Patent Application
20200138880
The present invention relates to a composition for use as a medicament, in the form of a gel with characteristics of a reversible thermogel, based on one or more poloxamers, water and optionally excipients, and an effective amount of an active substance. Furthermore, the present invention relates to a process for the preparation of said composition and uses thereof.
In the market, there exist various forms of administration of active ingredients, e.g. microorganisms such as bacteria, at least one flavonoid such as rutin, oxerutin, diosmin, hesperidin and troxerutin, chemotherapeutic agents and/or arctigenin, arctin, berberine, berbamine, sanguinarine and chelerythrine, or rutoxides, as such or in the form of plant extracts containing said compounds, or melatonin or derivatives thereof. The topical forms of administration can be patches, creams, gels or suspensions. An example of a gel composition is described in EP2520279 A1.
However, there continues to be a felt need to be able to have a form of administration for topical use for transdermal application that is economical and easy to prepare, stable, efficacious in dosing the effective amount of active substance contained therein, and easily absorbable at the level of the cutis, dermis and epidermis without leaving residues or streaks.
The Applicant has developed a composition in gel form for topical use which provides an adequate and advantageous response to the above-mentioned needs.
The present invention relates to a composition in the form of gel for topical use, having the features as claimed in the accompanying claims.
The present invention relates to a process for the preparation of said composition, having the features as claimed in the accompanying claims.
The present invention relates to a composition for use as a medicament in gel form or topical use, having the features as claimed in the accompanying claims.
The preferred embodiments of the present invention set forth in the description that follows are illustrated solely by way of example and in no way limit the broad scope of application of the present invention, which will appear clear to the person skilled in the art.
In the context of the present invention, composition(s) means a pharmaceutical composition, a composition for medical devices, a composition for dietary supplements or a food composition.
Advantageously, the composition of the present invention is capable of releasing the active substance or active ingredient over time, in a constant, gradual and lasting manner as a controlled transdermal release, in such a way as to prolong its activity and therapeutic effectiveness over time.
Advantageously, the composition of the present invention is a gel in the form of a reversible therrnogel thanks to the presence of a vehicle or carrier which contains specific selected polymers of the poloxamer type.
Advantageously, the composition of the present invention is for topical use for transdermal and/or transmucosal application, said composition is furthermore economical and easy to prepare, stable, efficacious in dosing the effective amount of active substance contained therein over time, and easily absorbable at the level of the cutis, dermis and epidermis without leaving residues or streaks.
The present invention relates to a composition (abbreviated as CMP) in the form of a gel for topical use. Said composition comprises:
In a preferred embodiment (CMP1), the composition of the present invention (CMP) comprises a poloxamer which is selected from the group comprising or, alternatively, consisting of Poloxamer 124 CAS N. 9003-11-6 with an average molecular weight of 2090-2360, Poloxamer 188 (Lutrol F66) CAS No. 9003-11-6 with an average molecular weight of 7680-9510, Poloxamer 237 CAS N, 9003-11-6 with an average molecular weight of 6840-8830, Poloxamer 338 CAS N 9003-11-6 with an average molecular weight of 12700-17400, Poloxamer 407 (Lutrol® F127 Prill) CAS No. 9003-11-6 with an average molecular weight of 9840-14600, or mixtures thereof; preferably said Poloxamer 188 is present in an amount comprised from 0.1% to 10% by weight, out of 100 g of composition, preferably in an amount comprised from 0.5% to 5% by weight, for example from 1% to 3% by weight; and/or preferably said Poloxamer 407 is present in an amount comprised from 1% to 40% by weight, out of 100 g of composition, preferably in an amount comprised from 5% to 30% by weight, for example from 10% to 20% by weight (composition CMP1). Advantageously, the composition CMP1 contains Poloxamer 188 (Lutrol F66) CAS No. 9003-11-6 with an average molecular weight of 7680-9510 and/or Poloxamer 407 (Lutrol F127 Prill) CAS No. 900-11-6 with an average molecular weight of 984014600.
