WO2023152672A1 - Composition comprising melatonin with high bioavailability - Google Patents

Composition comprising melatonin with high bioavailability Download PDF

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Publication number
WO2023152672A1
WO2023152672A1 PCT/IB2023/051160 IB2023051160W WO2023152672A1 WO 2023152672 A1 WO2023152672 A1 WO 2023152672A1 IB 2023051160 W IB2023051160 W IB 2023051160W WO 2023152672 A1 WO2023152672 A1 WO 2023152672A1
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Prior art keywords
melatonin
lpf
composition
weight ratio
tpgs
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PCT/IB2023/051160
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French (fr)
Inventor
Walter Pierpaoli
Alexey PIERPAOLI
Original Assignee
Pierpaoli Exelyas S.R.L.
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Publication of WO2023152672A1 publication Critical patent/WO2023152672A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/4045—Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/006—Oral mucosa, e.g. mucoadhesive forms, sublingual droplets; Buccal patches or films; Buccal sprays
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/145—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/20—Hypnotics; Sedatives

Definitions

  • composition comprising melatonin with high bioavailability
  • the present invention relates to a composition comprising melatonin having high bioavailability.
  • the present invention concerns a solid composition
  • melatonin tocopherol polyethylene glycol succinate, ascorbyl palmitate, and lysophospholipids and pharmaceutical, nutraceutical and cosmetic formulations comprising it.
  • Melatonin is a hormone produced by the pineal gland. It has been extensively demonstrated that melatonin carries out a fundamental role in the sleep-wake cycle regulation.
  • synthesis and secretion of melatonin are strongly influenced by alternating light ⁇ dark cycle, through a multi-synaptic signal that connects to the superior cervical ganglion of the orthosym pathetic.
  • production and secretion of melatonin decrease, as well as pulses flow that runs along vegetative innervation of the epiphysis.
  • melatonin has a low solubility and consequently a low bioavailability.
  • most liquid formulations based on melatonin, of synthetic or animal origin know in the art, comprise significant amounts of one or more solvents such as for example ethanol, sorbitol, and polypropylene glycol. These solvents are often contraindicated for use in specific population, for example in the paediatric and neonatal age.
  • formulations know in the art, both liquid and solid, are often characterized by a modest concentration of the active ingredient.
  • the Applicant has therefore set the objective of obtaining compositions comprising melatonin with high bioavailability.
  • the Applicant has set the objective to overcome at least in part the disadvantages of the formulations known in the art and to obtain a solid composition comprising a high percentage of melatonin that can be formulated both in solid and liquid form, without the use of undesired solvents.
  • a solid composition comprising melatonin, tocopherol polyethylene glycol succinate (TPGS), ascorbyl palmitate (AP), and lysophospholipids (LPF), characterized in that the weight ratio between AP and LPF is of between 0.5 and 1.5, and in that the weight ratio between TPGS and AP and/or LPF is of between 2 and 4.
  • TPGS tocopherol polyethylene glycol succinate
  • AP ascorbyl palmitate
  • LPF lysophospholipids
  • It is also an object of the present invention a formulation comprising the above solid composition and one or more additional excipients and/or a vehicle.
  • Figure 1 Permeation profile of melatonin through the oesophageal mucosa, (black circle, grey square) formulation according to the invention; (black square) comparative, 1 mg/ml solution; (grey circle) comparative, saturated solution. DETAILED DESCRIPTION OF THE INVENTION
  • It is a first object of the present invention a solid composition comprising melatonin, tocopherol polyethylene glycol succinate (TPGS), ascorbyl palmitate (AP), and lysophospholipids (LPF), characterized in that the weight ratio between AP and LPF is of between 0.5 and 1 .5, and in that the weight ratio between TPGS and AP and/or LPF is of between 2 and 4.
