Classifications

• • 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
  • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
  • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
  • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
  • A61K31/23—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms
  • A61K31/232—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms having three or more double bonds, e.g. etretinate
• • 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
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
• • 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
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/4406—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 3, e.g. zimeldine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/06—Antianaemics

Definitions

• the present invention relates to the use of a DHA ester as a prophylactic and / or curative treatment in sickle cell disease.
• Sickle cell disease also known as hemoglobin S or sickle cell anemia is a genetic disorder of hemoglobin, the protein that carries oxygen through the blood.
• Sickle cell disease is not a very rare disease. It is particularly common in populations of sub-Saharan African origin, the West Indies, India, the Middle East and the Mediterranean basin, particularly in Greece and Italy. It is estimated that more than 100 million people are affected worldwide. It is the first genetic disease in France, and probably in the world.
• Sickle cell disease is due to an abnormality of hemoglobin, the main constituent of the red blood cell also called erythrocyte. These are flattened disks, with a finer center than the edges. Their so-called biconcave shape is characteristic, it gives them a very great flexibility, essential to pass into the finest blood capillaries.
• the erythrocyte membrane consists of a lipid bilayer whose central part between the outer and inner surfaces is hydrophobic and contains fatty acids. The adhesion, aggregation and deformability of blood cells is greatly impacted by the fatty acid content of their membrane.
• Hemoglobin consists of four chains assembled together. Hemoglobin A, predominant in adults, consists of two so-called alpha chains and two so-called beta chains. In case of sickle cell disease, beta chains are abnormal. Hemoglobin formed from abnormal beta chains and normal alpha chains is a hemoglobin that "agglomerates" in red blood cells; it is called hemoglobin S, an abbreviation for sickle, which means sickle.
• a red blood cell normally has the shape of a disk which each face is a little hollow. In case of sickle cell disease, the agglomeration of hemoglobin S causes the red blood cells to take the form of a sickle or a crescent, especially when the amount of oxygen is lower.
• beta chain of hemoglobin depends on two genes, the "beta-globin" genes located on chromosome 11. At the molecular level, beta chains are abnormal because of a 6-position glutamic acid replaced by a valine.
• Hemoglobin S is distinguished from normal hemoglobin A by its slower electrophoretic mobility, but especially by the insolubility of its deoxygenated form, which crystallizes easily. Hemoglobin S is now the most common genetic abnormality in France. Heterozygous (A / S) forms, usually silent, should be distinguished from homozygous (S / S) or heterozygous composite forms (essentially S / C, S / beta thalassemia, S / D-Punjab, S / O-Arab) that are responsible for major sickle cell syndromes, which are always clinically and hematologically serious.
• the severity of sickle cell disease varies greatly between people and over time for the same person. The condition is noted in the infant, but is usually not manifest at birth because red blood cells of the newborn still contain 50-90% fetal hemoglobin. The symptoms of this disease can appear from the age of two to three months, date of appearance of the beta chain. The three main manifestations are anemia, vaso-occlusive attacks and less resistance to certain infections.
• Anemia refers to a lack of hemoglobin and results in excessive fatigue and a feeling of weakness.
• the red blood cells which are constantly renewed, are produced in the center of the bones, in the red bone marrow. From there, they pass into the general circulation where normally they remain 120 days in the bloodstream and are then destroyed in the spleen.
• sickle cell disease as the sickle-shaped red blood cells are abnormally fragile, they are easily destroyed which causes anemia.
• the severity of the anemia varies over time, it can worsen brutally in case of excessive operation of the spleen, it is called splenic sequestration.
• abnormal red blood cells are quickly eliminated by the body, and more specifically by the spleen.
• Sickle red blood cells are considered abnormal by the spleen that captures them (or sequesters) and then eliminates them, which increases anemia.
• the vaso-occlusive crises or painful crises are manifested by sharp and brutal pains.