In another preferred embodiment (CMP4), the composition CMP or CMP1 comprises said microorganisms at a concentration comprised from 1×106 CFU/g to 1×1012 CFU/g of composition, preferably from 1×107 CFU/g to 1×1011 CFU/g of composition, even more preferably from 1×108 CFU/g to 1×1010 CFU/g of composition, for example 1×109 CFU/g of composition, and wherein:
In another preferred embodiment (CMP5), the composition CMP, CMP1 or 4 comprises an effective amount of an active substance selected from:
(iii) at least one compound selected from the group comprising or, alternatively, consisting of a flavonoid such as rutin, rutoxides, arbutin, oxerutin, diosmin, hesperidin and troxerutin, chemotherapeutic agents, arctigenin, arctin, berberine, berbamine, sanguinarine and chelerythrine, as such or in the form of plant extracts containing said compounds, on their own or in combination with one or more chemotherapeutic agents or anti-tumour drugs; and
(ii) melatonin and/or the natural and/or synthetic derivatives thereof;
said active substance being present in said composition in an amount by weight comprised from 0.1% to 10% by weight, preferably in an amount comprised from 0.5% to 5% by weight, even more preferably from 1% to 3% by weight, relative to the total weight of the composition (CMP5).
In another embodiment (CMP6), the composition as described above is for use:
The present invention relates to a process for the preparation of a composition in accordance with the compositions CMP or CMP1 or 4 or 5 or 6, said process comprising the following steps:
The vehicle present in the composition of the present invention comprises water and a thickening viscous matrix comprising at least one poloxamer or a mixture of at least due poloxamers.
In the context of the present invention, the term “reversible thermogel” refers to a gel obtained from aqueous copolymer solutions, wherein the copolymer is a poloxamer, capable of gelling in a reversible manner according to the temperature.
Typically, the composition, i.e. the reversible thermogel, of the present invention is liquid at temperatures below room temperature and becomes gelatinous at temperatures close to human body temperature. For example, said composition is liquid at temperatures approximately comprised from 3° C. to 15° C.; in particular, at about 5° C. Said composition in turn gels, that is, becomes a gel, starting from about 20° C. until about 37°; in particular it gels between about 20° and 25° C., preferably, between about 21° and 23° C.
In the context of the present invention, the active agent, for example the melatonin, is present in the composition of the invention in an amount comprised from 0.1% to 3% by weight, out of 100 g of composition; preferably, comprised from 0.5% to 2% by weight, out of 100 g of composition: more preferably, about 1% by weight, out of 100 g of composition; even more preferably, 1% by weight, out of 100 g of composition. The melatonin can be added into the composition as such, in powder, with a purity comprised from 96% to 99.9%; in particular, with a purity comprised from 97% to 99%; for example, with a purity of the 98%. Alternatively, the melatonin can be added into the composition also in the form of microparticles, for example of a size comprised from 50 to 100 μm.
In the context of the present invention, the poloxamers are a series of block copolymers of ethylene oxide and propylene oxide in accordance with the following structural formula;
HO(C2H4O)8(C3H6O)b(C2H4O)aH,
where a and b are whole numbers expressing the number of the oxyethylene and oxypropylene residues present in the molecule. Poloxamers are non-ionic copolymers of polyoxyethylene-polyoxypropylene used primarily in pharmaceutical formulations as emulsifying, solubilising or welling agents.
The poloxamers of the present invention are capable of thickening the aqueous solutions and giving rise to reversible thermogels possessing rheological properties that vary according to their concentration and molecular weight. A large number of poloxamers (whose molecular weight varies widely according to the values of a and b) can be used for the purposes of the present invention. Among them, the five poloxamers (characterised by the codes 124, 188, 237, 338, 407, respectively) included in the US Pharmacopeia USP NF XVII, in the chapter headed “Poloxamer”, whose contents are incorporated herein in their entirety, have proven to be preferable. Among these, the poloxamers are preferably selected from the group consisting of Poloxamer 188 (Lutrol® F 68, BASF SE, Ludwigshafen, DE), CAS 9003-11-6; Poloxamer 407 (Lutrol® F 127, BASF SE, Ludwigshafen, DE), CAS 900-11-6; or mixtures thereof. In a preferred embodiment of the invention, a mixture of Poloxamer 188 and Poloxamer 407 is used.