  • TPGS tocopherol polyethylene glycol succinate
  • AP ascorbyl palmitate
  • LPF lysophospholipids
  • said weight ratio between AP and LPF is of between 0.8 and 1 .2, and the weight ratio between TPGS and AP and/or LPF is of between 2.5 and 3.5. In one embodiment, said weight ratio between AP and LPF is of about 1 , and the weight ratio between TPGS and AP and/or LPF is of about 3.
  • the weight ratio between TPGS, AP and LPF is of about 3:1 :1.
  • the weight ratio between melatonin and TPGS is of between 2.5 and 3.5, preferably said weight ratio is of about 3:1 .
  • the weight ratio between melatonin and AP and/or LPF is independently of between 9 and 11 , preferably said weight ratio is of about 10:1 .
  • the solid composition according to the present invention further comprises maltodextrin.
  • the maltodextrin is present in an amount of at least 70% by weight with respect to the total weight of the composition.
  • the solid composition according to the present invention comprises melatonin in an amount of 15-25% by weight with respect to the total weight of the composition, preferably 15-20% by weight, even more preferably 17-19% by weight.
  • the solid composition according to the present invention comprises AP and LPF independently in an amount of 1.5-2.5% by weight with respect to the total weight of the composition.
  • the solid composition according to the present invention comprises TGPS in an amount of 5-7% by weight with respect to the total weight of the composition.
  • the solid composition according to the present invention consists of: 17-19% melatonin, 5-6% TPGS, 1.5-2% AP, 1.5- 2% LPF and 70-75% maltodextrin, wherein the percentages are by weight with respect to the total weight of the composition.
  • composition is obtained according to methods of the known art, for example it is obtained by spray drying, freeze drying, grinding.
  • composition according to the present invention has good stability and dissolution characteristics.
  • said formulation can be in the form of powder, tablet, capsule, dragee, or granules.
  • said formulation can be administered orally as such or after dissolution/suspension in water.
  • Said formulation ensures rapid absorption when administered orally.
  • the formulation according to the present invention can be a pharmaceutical, nutraceutical or cosmetic formulation.
  • the expert in the field will be able to select, among those known in the art, the suitable and pharmaceutically, nutraceutically or cosmetically acceptable excipients and/or vehicles to obtain the final formulation.
  • a solid composition comprising melatonin, tocopherol polyethylene glycol succinate (TPGS), ascorbyl palmitate (AP), and lysophospholipids (LPF), characterized in that the weight ratio between AP and LPF is of between 0.5 and 1 .5, and in that the weight ratio between TPGS and AP and/or LPF is of between 2 and 4 for the use as dietary supplement.
  • TPGS tocopherol polyethylene glycol succinate
  • AP ascorbyl palmitate
  • LPF lysophospholipids
  • a formulation comprising this solid composition and one or more additional excipients and/or a vehicle for the use as dietary supplement.
  • a solid composition comprising melatonin, tocopherol polyethylene glycol succinate (TPGS), ascorbyl palmitate (AP), and lysophospholipids (LPF) characterized in that the weight ratio between AP and LPF is of between 0.5 and 1 .5, and in that the weight ratio between TPGS and AP and/or LPF is of between 2 and 4 for the use as a medicament.
  • TPGS tocopherol polyethylene glycol succinate
  • AP ascorbyl palmitate
  • LPF lysophospholipids
  • LPF Lysophospholipids
  • Solid powder compositions comprising melatonin, maltodextrin and one or more of TGPS, AP and LPF were prepared according to the method described below.
  • the quali-quantitative composition (in grams) of the prepared samples is reported in the following Table 1 .
  • compositions were prepared by dissolving melatonin and one or more of TGPS, AP and LPF in ethyl acetate, subsequently suspending the maltodextrin in the mixture thus obtained and then removing the solvent under reduced pressure until a viscous suspension was obtained. The drying was then completed in a cabinet and the obtained powder classified through sieves.