• the sickle-shaped red blood cells block circulation in the blood vessels, which prevents the optimal distribution of oxygen in the body. This process can occur in different parts of the body (bone, abdomen, kidney, brain, retina ). These crises can be very painful. These pains are the most common manifestations of the disease, they can be sudden and transient or chronic. They are favored by dehydration, cold, stress, altitude ... All parts of the body may be involved, but osteo-articular involvement is very common. In the long term, bone infarctions can occur causing problems in the joints. Ocular involvement is also common, intraocular hemorrhages can occur. They limit the field of vision more or less completely.
• Infections are one of the most common complications of sickle cell disease. They can occur throughout the life of sickle cell disease and can put life at risk, especially in infants and young children. Bacterial infection is susceptible to rapid spread and severe localization such as meningitis or osteomyelitis. Pneumococcus and salmonella are the most common bacteria. This increased susceptibility to infections is due to the fact that the spleen, which plays an important role in the defense process against bacteria, is almost always damaged in patients.
• anemia evolves by relapses, or "haemolytic seizures," which are promoted or triggered by infections. Painful vaso-occlusive attacks occur at varying intervals, more or less markedly. The evolution is even better than the access and the quality of the care are good.
• nonsteroidal anti-inflammatory drugs paracetamol, codeine, tramadol, buprenorphine, nalbuphine, orphan, fentanyl, hydromorphone, oxycodone.
• Oxygen therapy is often established during hospitalization, it consists of daily inhalation of oxygen enriched air to increase oxygenation of the organs and thus relieve pain.
• a drug treatment can be proposed to patients with severe sickle cell disease, it is hydroxyurea (or hydroxycarbamide), a product used in leukemias.
• This molecule acts on ribonucleotide reductase. It is the key enzyme in the transformation of the four ribonucleotides into deoxyribonucleotides essential for DNA synthesis.
• This molecule is able to increase in the adult the production of a hemoglobin normally present in the fetus and in a small quantity at birth (hemoglobin F). The forced production of this fetal hemoglobin F makes it possible to reduce the agglomeration of hemoglobin S.
• this molecule does not act on pulmonary or bone infections nor does it protect secondary bone lesions.
• hydroxyurea is not devoid of undesirable effects, such as an influence on male fertility.
• bone marrow transplant healthy bone marrow will make healthy erythrocytes. This procedure is, however, reserved for only a very small part of the patients. It is an operation that requires extremely heavy treatment and can lead to serious life-threatening complications.
• the polyunsaturated fatty acids of the Omega 3 series in particular docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), advantageously purified and concentrated in the form of ethyl ester are known for their potential use in the treatment of certain cardiovascular diseases and the modulation of the corresponding risk factors. In particular, they are known in the treatment of hyperlipidemia, hypercholesterolemia and arterial hypertension. Clinical trials conducted with formulations containing a high concentration of DHA ethyl ester in patients who had suffered from myocardial infarction have been shown to be effective in reducing mortality and in particular sudden death.
• DHA docosahexaenoic acid
• EPA eicosapentaenoic acid
• DHA which plays a preponderant role in this pathology.
• An intake of DHA could increase the erythrocyte rate in DHA in people with sickle cell disease and therefore reduce the damage of these red blood cells, these cells being the real hub of sickle cell disease but also the alteration of other cells involved in the physiopathology of the sickle cell disease such as endothelial cells, platelets, mononuclear cells.
• Vitamins or provitamins of group B have the advantages related to their function.
• nicotinol is the alcohol derived from nicotinic acid (vitamin B3). It is rapidly converted to nicotinic acid in the human body.
• Nicotinic acid also known as niacin, is a group B water soluble vitamin that can be synthesized from tryptophan. Vitamin B3 plays an important role in the release of energy from food but also in the reduction of cholesterol.
• the effective therapeutic doses for hypocholesterolemic and hypolipidemic purposes are greater than the quantities synthesized by the body and oral supplementation is necessary in a hypocholesterolemic and / or hypotriglyceridemic aiming.
• vitamin B3 intake Deficiencies of vitamin B3 intake still exist in some countries in Asia and Africa, ie in areas where sickle cell disease is highly prevalent. Vitamin B3 deficiency leading to general fatigue, vitamin B3 intake could be a real benefit for people anemic and therefore getting tired already faster.