In the context of the present invention, the at least one poloxamer or mixture of poloxamers is present in a total amount comprised from 1% to 35% by weight, out of 100 g of composition.
In a preferred embodiment of the invention,
the Poloxamer 188 is present in an amount comprised from 1% to 5% by weight, out of 100 g of composition; preferably, comprised from 1% to 3% by weight, out of 100 g of composition; more preferably, comprised from 1% to 2% by weight, out of 100 g of composition; even more preferably, of 1% by weight, out of 100 g of composition; and
the Poloxamer 407 is present in an amount comprised from 15% to 30% by weight, out of 100 g of composition; preferably, comprised from 18% to 25% by weight, out of 100 g of composition; more preferably, comprised from 20% to 23% by weight, out of 100 g of composition; even more preferably, of 21% by weight, out of 100 g of composition.
In the context of the present invention, the water is preferably purified water and is present in an amount such as to reach a balance at 100% by weight of the composition.
The composition of the present invention further comprises known additives/excipients/adjuvants commonly used in the pharmaceutical formulation technique; in particular, said additives/excipients/adjuvants are selected from among those particularly advantageous for the formation and the stabilisation of the reversible thermogel of the invention. In a preferred embodiment of the invention, said additives/excipients/adjuvants are selected from the group consisting of acid salts, such as sodium sorbate, potassium sorbate, sodium benzoate, potassium benzoate; and glycols, such as ethylene glycol and propylene glycol.
In a particularly preferred embodiment, said additives/excipients/adjuvants consist of potassium sorbate, sodium benzoate and propylene glycol, in a total amount comprised from 1 to 5% by weight, out of 100 g of composition.
The pharmaceutical composition for topical use in the form of a reversible thermogel of the present invention can be prepared by adopting well-known apparatus and processing conditions commonly used in the industry for the preparation of a reversible thermogel.
Essentially, the various ingredients making up the desired composition, added in succession or pre-mixed, undergo cold mixing using a suitable mixer provided with a refrigeration temperature control and adjustment means and a stirring means. The mixing temperature is set on values below room temperature, preferably between 3 and 8° C., and the mixture of components is kept under cold stirring for an amount of time sufficient to obtain a completely homogeneous mixture which, in an amount of time that will vary depending on the type of poloxamers used, will transform, still cold, into a completely liquid solution.
The product thus obtained is then packaged in suitable containers, e.g. sealed tubes, and subsequently, on reaching room temperature, will transform into a gel, in particular at the moment when it is applied on the skin.
In one embodiment (FR1), the present invention relates to a composition in the form of a gel for topical use comprising:
In a preferred embodiment (FR2), the present invention relates to the composition for use according to FR1, wherein said poloxamer is selected from the group comprising or, alternatively, consisting of Poloxamer 124 with an average molecular weight of 2090-2360, Poloxamer 188 (Lutrol F66) CAS No. 9003-11-6 with an average molecular weight of 7680-9510, Poloxamer 237 with an average molecular weight of 6840-8830, Poloxamer 338 with an average molecular weight of 12700-17400, Poloxamer 407 (Lutrol F127 Prill) CAS No. 900-11-6, with an average molecular weight of 984014600, or mixtures thereof; preferably said Poloxamer 188 is present in an amount comprised from 0.1% to 10% by weight, out of 100g of composition, preferably in an amount comprised from 0.5% to 5% by weight, for example from 1% to 3% by weight; and/or preferably said Poloxamer 407 is present in an amount comprised from 1% to 40% by weight, out of 100g of composition, preferably in an amount comprised from 5% to 30% by weight, for example from 10% to 20% by weight.
