous bolus injection (in PBS) were determined. Between the cycles, a wash-out phase of one week was performed. Bioavailability of the SEDDS formulation was 2.8%, whereas free drug bioavailability was 2.1% (FIG. 18). Thus, performance of the SEDDS formulation was about 25% improved by the SEDDS.

[0170] 9. Preparation of C16-TAT Micelles

[0171] The C16-TAT lipid-conjugate (see FIG. 16) was used in lyophilized form. The lyophilized lipid-conjugate was blended with lyophilized octreotide and suspended in citrate buffer (pH=5.5). Upon suspension, the lipid conjugate formed micelles. The particles had sizes of about 40 to 50 nm with a PDI in the range of from 0.3 to 0.5 (FIGS. 17A and B). The zeta potential was in a range of about +10 mV to about +15 mV (FIG. 17C). FIG. 17 shows the results of measurement of size, PDI and zetapotential of C16-TAT-micelles (c=1 mg/ml), containing 0.2 mg/ml octreotide), n=3.

[0172] 10. Preparation of Exenatide Formulations

[0173] The co-administration of lipid conjugate with the peptide therapeutic exenatide was investigated in a study in beagle dogs at Charles River, Evreux, France (Study No 48482 PAC). Individual systemic absolute bioavailability of exenatide in SEDDS (dose: 1 mg/animal) and micellar formulation (only lipid-conjugate in lyophilized form; dose: 1 mg/animal) was evaluated by comparison to intravenous administration of 0.1 mg/animal of free drug, and oral administration of free drug at a dose of 1 mg/animal. Animals were treated with a combination of exenatide/lipid conjugate, exenatide in SEDDs formulation, or exenatide alone, prepared in citrate buffer (100 mM, pH 5.5), by single oral administration via gavage. For determination of the absolute bioavailability plasma concentrations after intravenous bolus injection (in PBS) were determined. Between the cycles, a wash-out phase of one week was performed. Compared to free drug, the bioavailability of exenatide in a lipid-conjugate micelle formulation with Aprotinin was increased by factor 3.9 (F=0.093%), and in lipid-conjugate SEDDS formulation by a factor of 4.2 (F=0.10%) in beagle dogs (FIG. 19).

[0174] The SEDDS were prepared as described in example 7. In the bioavailability study in beagles, each dog received 7 ml of the SEDDS formulation.

[0175] For preparation of the micellar formulation, the lipid-conjugate was used in lyophilized form. The lyophilized lipid-conjugate was blended with lyophilized exenatide (c=0.14 mg/ml) and the protease inhibitor Aprotinin (c=3.5 mg/ml) and suspended in citrate buffer (pH=5.5). Upon suspension, the lipid conjugate formed micelles. In the bioavailability study in beagles, each dog received 7 ml of the micelle formulation.

[0176] Plasma concentrations of all investigated peptide therapeutics were determined by quantification using a validated UPLC-MS/MS assay (according to the pertinent recommendations for bioanalytical method development of the US FDA and EMA). All assays were performed on a Waters Xevo TQ-XS triple quadrupole tandem mass spectrometer coupled to a Waters Acquity classic UPLC using C18 columns, heated electrospray ionization (ESI), and selected ion monitoring in the positive ion mode.