• Panthenol is the alcohol derived from pantothenic acid, better known as vitamin B5. In the body, panthenol is converted into pantothenic acid which then becomes an important part of the compound "coenzyme A", which is particularly interesting in cell metabolism. Indeed, it takes part in the metabolism of lipids, carbohydrates and proteins. Panthenol also participates in the formation of acetylcholine and adrenal steroids. It is also involved in the detoxification of foreign bodies and in the resistance to infections which is particularly interesting in people with sickle cell disease.
• Inositol or vitamin B7 mobilizes fats avoiding their accumulation. It also has an anxiolytic effect. It tones the nervous system and the liver. It also reduces the level of cholesterol in the blood. She is involved in the enhancement of serotonin activity, control of intracellular calcium concentration, maintenance of cell membrane potential and cytoskeletal assembly. Inositol deficiency can lead to muscle pain and eye diseases. Consequently, an intake of inositol can only be favorable for sickle cell disease.
• Isosorbide in particular isosorbide mononitrate, is a potent peripheral vasodilator. It also has diuretic properties relieving the work of the kidneys, this organ being a preferred target during vaso-occlusive crises, an intake of isosorbide can also be beneficial in sickle cell patients.
• vitamins B3 and B5 are involved in the production of red blood cells.
• the provision of one or other of these vitamins in people with sickle cell disease therefore makes them the preferred alcohols of this invention.
• the subject of the present invention is therefore an ester of docosahexaenoic acid with an alcohol chosen from the group consisting of:
• the DHA ester with an alcohol selected from the group consisting of nicotinol, panthenol, inositol, isosorbide or isosorbide mononitrate is used as a medicament for preventing and / or relieve vaso-occlusive seizures in a patient with sickle cell disease.
• the DHA ester with an alcohol selected from the group consisting of nicotinol, panthenol, inositol, isosorbide or isosorbide mononitrate is used as a medicament for prevent and / or treat anemia in a patient with sickle cell disease.
• sickle cell disease is understood to mean all the genetic forms of sickle cell disease, homozygous or heterozygous sickle cell disease.
• prophylactic treatment is understood to mean treatment intended to prevent the onset or spread of the disease.
• Curative treatment is a treatment that is intended to cure, minimize or relieve symptoms.
• enantiomers is intended to denote optical isomeric compounds which have identical molecular formulas but which differ in their spatial configuration and which are non-superimposable mirror images.
• diastereoisomers means optical isomers which are not images in a mirror of each other.
• a “racemic mixture” is a mixture in equal proportions of the levorotatory and dextrorotatory enantiomers of a chiral molecule.
• the term "pharmaceutically acceptable” or “pharmaceutically acceptable” is intended to mean that which is useful in the preparation of a pharmaceutical composition which is generally safe, non-toxic and neither biologically nor otherwise undesirable and which is acceptable for veterinary as well as human pharmaceutical use.
• salts of a compound is intended to mean salts which are pharmaceutically acceptable, as defined herein, and which possess the desired pharmacological activity of the parent compound.
• Such salts include: acid addition salts formed with mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or formed with organic acids such as acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydoxynaphthoic acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, acid methanesulfonic acid, muconic acid, 2-naphthalenesulfonic acid, propionic acid, salicylic acid, succinic acid, dibenzo
• the salts formed when an acidic proton present in the parent compound is replaced by a metal ion, for example an alkali metal ion, an alkaline earth metal ion or an aluminum ion; either coordinates with an organic or inorganic base.
• a metal ion for example an alkali metal ion, an alkaline earth metal ion or an aluminum ion; either coordinates with an organic or inorganic base.
• Acceptable organic bases include diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, tromethamine and the like.
• Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate and sodium hydroxide.
• Preferred pharmaceutically acceptable salts are salts formed from hydrochloric acid, trifluoroacetic acid, dibenzoyl-L-tartaric acid and phosphoric acid.
• references to pharmaceutically acceptable salts include solvent addition forms (solvates) or crystalline forms (polymorphs) as defined herein, of the same acid addition salt.