In a preferred embodiment (FR3), the present invention relates to the composition for use according to FR1, wherein:
In a preferred embodiment (FR4), the present invention relates to the composition for use according to one of FR1-FR3, wherein said at least one microorganism is selected from the group comprising or, alternatively, consisting of the microorganisms in Table 1; preferably said at least one microorganism is selected from the group comprising or, alternatively, consisting of the strains: Lactobacillus salivarius (LS01) DSM 22775, deposited on 23 Jul 2009; Bifidobacterium breve (BR03) DSM 16604, deposited on 20.072004; Lactobacillus pentosus (LPS01) DSM 21980, deposited on 14 Nov. 2008; Streptococcus thermophilus (FP4) DSM 18616, deposited on 13 Sep. 2006; Lactobacillus casei ssp. rhamnosus (LR04) DSM 16605, deposited on 20 Jul. 2004; and Lactobacillus acidophilus (LA02) DSM 21717, deposited on 6 Aug. 2008; even more preferably the strain is Lactobacillus salivarius (LS01) DSM 22775, and/or Bifidobacterium breve (BR03) DSM 16604, and/or L. pentosus (LPS01) DSM 21980.
In a preferred embodiment (FR5), the present invention relates to the composition for use according to FR4, wherein said microorganisms are present at a concentration comprised from 1×106 CFU/g to 1×1012 CFU/g of composition, preferably from 1×107 CFU/g to 1×1011 CFU/g of composition, even more preferably from 1×108 CFU/g to 1×1010 CFU/g of composition, for example 1×109 CFU/g of composition, and wherein:
In a preferred embodiment (FR6), the present invention relates to the composition for use according to FR1 or the FR2, wherein:
In a preferred embodiment (FR7), the present invention relates to the composition for use according to one of FR1-FR6, for use:
In a preferred embodiment (FR8), the present invention relates to a process for the preparation of a composition in accordance with any one of FR1-FR7, said process comprising the following steps:
Solely by way of example, which does not limit the various possibilities of production, an example of preparation of a reversible thermogel/composition of the present invention is described here below.
Mixture (1) consisting of only Lactobacillus salivarius (LS01) DSM 22775, or only Bifidobacterium breve (BR03) DSM 16604, or only L. pentosus (LPS01) OSM 21980.
Mixture (2) consisting of Lactobacillus salivarius (LS01) DSM 22775, Bifidobacterium breve (BR03) DSM 16604 and L. pentosus (LPS01) DSM 21980, in a ratio by weight of 1:1:1.
Mixture (3) consisting of Lactobacillus salivarius (LS01) DSM 22775 and Bifidobacterium breve (BR03) DSM 16604 in a ratio by weight of 1:1.
Mixture (4) consisting of Lactobacillus salivarius (LS01) DSM 22775 and L. pentosus (LPS01) OSM 21980, in a ratio by weight of 1:1.
Mixture (5) consisting of Bifidobacterium breve (BR03) DSM 16604 and L. pentosus (LPS01) DSM 21980, in a ratio by weight of 1:1.
The cold purified water (at 5° C.) is weighed into a glass container (for example a beaker) with magnetic stirring, a thermometer, and an external ice bath and the potassium sorbate and sodium benzoate are added under stirring. The mixture is maintained under stirring at 5° C. until complete dissolution of the salts. Then the following are added, again under stirring: the two poloxamers (crystalline), in succession or in a mixture, the methionine (in powder, or in the form of micro-/nanoparticles) and the propylene glycol and cold stirring is continued for at least 15 minutes until a homogeneous appearance is obtained. The container is then placed in a refrigerator at 5° C. for at least 24 h, until obtaining complete dissolution of the ingredients.
After this, the resulting product (liquid) is packaged in 18-20 g tubes and sealed pending use/experimentation. On reaching room temperature and in contact with the skin the product transforms completely into a gel, which is rapidly absorbed following cutaneous and/or transmucosal administration. The pH of the liquid thermogel is 7.40. The gelling temperature is 21°-22° C.
The composition in the form of a thermogel of the present invention, topically administered, in particular on wrists and/or forearms, has shown an excellent cutaneous permeability, triggering a fast onset of the pharmacological action. Furthermore, thanks to the fact of going directly into circulation without passing through the gastrointestinal tract (no metabolism by the liver), it has also shown high blood levels of the active ingredient, for example, melatonin, for a prolonged period of time (slow release). This fact has made it possible to apply low dosages of the drug, thus obtaining the desired advantageous results (modest dosage/high, long-lasting activity) compared to the traditional forms of administration thereof.
For example, a study was conducted on 10 healthy volunteers with different formulations of melatonin, observing the accumulation of melatonin in the saliva over time (it is well known that melatonin spreads passively into saliva via the bloodstream, and its concentration therein represents 24%-33% of its plasma levels, that is to say, the amount of free melatonin not bound to globulins).