  • the powders thus obtained were characterized in terms of content of melatonin, apparent solubility, dissolution rate and permeation of active ingredient through the mucosa, as will be detailed below.
  • Table 2 shows the values of melatonin content, expressed as percentage by weight with respect to the total weight of the solid composition, calculated and experimentally measured. As can be seen from what is shown in the table, the theoretical and experimental titers are comparable for all the samples prepared.
  • the thermal profile of the samples was determined using a Differential Scanning Calorimetry (DSC) device Mettler Toledo DSC2 Stare system (Switzerland). DSC calibration was performed using certified indium reference materials. For each DSC measurement, a sample of about 5 mg was placed in an aluminium standard pan of 40 pL sealed, on the lid of which a small hole has been made. An empty pan of the same type was used as a reference. All the experiments have been carried out at heating speed of 10 K/min under a constant nitrogen flow (80 ml/min). The temperature range studied was between 25 and 150°C. DSC analysis has allowed to conclude that there are no substantial changes in the solid state of the active ingredient in the samples obtained.
  • DSC Differential Scanning Calorimetry
  • Apparent solubility of melatonin in water was determined dispersing an excess of the different compositions in water and keeping the powder under stirring for 24 hours.
  • the suspension thus obtained was then filtered and diluted with an equal amount of a mobile phase and analysed via high performance liquid chromatography (HPLC) to determine the amount of melatonin.
  • HPLC high performance liquid chromatography
  • a reversed phase C18 column 150 mm x 4,6 mm, 3 pm; Luna Phenomenex®) thermostated at 40°C and a mobile phase composed of a water/acetonitrile mixture (60:40, v/v) were used.
  • the injected sample volume was 10 pl, the flow rate of the mobile phase was 1 ml/min, and the detection was performed at 280 nm.
  • Standard solutions of melatonin were prepared dissolving melatonin in a water/acetonitrile mixture (60:40, v/v).
  • Table 3 shows the apparent solubility values of melatonin obtained for the different compositions.
  • Table 4 shows the solubility values of a sample of pure melatonin in water (sample A) and in a vehicle simulating gastric pH (sample B).
  • Dissolution profile has been evaluated under sink condition to determine the necessary time to ensure the dissolution of 80% of the melatonin introduced into the paddle apparatus (T80%).
  • the data in table 5 show how the composition according to the present invention has a higher dissolution rate than the tested control compositions.
  • composition 14 was selected for skin permeation studies through porcine oesophageal mucosa in Franz cell.
  • a Franz cell consists of two compartments, one into which the composition containing the active component is loaded (donor compartment) and the other containing a receiving solution. Porcine oesophageal mucosa is placed between the two compartments.
  • the studies were performed using Franz diffusion cells having a diffusion area of 0.636 cm 2 and a receiving compartment of about 3 ml, equipped with an individually calibrated magnetic stirrer.
  • Porcine oesophagus samples were obtained from pigs slaughtered in a local slaughterhouse. Oesophageal mucosa samples were prepared by mechanical scalpel separation according to a standard procedure. Samples with an area of about 2.5 cm 2 were then placed in aluminium foil and frozen at -20°C (Labor 2T 500 ECT-F Touch, Fiocchetti, Italia). The procedure was completed within 2 hours of explant; samples of oesophageal mucosa were used for permeation tests within the next 24 hours.
  • Each oesophageal mucosa sample was thawed and mounted on the receiving compartment of a Franz diffusion cell filled with 0.9% sodium chloride solution, previously filtered with a 0.2 pm filter.
  • the mucosa surface facing the lumen of the oesophagus was placed in such a way as to contact the donor compartment. The operation was carried out paying particular attention not to create air bubbles between the solution and the membrane.
  • the system was maintained at 37 ⁇ 1 °C, while the receiving compartment was maintained under magnetic stirring at 1500 rpm/min to ensure a uniform concentration of the permeant.