• the present invention further relates to a pharmaceutical composition
• a pharmaceutical composition comprising the DHA ester with an alcohol selected from the group consisting of nicotinol, panthenol, inositol, isosorbide or isosorbide mononitrate, and at least one pharmaceutically acceptable excipient for use as a medicament for the prophylactic and / or curative treatment of sickle cell disease.
• the pharmaceutical composition according to the present invention can be used as a medicament for preventing and / or alleviating vaso-occlusive attacks in a patient with sickle cell disease.
• the pharmaceutical composition according to the present invention can be used as a medicament for preventing and / or treating anemia in a patient with sickle cell disease.
• composition according to the present invention can be administered orally or any other pharmaceutical route of administration.
• compositions according to the present invention can be formulated for administration to mammals, including humans. These compositions are made so that they can be administered orally, sublingually, subcutaneously, intramuscularly, intravenously, transdermally, locally or rectally.
• the active ingredient can be administered in unit dosage forms, in admixture with conventional pharmaceutical carriers, to animals or humans.
• Suitable unit dosage forms include oral forms such as tablets, capsules, powders, granules and oral solutions or suspensions, sublingual and oral forms of administration, subcutaneous forms of administration , topical, intramuscular, intravenous, intranasal or intraocular and forms of rectal administration.
• the main active ingredient is mixed with a pharmaceutical vehicle such as gelatin, starch, lactose, magnesium stearate, talc, gum arabic, silica or the like.
• a pharmaceutical vehicle such as gelatin, starch, lactose, magnesium stearate, talc, gum arabic, silica or the like.
• the tablets can be coated sucrose or other suitable materials or they can be treated in such a way that they have prolonged or delayed activity and that they continuously release a predetermined quantity of active ingredient.
• a capsule preparation is obtained by mixing the active ingredient with a diluent (optional step) and pouring the resulting mixture into soft or hard gelatin capsules.
• a syrup or elixir preparation may contain the active ingredient together with a sweetener, an antiseptic, as well as a flavoring agent and a suitable colorant.
• Water-dispersible powders or granules may contain the active ingredient in admixture with dispersing agents or wetting agents, or suspending agents, as well as with taste correctors or sweeteners.
• suppositories are used which are prepared with binders melting at the rectal temperature, for example cocoa butter or polyethylene glycols.
• aqueous suspensions for parenteral (intravenous, intramuscular, etc.), intranasal, or intraocular administration, aqueous suspensions, isotonic saline solutions, or sterile and injectable solutions containing dispersing agents and / or pharmacologically compatible wetting agents are used.
• the active ingredient may also be formulated as microcapsules, optionally with one or more additive carriers.
• the pharmaceutical composition according to the present invention is intended for oral or intravenous administration, more advantageously orally.
• Dosages of pharmaceutical compositions containing a DHA ester with an alcohol selected from the group consisting of nicotinol, panthenol, inositol, isosorbide or isosorbide mononitrate in the compositions of the invention are adjusted to obtain an amount of active substance that is effective in achieving the desired therapeutic response for a particular composition in the method of administration.
• the chosen level of dosage therefore depends on the desired therapeutic effect, the route of administration chosen, the desired duration of treatment, the weight, age and sex of the patient, the sensitivity of the individual to be treated . Consequently, the optimal dosage should be determined according to the parameters deemed relevant by the specialist in the field.
• the DHA ester is administered in acceptable pharmaceutical compositions in which the daily dose is between 250 mg and 10 g per day, more preferably the daily dose is between 1 and 6 g per day, for example 1 g, 2 g or 4 g / day. It may be necessary to use larger doses (called loading dose) at the beginning of prophylactic and / or curative treatment and then reduce the doses (maintenance dose) during treatment.
• loading dose larger doses
• maintenance dose maintenance dose
• the pharmaceutical composition according to the present invention may further comprise at least one other active ingredient, such as an analgesic and / or hydroxyurea leading to a complementary or possibly synergistic effect.
• at least one other active ingredient such as an analgesic and / or hydroxyurea leading to a complementary or possibly synergistic effect.