The following treatments were administered to the volunteers, randomly divided into 3 groups:
A saliva sample was taken at time 0 (basal) and at subsequent times (up to 7 h) after administration, using a Cortisol-Salivette® device (SARSTEDT S.r.l., Verona, IT); the melatonin levels in the saliva were determined by LC-MS (liquid chromatography-mass spectrometry) using a triple quadrupole mass spectrometer (ABSciex QTrap 3200) and substantially following the method of Khan et al. 2013.
The study showed that the formulations of melatonin in a reversible thermogel in accordance with the present invention possess an excellent thermal permeability, a fast onset of the pharmacological action and a lasting duration thereof compared to the other formulations tested.
The pharmaceutical compositions of the present invention also showed to act effectively on all of the patients treated, whereas the commonly used formulations, such as, for example, oral ones, do not act on all individuals.
Furthermore, the pharmaceutical compositions of the present invention have also shown to possess good stability over time.
The pharmaceutical compositions of the present invention are particularly recommended both for transdermal administration and transmucosal administration.
The pharmaceutical compositions of the present invention have shown to be advantageously active, or in any case very promising in the treatment of a number of disorders/diseases due to dysfunctions of physiological functions connected with circadian rhythms such as, for example, the sleep-wake balance; in the regulation of blood pressure; in eliminating of free radicals; in interacting with the immune system, for example, in the treatment of inflammatory states, in the treatment of acquired immunodeficiencies and in the treatment of viral and bacterial infectious diseases and cancer.
Furthermore, the compositions of the present invention have shown to be useful: in the treatment of sleep disorders in all the treated subjects, by favouring the quality of sleep, in particular of REM sleep; in the treatment of the autism and Down syndrome, particularly in children, by favouring their balance; in the treatment of Alzheimer's disease; in the treatment of Parkinson's disease, by improving sleep and tremors: in the treatment of the vision disorders, for example in maculopathy of the retina, via external applications in the vicinity of the eye; in the treatment of the pulmonary manifestations of cystic fibrosis in children, where they provide an anti-inflammatory action; in the treatment of prostate cancer, where they are capable of preventing the propagation thereof; in the treatment of the tumours, as anti-inflammatory, anti-tumour adjuvants.
Lactobacillus casei
Lactobacillus gasseri
Lactobacillus crispatus
Lactobacillus fermentum
Lactobacillus fermentum
Lactobacillus casei ssp. pseudoplantarum
Streptococcus thermophilus B39
Streptococcus thermophilus T003
Lactobacillus pentosus 9/1 ei
Lactobacillus plantarum 776/1 bi
Lactobacillus plantarum 476LL 20 bi
Lactobacillus plantarum PR ci
Lactobacillus plantarum 776/2 hi
Lactobacillus casei ssp. paracasei 181A/3 aiai
Lactobacillus belonging to the acidophilus
Bifidobacterium longum 175A/1 aiai
Bifidobacterium breve 195A/1 aici
Bifidobacterium lactis 32A/3 aiai
Lactobacillus plantarum 501/2 gi
Lactococcus lactis ssp. lactis 504/4 ci
Lactococcus lactis ssp. lactis 501/4 hi
Lactococcus lactis ssp. lactis 501/4 ci
Lactobacillus plantarum 501/4 li
Lactobacillus acidophilus
Lactobacillus paracasei ssp. paracasei
Streptococcus thennophilus
Streptococcus thermophilus