  • the donor and receiving compartments were then sealed with Teflon (VWR International, I) e Parafilm® (Pechiney Plastic Packaging Company, USA) and maintained in position with a clamp. Aliquots (0.3 ml) of the compositions to be tested were loaded into the donor compartments of the Franz cells. The experiment was conducted under occlusive conditions by applying a Teflon cap to close the donor compartment.
  • the permeation profile of melatonin through the oesophageal mucosa appears to be mainly influenced by the concentration of the active ingredient in the donor compartment.
  • the higher the concentration of melatonin in the composition the higher the permeation profile. This indicates that the tested compositions do not affect the permeability of oesophageal membrane.
  • the composition comprising PPGS, AP and LPF according to the present invention ensuring a high and quick solubility of melatonin, ensures an optimal membrane permeation.
  • the Form. 14 ensures a permeated quantity equal to 33.57 ⁇ 5.42 pg/cm 2 after 6 hours.
  • Quantification of melatonin was performed by high performance liquid chromatography (HPLC) using a C18 reversed phase column (150 mm x 4.6 mm, 3 pm; Luna Phenomenex®) thermostated at 40°C and a mobile phase composed of a water/acetonitrile mixture (60:40, v/v).
  • HPLC high performance liquid chromatography
  • the injected sample volume was 10 pl
  • mobile phase flow rate was 1 mL/min
  • detection was performed at 280 nm.
  • Standard solutions of melatonin were prepared by dissolving melatonin in a water/acetonitrile mixture (60:40, v/v).
  • composition 14 was subjected to stability tests under accelerated conditions. The powder was stored at 40°C and some aliquots were taken up and analysed by HPLC at time 0 and after 3 months of storage. In the chromatograms there are no peaks with retention times associated with melatonin impurities, confirming the stability of the composition according to the present invention.

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Abstract

It is an object of the present invention a solid composition comprising melatonin, tocopherol polyethylene glycol succinate (TPGS), ascorbyl palmitate (AP), and lysophospholipids (LPF), characterized in that the weight ratio between AP and LPF is of between 0.5 and 1.5, and in that the weight ratio between TPGS and AP and/or LPF is of between 2 and 4.

Description

Composition comprising melatonin with high bioavailability
The present invention relates to a composition comprising melatonin having high bioavailability.
In particular, the present invention concerns a solid composition comprising melatonin, tocopherol polyethylene glycol succinate, ascorbyl palmitate, and lysophospholipids and pharmaceutical, nutraceutical and cosmetic formulations comprising it.
PRIOR ART
Melatonin is a hormone produced by the pineal gland. It has been extensively demonstrated that melatonin carries out a fundamental role in the sleep-wake cycle regulation.
In vivo, synthesis and secretion of melatonin are strongly influenced by alternating light\dark cycle, through a multi-synaptic signal that connects to the superior cervical ganglion of the orthosym pathetic. During the diurnal-lighting phase, production and secretion of melatonin decrease, as well as pulses flow that runs along vegetative innervation of the epiphysis. After sunset, with the progress of darkness, its blood concentrations rapidly increase and reach maximum between 2.00 AM and 4.00 AM and then gradually decrease as morning approach.
In addition to this fundamental role, several studies on melatonin are known in the art that have highlighted other extremely interesting properties from a nutraceutical and\or pharmaceutical point of view, such as for example the high antioxidant power and the free radical’s scavenger.
However, it’s known in the art that melatonin has a low solubility and consequently a low bioavailability. For this reason, most liquid formulations based on melatonin, of synthetic or animal origin, know in the art, comprise significant amounts of one or more solvents such as for example ethanol, sorbitol, and polypropylene glycol. These solvents are often contraindicated for use in specific population, for example in the paediatric and neonatal age.
Furthermore, the formulations know in the art, both liquid and solid, are often characterized by a modest concentration of the active ingredient.
SUMMARY OF THE INVENTION
The Applicant has therefore set the objective of obtaining compositions comprising melatonin with high bioavailability.