• Example 1 Effect of nicotinol DHA on the fatty acid composition of plasma and red blood cells of dogs treated orally.
• the purpose of this first study is to measure the total DHA in the blood (plasma and red blood cells) of dogs receiving oral nicotinol DHA.
• Group 2 nicotinol of DHA at 2g per day.
• All animals receive orally for 28 days, either a placebo or nicotinol DHA at 2g per day.
• the Blood samples are taken at (control), D7, D14, D21 and D28.
• the total lipids of plasma (500 L) and red blood cells ("500 mg, weighing red blood cells is more accurate than measuring a volume) are extracted with 4 mL of a mixture of hexane and isopropanol (2/1, v / v), in acid medium (3M HCl, 500 L) in the presence of margaric acid as internal standard (100 ⁇ g). After stirring and centrifugation (2000g, 15 minutes, 10 ° C) the organic phase is separated. A second extraction with 2 ml of the same solvent is carried out under the same conditions. The organic phases are washed with 2 mL of salt water (NaCl 9 ° a). The solvents are evaporated under a stream of nitrogen at 40 ° C.
• the total lipids from the plasma and the red blood cells are then saponified (1 mL of 0.5M NaOH in methanol, 70 ° C., 30 minutes) and then converted into methyl esters (1 mL, 14% BF 3 in methanol, 70% strength). ° C, 15 minutes). After hydrolysis (4 mL NaCl 9 °) they are extracted with 4 then 2 mL of pentane. The organic phases are washed with 2 mL of salt water (9Cl NaCl). The solvents are evaporated under a stream of nitrogen at 40 ° C. The methyl esters are taken up in 200 L of hexane for plasma and red blood cells. The extracted fatty acid methyl esters are analyzed by gas chromatography.
• the chromatograph (Agilent Technologies 6890N) is equipped with a split injector heated to 260 ° C (1:10 division), a capillary column (60 m length, 0.25 mm diameter) with a stationary phase BPX70 (70% cyanopropylpolyphenylene siloxane, thickness 0.25 ⁇ ) and a flame ionization detector heated to 260 ° C (hydrogen: 40 mL / min, air: 450 mL / min).
• the carrier gas is helium (constant flow rate 1.5 mL / min).
• the temperature of the column is initially 150 ° C. and then rises according to a temperature gradient of 1.3 ° C./min up to 220 ° C.
• Table 1 Evolution of the plasma DHA level during treatment with nicotinol DHA at 2 g / day.
• DHA levels are expressed in g / mL, Avg: mean value; SD: standard deviation; G 1: group 1; G2: group 2. The differences between the 2 groups are statistically significant regardless of the treatment time.
• Plasma DHA levels are equivalent between the 2 groups at the beginning of the experiment. In contrast, throughout the course of treatment, the plasma DHA content is higher in the "nicotinol DHA" group compared to the control group.
• Table 2 shows DHA levels of red blood cells.
• Table 2 Evolution of red blood cell DHA during treatment with nicotinol DHA at 2 g / day.
• DHA levels of red blood cells are equivalent between the 2 groups at the beginning of the experiment. In contrast, throughout the course of treatment, the DHA content of red blood cells is higher in the "nicotinol DHA" group compared to the control group.
• nicotinol DHA induces an increase in plasma DHA but above all induces a rise in the red blood cell DHA level.
• Example 2 incorporation of plasma DHA and into the red blood cells of rats receiving orally panthenol DHA.
• the purpose of this study is to measure the total DHA in the blood (plasma and red blood cells) of rats receiving panthenol DHA by oral gavage for 7 days.
• Group 1 vehicle group (olive oil)
• Group 2 panthenol DHA at 300 mg / kg daily.
• Group 3 Panthenol DHA at 1000 mg / kg per day.
• the total lipids of the plasma (500 L) and red blood cells are extracted with a mixture of hexane and isopropanol (3/2, v / v), in acidic medium (3M HCl, 1 mL) in the presence of acid. margaric as internal standard.
• the total lipids from the plasma and the red blood cells are then saponified (1 mL of 0.5M NaOH in methanol, 70 ° C., 30 minutes) and then converted into methyl esters (1 mL, 14% BF 3 in methanol, 70% strength). ° C, 15 minutes).