Streptococcus thermophilus
Streptococcus thermophilus
Streptococcus thermophilus
Streptococcus thermophilus
Bifidobacterium adolescentis
Bifidobacterium adolescentis
Bifidobacterium breve
Bifidobacterium pseudocatenulatum
Bifidobacterium pseudocatenulatum
Bifidobacterium longum
Bifidobacterium breve
Lactobacillus casei ssp. rhamnosus
Lactobacillus delbrueckii ssp. bulgaricus
Lactobacillus delbrueckii ssp. bulgaricus
Staphylococcus xylosus
Bifidobacterium adolescentis
Lactobacillus plantarum
Streptococcus thermophilus
Streptococcus thermophilus
Streptococcus thermophilus
Lactobacillus fermentum
Lactobacillus fermentum
Lactobacillus fermentum
Lactobacillus fermentum
Lactobacillus gasseri
Lactobacillus gasseri
Lactobacillus gasseri
Lactobacillus gasseri
Bifidobacterium adolescentis EI-3
Bifidobacterium catenulatum
sp./pseudocatenulatum EI-3I, ID 09-255
Bifidobacterium adolescentis EI-15
Bifidobacterium adolescentis EI-18
Bifidobacterium animalis subsp. lacts EI-18,
Bifidobacterium catenulatum EI-20
Streptococcus thermophilus FRai
Streptococcus thermophilus LB2bi
Streptococcus thermophilus LRci
Streptococcus thermophilus FP4
Streptococcus thermophilus ZZ5F8
Streptococcus thermophilus TEO4
Streptococcus thermophilus S1ci
Streptococcus thermophilus 641bi
Streptococcus thermophilus 277A/1ai
Streptococcus thermophilus 277A/2ai
Streptococcus thermophilus IDC11
Streptococcus thermophilus ML3di
Streptococcus thermophilus TEO3
Streptococcus thermophilus G62
Streptococcus thermophilus G1192
Streptococcus thermophilus GB18
Streptococcus thermophilus CCR21
Streptococcus thermophilus G92
Streptococcus thermophilus G69
Streptococcus thermophilus
Streptococcus thermophilus
Streptococcus thermophilus
Streptococcus thermophilus
Weissella ssp. WSP 01
Weissella ssp. WSP 02
Lactobacillus ssp. WSP 03
Lactobacillus plantarum LP 09
Lactobacillus plantarum LP 10
Lactococcus lactis
Lactobacillus fermentum
Lactobacillus fermentum
Lactobacillus casei ssp. rhamnosus
Bifidobacterium bifidum
Lactobacillus delbrueckii subsp.
bulgaricus LD 01
Lactobacillus delbrueckii subsp.
bulgaricus LD 02
Lactobacillus delbrueckii subsp.
bulgaricus LD 03
Lactobacillus delbrueckii subsp.
bulgalicus LD 04
Lactobacillus delbrueckii subsp.
bulgaricus LD 05
Bifidobacterium pseudocatenulatum
Lactobacillus acidophilus
Lactobacillus paracasei
Lactobacillus pentosus
Lactobacillus rahmnosus
Lactobacillus delbrueckii ssp. delbrueckii
Lactobacillus plantarum
Lactobacillus salivarius
Lactobacillus salivarius
Bifidobacterium bifidum
Bifidobacterium bifidum
Bifidobacterium bifidum
Bifidobacterium lactis
Lactobacillus acidophilus
Lactobacillus brevis
Bifidobacterium animalis ssp. lactis
Bifidobacterium longum
Bifidobacterium longum
Bifidobacterium bifidum
Bifidobacterium breve
Bifidobacterium lactis
Lactobacillus reuteri
Lactobacillus reuteri
Lactobacillus reuteri
Lactobacillus reuteri
Lactobacillus paracasei ssp. paracasei
Lactobacillus acidophilus
Bifidobacterium bifidum
Lactobacillus crispatus
Lactobacillus crispatus
Lactobacillus paracasei
Lactobacillus Salivarius
Lactobacillus gasseri
Lactobacillus acidophilus
Lactobacillus salivarius
Lactobacillus crispatus
Lactobacillus crispatus
Lactobacillus acidophilus
Lactobacillus gasseri
Lactobacillus paracasei
Bifidobacterium infantis
Bifidobacterium bifidum
Bifidobacterium longum
Bifidobacterium lactis
Bifidobacterium longum
Bifidobacterium breve
Bifidobacteriura breve
Bifidobacterium breve
Bifidobacterium longum
Lactobacillus salivarius
Lactobacillus
reuteri
Lactobacillus
reuteri
Lactobacillus
reuteri
Streptococcus thermophilus