In particular, the Applicant has set the objective to overcome at least in part the disadvantages of the formulations known in the art and to obtain a solid composition comprising a high percentage of melatonin that can be formulated both in solid and liquid form, without the use of undesired solvents.
It is an object of the present invention a solid composition comprising melatonin, tocopherol polyethylene glycol succinate (TPGS), ascorbyl palmitate (AP), and lysophospholipids (LPF), characterized in that the weight ratio between AP and LPF is of between 0.5 and 1.5, and in that the weight ratio between TPGS and AP and/or LPF is of between 2 and 4.
It is also an object of the present invention a formulation comprising the above solid composition and one or more additional excipients and/or a vehicle.
It is finally an object of the present invention the formulation or composition as described above for the use as dietary supplement.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 : Permeation profile of melatonin through the oesophageal mucosa, (black circle, grey square) formulation according to the invention; (black square) comparative, 1 mg/ml solution; (grey circle) comparative, saturated solution. DETAILED DESCRIPTION OF THE INVENTION
It is a first object of the present invention a solid composition comprising melatonin, tocopherol polyethylene glycol succinate (TPGS), ascorbyl palmitate (AP), and lysophospholipids (LPF), characterized in that the weight ratio between AP and LPF is of between 0.5 and 1 .5, and in that the weight ratio between TPGS and AP and/or LPF is of between 2 and 4.
In one embodiment, said weight ratio between AP and LPF is of between 0.8 and 1 .2, and the weight ratio between TPGS and AP and/or LPF is of between 2.5 and 3.5. In one embodiment, said weight ratio between AP and LPF is of about 1 , and the weight ratio between TPGS and AP and/or LPF is of about 3.
In a particularly preferred embodiment the weight ratio between TPGS, AP and LPF is of about 3:1 :1.
In one embodiment the weight ratio between melatonin and TPGS is of between 2.5 and 3.5, preferably said weight ratio is of about 3:1 .
In one embodiment the weight ratio between melatonin and AP and/or LPF is independently of between 9 and 11 , preferably said weight ratio is of about 10:1 .
In a preferred embodiment the solid composition according to the present invention further comprises maltodextrin. Preferably, the maltodextrin is present in an amount of at least 70% by weight with respect to the total weight of the composition.
The solid composition according to the present invention comprises melatonin in an amount of 15-25% by weight with respect to the total weight of the composition, preferably 15-20% by weight, even more preferably 17-19% by weight.
The solid composition according to the present invention comprises AP and LPF independently in an amount of 1.5-2.5% by weight with respect to the total weight of the composition. The solid composition according to the present invention comprises TGPS in an amount of 5-7% by weight with respect to the total weight of the composition.
In one embodiment, the solid composition according to the present invention consists of: 17-19% melatonin, 5-6% TPGS, 1.5-2% AP, 1.5- 2% LPF and 70-75% maltodextrin, wherein the percentages are by weight with respect to the total weight of the composition.
Said composition is obtained according to methods of the known art, for example it is obtained by spray drying, freeze drying, grinding.
Advantageously, the composition according to the present invention has good stability and dissolution characteristics.
It is further an object of the present invention a formulation comprising the above solid composition and one or more additional excipients and/or a vehicle.
In particular, said formulation can be in the form of powder, tablet, capsule, dragee, or granules. In embodiments, said formulation can be administered orally as such or after dissolution/suspension in water.
Said formulation ensures rapid absorption when administered orally.
The formulation according to the present invention can be a pharmaceutical, nutraceutical or cosmetic formulation. The expert in the field will be able to select, among those known in the art, the suitable and pharmaceutically, nutraceutically or cosmetically acceptable excipients and/or vehicles to obtain the final formulation.