• the fatty acid methyl esters are extracted with pentane and analyzed by gas chromatography.
• the chromatograph (Agilent Technologies 6890N) is equipped with an injector with split heated to 250 ° C (split at 1:10), a capillary column (length 60 m, diameter 0.25 mm) with a stationary phase BPX70 (70% cyanopropylpolyphenylene siloxane, thickness 0.25 ⁇ ).
• the carrier gas is helium.
• the temperature of the column is initially 150 ° C. and then rises according to a temperature gradient of 1.3 ° C./min up to 220 ° C. and then remains at 220 ° C. for 10 minutes.
• the retention times of standard methyl esters make it possible to identify the methyl esters of extracted fatty acids.
• DHA is quantified relative to the internal standard (C17: 0) added in known quantity to the sample before extraction of total lipids. It is expressed in g / mL for plasma, in g / g for red blood cells. Values are presented as mean ⁇ standard deviation.
• Figure 1 represents the plasma levels of DHA (top panels) in male rats (left panels) and in female animals (right panels) as well as DHA levels in red blood cells (bottom panels), in the control group (G1), in rats receiving DHA panthenol at 300 mg / kg / day (G2) and in rats receiving 1000 mg / kg / day of DHA panthenol (G3).
• the amount of DHA found in red blood cells and in the plasma depends on the dose of panthenol of DHA that the animals received.
• the amount of DHA found in red blood cells and plasma is increased only with the highest dose of panthenol DHA.
• panthenol of DHA makes it possible to release DHA at the plasma level but, above all, to incorporate DHA into the red blood cells in the rat.
• Example 3 Concentration of DHA in human red blood cells after absorption of panthenol DHA.
• the objective of this clinical study was to determine the total DHA levels in red blood cells in volunteers receiving orally once daily panthenol DHA for 28 days.
• Three doses of panthenol of DHA were tested in this study, 1, 2 and 4 g / day. Twelve subjects were included in this study, 3 subjects received placebo (without panthenol DHA) and 9 received panthenol DHA.
• Blood samples were taken prior to administration of panthenol DHA (baseline level) and on days 4, 7, 10, 14, 15, 19, 22, 25 and 29 to determine DHA levels in the blood.
• red blood cells Two blood samples of 4 ⁇ L each were made in tubes containing EDTA. The tubes are centrifuged at 3000 g for 15 minutes at room temperature within 30 minutes after the sampling. The red blood cells were stored at 4 ° C and sent under refrigerated conditions (2 ° C to 8 ° C) in the laboratory that performed the analyzes.
• the lipids were extracted from the red blood cell samples ( ⁇ 500 mg) with a mixture of hexane / isopropanol (3/2 v / v) in an acid medium in the presence of margaric acid as internal standard (100 ⁇ g). .
• Total lipid extracts are saponified and converted to methyl esters.
• the methyl esters of extracted fatty acids are analyzed by gas chromatography.
• the chromatograph (Agilent Technologies 6890N) is equipped with a split injector heated to 250 ° C, a capillary column (length 60 m, diameter 0.25 mm).
• the carrier gas is helium (constant flow rate 1.5 mL / min).
• the temperature of the column is initially 150 ° C.
• the flame ionization detector is heated to 250 ° C (hydrogen: 40 mL / min, air: 450 mL / min).
• the retention times of standard methyl esters make it possible to identify the methyl esters of extracted fatty acids.
• DHA is quantified relative to the internal standard (C17: 0) added in known quantity to the sample before extraction of the total lipids. Values are presented as mean ⁇ standard deviation.
• FIG. 2 shows the DHA level at the end of the study, calculated as a percentage of fatty acid in human red blood cells according to the doses of panthenol of DHA administered. Regardless of the dose of panthenol DHA administered, the level of DHA in red blood cells is increased compared to the placebo group. At 28 days of treatment, a dose-dependent effect is demonstrated, the maximum effect seems to be reached from 2 g / day even if the variability is lower with a dose of 4 g / day.

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