Streptococcus thermophilus
Streptococcus thermophilus
Lactobacillus salivarius
Bifidobacterium longum
Bifidobacterium longum
Bifidobacterium longum
Bifidobacterium longum
Bifidobacterium longum
Bifidobacterium longum
Bifidobacterium longum
Bifidobacterium longum
Bifidobacterium longum
Bifidobacterium longum
Bifidobacterium longum
Lactobacillus johnsonii
Lactobacillus rhamnosus
Lactobacillus rhamnosus
Lactobacillus reuteri
Lactobacillus reuteri
Lactobacillus reuteri
Bifidobacterium longum
Bifidobacterium infantis
Lactobacillus plantarum
Bifidobacterium longum
Bifidobacterium longum
Lactobacillus salivarius
Lactobacillus salivatius
Lactobacillus pentosus
Bifidobacterium pseudolongum
Lactobacillus fermentum
Lactobacillus fermentum
Lactobacillus casei
Lactobacillus crispatus
Lactobacillus jensenii
Lactobacillus helveticus ID 922
Lactobacillus helveticus ID 923
Lactococcus lactis ssp. cremoris ID 1612
Lactococcus lactis ssp. cremoris ID 1252
Lactococcus lactis ssp. Lactis ID 1254
Bifidobacterium longum
Bifidobacterium lungum
Bifidobaterium animalis ssp. lactis
Streptococcus thermophilus
Bifidobacterium infantis
Bifidobacterium infantis
Streptococcus thermophilus
Streptococcus thermophilus
Streptococcus thermophilus
Lactobacillus fermentum
Lactobacillus fermentum
Leuconostoc sp.
Leuccnostoc sp.
Leuconostoc sp.
Leuconostoc sp.
Lactobacillusplantarum
Lactobacillusplantarum
Lactobacillusplantarum
Lactobacillusplantarum
Lactobacillus pentosus
Lactobacillus reuteri
Lactobacillus brevis
Lactobacillus salivarius
Bifidobacterium breve
Lactococcus lactis ssp. cremoris
Bifidobacterium longum
Lactobacillus rhamnosus
Lactobacillus kefiri
Lactobacillus kefiri
Lactobacillus acidophilus
Lactobacillus kefiranofaciens
Lactobacillus kefiri
Lactobaciullus kefiri
Streptococcus thermophilus
Streptococcus thermophilus
Streptococcus thermophilus
Streptococcus thermophilus
Streptococcus thermophilus
Streptococcus thermophilus
Streptococcus thermophilus
Lactobacillus salivarius
Weissella confusa
Weissella confusa
Lactobacillus curvatus
Lactobacillus plantarum
Lactobacillus reuteri
Lactobacillus parasei
Lactobacillus reuteri
Lactobacillus rhamnosus
Lactobacillus rhamnosus
Lactobacillus paracasei
Lactobacillus reuteri
Lactobacillus rhamnosus
Lactobacillus fermentum
Lactobacillus fermentum
Lactobacillus fermentum
The experiment was conducted on a group of 6 volunteers, all female and aged between 29 and 41 years (mean age 34.5 years).
Each volunteer tested 5 formulations of melatonin (gel 0.1%, gel 0.2%, gel 1%, cream, Circadin 2 mg), with a wash-out period of one week after each application.
The saliva samples were taken according to the following experimental scheme:
h 10.30 30′
h 11.00 1 h
h 13.00 3 h
h 16.00 6 h
The saliva was collected with Salivette (Sarstedt), following the instructions attached thereto (mouth rinsing before each sampling, chewing on the swab for 1 minute). The saliva was extracted from the swab by 2 min centrifugation at 4000 rpm, then divided into 300 μl aliquots and frozen at −20° C. until the time of analysis.
The melatonin assays were performed with the ELISA (Enzyme-Linked Immunosorbent Assay) technique, using the Melatonin (direct) Saliva kit (SLV-4779-DRG Instruments, Germany). The analytical sensitivity and inter- and intra-assay coefficients of variation are the following: 0.3 pg/ml, 7.6-13.0% and 6.1-10.8%.
The kit in question is certified for in-vitro diagnostic use.
| Number | Date | Country | Kind |
|---|---|---|---|
| 102016000057122 | Jun 2016 | IT | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2017/053206 | 5/31/2017 | WO | 00 |