It is a further object of the present invention a solid composition comprising melatonin, tocopherol polyethylene glycol succinate (TPGS), ascorbyl palmitate (AP), and lysophospholipids (LPF), characterized in that the weight ratio between AP and LPF is of between 0.5 and 1 .5, and in that the weight ratio between TPGS and AP and/or LPF is of between 2 and 4 for the use as dietary supplement. It is a further object of the present invention a formulation comprising this solid composition and one or more additional excipients and/or a vehicle for the use as dietary supplement.
It is a further object of the present invention a solid composition comprising melatonin, tocopherol polyethylene glycol succinate (TPGS), ascorbyl palmitate (AP), and lysophospholipids (LPF) characterized in that the weight ratio between AP and LPF is of between 0.5 and 1 .5, and in that the weight ratio between TPGS and AP and/or LPF is of between 2 and 4 for the use as a medicament.
EXPERIMENTAL PART
The present description will be better illustrated in the following examples which have only an exemplifying and non-limiting purpose.
Materials:
The melatonin and the excipients used were purchased and used without further purification:
• Melatonin (MT): A.C.E.F
• Maltodextrin (MDX): Glucidex IT6 (Roquette)
• Tocopherol polyethylene glycol succinate 1000 (TPGS): PMC Isochem
• Ascorbyl palmitate (AP): Ph. Eur. (A.C.E.F)
• Lysophospholipids (LPF): Lipoid P LPC 80 (Lipoid)
Preparation of the compositions:
Solid powder compositions comprising melatonin, maltodextrin and one or more of TGPS, AP and LPF were prepared according to the method described below. The quali-quantitative composition (in grams) of the prepared samples is reported in the following Table 1 .
The compositions were prepared by dissolving melatonin and one or more of TGPS, AP and LPF in ethyl acetate, subsequently suspending the maltodextrin in the mixture thus obtained and then removing the solvent under reduced pressure until a viscous suspension was obtained. The drying was then completed in a cabinet and the obtained powder classified through sieves.
TABLE 1
Figure imgf000007_0001
The powders thus obtained were characterized in terms of content of melatonin, apparent solubility, dissolution rate and permeation of active ingredient through the mucosa, as will be detailed below.
Table 2 below shows the values of melatonin content, expressed as percentage by weight with respect to the total weight of the solid composition, calculated and experimentally measured. As can be seen from what is shown in the table, the theoretical and experimental titers are comparable for all the samples prepared.
TABLE 2
Figure imgf000007_0002
The thermal profile of the samples was determined using a Differential Scanning Calorimetry (DSC) device Mettler Toledo DSC2 Stare system (Switzerland). DSC calibration was performed using certified indium reference materials. For each DSC measurement, a sample of about 5 mg was placed in an aluminium standard pan of 40 pL sealed, on the lid of which a small hole has been made. An empty pan of the same type was used as a reference. All the experiments have been carried out at heating speed of 10 K/min under a constant nitrogen flow (80 ml/min). The temperature range studied was between 25 and 150°C. DSC analysis has allowed to conclude that there are no substantial changes in the solid state of the active ingredient in the samples obtained.
Apparent solubility determination
Apparent solubility of melatonin in water was determined dispersing an excess of the different compositions in water and keeping the powder under stirring for 24 hours. The suspension thus obtained was then filtered and diluted with an equal amount of a mobile phase and analysed via high performance liquid chromatography (HPLC) to determine the amount of melatonin. A reversed phase C18 column (150 mm x 4,6 mm, 3 pm; Luna Phenomenex®) thermostated at 40°C and a mobile phase composed of a water/acetonitrile mixture (60:40, v/v) were used. The injected sample volume was 10 pl, the flow rate of the mobile phase was 1 ml/min, and the detection was performed at 280 nm. Standard solutions of melatonin were prepared dissolving melatonin in a water/acetonitrile mixture (60:40, v/v).
Table 3 below shows the apparent solubility values of melatonin obtained for the different compositions.
TABLE 3
Figure imgf000008_0001
Figure imgf000009_0001
For comparison Table 4 below shows the solubility values of a sample of pure melatonin in water (sample A) and in a vehicle simulating gastric pH (sample B).
TABLE 4
Figure imgf000009_0002
The data shown in the two tables confirm the advantage obtained with the composition according to the present invention in terms of melatonin solubility.
Dissolution profile
Dissolution profile has been evaluated under sink condition to determine the necessary time to ensure the dissolution of 80% of the melatonin introduced into the paddle apparatus (T80%).
An amount of each sample containing an amount of melatonin equal to 33% of its water solubility (about 1.5 g) was dispersed in 500 ml of MilliQ® water and maintained at a temperature of 37 ± 0.5 °C. The mixtures thus obtained were placed in a paddle apparatus with a rotation speed of the paddles equal to 25 rpm.
At pre-set times (2.5, 5, 7.5, 10, 15, 30 and 45 minutes), 5 ml of each mixture was taken from each vessel and reconstituted with 5 ml of fresh solvent. The amount of melatonin released was determined by HPLC analysis according to method described above. The T80 values for each compositions tested are summarized in the following Table 5. TABLE 5
Figure imgf000010_0001
The data in table 5 show how the composition according to the present invention has a higher dissolution rate than the tested control compositions.
Permeation studies
Based on the previous results, composition 14 was selected for skin permeation studies through porcine oesophageal mucosa in Franz cell. A Franz cell consists of two compartments, one into which the composition containing the active component is loaded (donor compartment) and the other containing a receiving solution. Porcine oesophageal mucosa is placed between the two compartments.
To investigate the impact of the excipients both on its solubilization and on the permeation through the oesophageal mucosa, permeation tests were carried out using 1 mg/ml concentration and saturated solutions (Control: 1 .665 ± 0.051 mg/ml; Form. 14: 2.995 ± 0.240 mg/ml).
The studies were performed using Franz diffusion cells having a diffusion area of 0.636 cm2 and a receiving compartment of about 3 ml, equipped with an individually calibrated magnetic stirrer.
Porcine oesophagus samples were obtained from pigs slaughtered in a local slaughterhouse. Oesophageal mucosa samples were prepared by mechanical scalpel separation according to a standard procedure. Samples with an area of about 2.5 cm2 were then placed in aluminium foil and frozen at -20°C (Labor 2T 500 ECT-F Touch, Fiocchetti, Italia). The procedure was completed within 2 hours of explant; samples of oesophageal mucosa were used for permeation tests within the next 24 hours.
Each oesophageal mucosa sample was thawed and mounted on the receiving compartment of a Franz diffusion cell filled with 0.9% sodium chloride solution, previously filtered with a 0.2 pm filter. The mucosa surface facing the lumen of the oesophagus was placed in such a way as to contact the donor compartment. The operation was carried out paying particular attention not to create air bubbles between the solution and the membrane. During all the experiment the system was maintained at 37±1 °C, while the receiving compartment was maintained under magnetic stirring at 1500 rpm/min to ensure a uniform concentration of the permeant. The donor and receiving compartments were then sealed with Teflon (VWR International, I) e Parafilm® (Pechiney Plastic Packaging Company, USA) and maintained in position with a clamp. Aliquots (0.3 ml) of the compositions to be tested were loaded into the donor compartments of the Franz cells. The experiment was conducted under occlusive conditions by applying a Teflon cap to close the donor compartment.
At pre-set times (15, 45, 90, 135, 270, 360 min), 200 pl samples were taken from the receiving compartment and replaced with a fresh receiving solution to maintain sink conditions during all the experiment. Aliquots taken were then analysed in HPLC according to the method described above.
As can be seen from Figure 1 , the permeation profile of melatonin through the oesophageal mucosa appears to be mainly influenced by the concentration of the active ingredient in the donor compartment. In fact, the higher the concentration of melatonin in the composition, the higher the permeation profile. This indicates that the tested compositions do not affect the permeability of oesophageal membrane. The composition comprising PPGS, AP and LPF according to the present invention, ensuring a high and quick solubility of melatonin, ensures an optimal membrane permeation.
As can be see from the graph, advantageously the Form. 14 (Sat.) ensures a permeated quantity equal to 33.57 ± 5.42 pg/cm2 after 6 hours.
Composition stability
Analytical method
Quantification of melatonin was performed by high performance liquid chromatography (HPLC) using a C18 reversed phase column (150 mm x 4.6 mm, 3 pm; Luna Phenomenex®) thermostated at 40°C and a mobile phase composed of a water/acetonitrile mixture (60:40, v/v). The injected sample volume was 10 pl, mobile phase flow rate was 1 mL/min, and detection was performed at 280 nm. Standard solutions of melatonin were prepared by dissolving melatonin in a water/acetonitrile mixture (60:40, v/v). In these conditions the retention time of melatonin is 4.50 min., while the impurities have a RT of 1.25, 1.67, 2.41 , 3.04, 3.43, and 5.72 min. Composition 14 was subjected to stability tests under accelerated conditions. The powder was stored at 40°C and some aliquots were taken up and analysed by HPLC at time 0 and after 3 months of storage. In the chromatograms there are no peaks with retention times associated with melatonin impurities, confirming the stability of the composition according to the present invention.
Solid powder compositions
The following solid powder compositions (Table 6) according to the present invention were prepared and analysed as previously described, showing similar behaviour to composition 14.
TABLE 6
Figure imgf000012_0001
Figure imgf000013_0001

Claims

1. Solid composition comprising melatonin, tocopherol polyethylene glycol succinate (TPGS), ascorbyl palmitate (AP) and lysophospholipids (LPF), characterized in that the weight ratio between AP and LPF is of between 0.5 and 1 .5, and in that the weight ratio between TPGS and AP and/or LPF is of between 2 and 4.
2. The composition according to claim 1 , wherein said weight ratio between AP and LPF is of between 0.8 and 1.2, and the weight ratio between TPGS and AP and/or LPF is of between 2.5 and 3.5.
3. The composition according to claim 1 or 2, wherein said weight ratio between AP and LPF is of about 1 , and the weight ratio between TPGS and AP and/or LPF is of about 3.
4. The composition according to any one of claims 1 to 3 further comprising maltodextrin.
5. The composition according to any one of claims 1 to 4 wherein melatonin is present in an amount of 15-25% by weight with respect to the total weight of the composition.
6. The composition according to any one of claims 1 to 4 wherein TPGS is present in an amount of 5-7% by weight with respect to the total weight of the composition.
7. The composition according to any one of claims 1 to 4 wherein AP and LPF are independently present in an amount of 1.5-2.5% by weight with respect to the total weight of the composition.
8. The composition according to any one of claims 1 to 4 wherein maltodextrin is present in an amount of at least 70% by weight with respect to the total weight of the composition.
9. Formulation comprising the composition according to any one of claims 1 to 8 and one or more additional excipients and/or a vehicle.
10. The formulation according to claim 9 in the form of powder, tablet, capsule, dragee, or granules.
11. The composition according to any one of claims 1 to 8, or the formulation according to any one of claims 9-10, for the use as dietary supplement.
12. The composition according to any one of claims 1 to 8, or the formulation according to any one of claims 9-10, for the use as a medicament.
13. The composition or the formulation for the use according to claim 11 or 12, wherein the use is by oral administration.
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Citations (3)

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WO1998008490A1 (en) * 1996-09-01 1998-03-05 Pharmos Corporation Solid coprecipitates for enhanced bioavailability of lipophilic substances
WO2001003695A1 (en) * 1999-07-13 2001-01-18 Avmax, Inc. Improvement of bioavailability with vitamin c fatty acid esters
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