The present invention relates to the use of an ester of DHA as a prophylactic and / or curative treatment in sickle cell disease.
Sickle cell Hb also called S or sickle cell anemia is a genetic disorder of hemoglobin, the protein providing the transport of oxygen in the blood. Sickle cell disease is not a rare disease. It is particularly common in sub-Saharan populations of African origin, the Caribbean, India, the Middle East and the Mediterranean especially in Greece and Italy. It is estimated that over 100 million individuals are infected worldwide. This is the first genetic disease in France, and probably in the world.
Sickle cell disease is caused by abnormal hemoglobin, the main component of red blood cells, also called erythrocytes. These are flattened disks with a thinner center than the edges. Their shape is characteristic biconcave said, it gives them great flexibility, necessary to pass through the finest capillaries. Erythrocyte membrane consists of a lipid bilayer having a central part between the outer and inner surfaces is hydrophobic and contains fatty acids. Membership, aggregation and deformability of blood cells is affected by the fatty acid content of their membrane.
Hemoglobin comprises four chains joined together. Hemoglobin A, the majority in adults, thus consists of two chains called alpha chains and two beta say. In case of sickle cell disease, the beta chains are abnormal. Hemoglobin formed from abnormal beta chains and alpha chains is a normal hemoglobin that "agglomerates are" in the red blood cells, it is called hemoglobin S, an abbreviation for the English word "sickle" which means sickle. A red blood cell normally has the form of a disk, each face is a little hollow. In case of sickle cell anemia, S agglomeration of hemoglobin leads to red blood cells take the form of a sickle or crescent, especially when the amount of oxygen is lower. Deformation "sickle" is called sickling and deformed red blood cells are called "sickle". In the blood, there is a majority of red blood cells of normal appearance and sickled red blood cells. In addition to being deformed, the sickled red blood cells are more fragile and more rigid than normal red blood cells. They circulate in the vessels evil, which prevents them from playing their full role of oxygen carrier, they easily hémolysent in fine capillaries. The manufacture of the beta chain of hemoglobin depends on two genes, genes "beta globin" located on chromosome 11. At the molecular level, the beta chains are abnormal because of a glutamic acid at position 6 replaced by an valine.
Hemoglobin S differs from hemoglobin A, normal, by its electrophoretic mobility slower, but especially by the insolubility of the deoxygenated form that crystallizes easily. The Hb S is the most frequent today genetic defects in France. We must distinguish heterozygous forms (A / S), usually silent, shapes homozygous (S / S) or compound heterozygotes (mostly S / C, S / beta thalassemia, S / D-Punjab, S / O-Arab) who are responsible for major sickle cell syndromes, always serious clinical and haematological.
The severity of sickle cell disease varies from person to person and over time for the same person. The disease reported in infants, but is not usually evident at birth because the red blood cells of newborns still contain 50-90% fetal hemoglobin. The symptoms of this disease can appear from the age of two to three months, date of onset of the beta chain. The three main manifestations are anemia, vaso-occlusive crises and less resistance to certain infections.
Anemia refers to a lack of hemoglobin and results in excessive fatigue and a feeling of weakness. Red blood cells, which are constantly renewed, are produced in bone center in the red bone marrow. From there they go into general circulation where normally they stay 120 days in the bloodstream and are destroyed in the spleen. In sickle cell disease, such as red blood cells in sickle are abnormally fragile, they are easily destroyed causing anemia. The severity of anemia varies over time, it can worsen suddenly in case of excessive functioning spleen, is called splenic sequestration. Indeed abnormal red blood cells are rapidly eliminated by the body, and more specifically by the spleen. Sickle red blood cells are considered abnormal by the spleen that the capture (or receiver) then eliminates, accentuating anemia.
Other cells involved in the pathophysiology of vaso-oclusives crises endothelial cells, reticulocytes, neutrophils, platelets. However, mononuclear cells and platelets are abnormally polyunsaturated fatty acid composition in sickle cell patients.
The vaso-occlusive crises or painful crises, manifested by sharp and sudden pain. Red blood cells in sickle-shaped block the flow in the blood vessels which prevents optimal distribution of oxygen in the organism. This process can occur in different parts of the body (bones, abdomen, kidney, brain, retina ...). These attacks can be very painful. These pains are the most frequent 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 can be affected, but the osteo-articular involvement is very common. Eventually, the bone infarction may occur causing problems in the joints. Ocular involvement is also common, intraocular bleeding can occur. They limit more or less completely the visual field.
Infections are one of the most common complications of sickle cell disease. They can occur throughout the life of the sickle cell and may endanger the life, especially in infants and young children. Bacterial infection is capable of rapid diffusion and serious locations such as meningitis or osteomyelitis. Pneumococcus and salmonella are the most common bacteria. This increased susceptibility to infection is due to the fact that the spleen, which plays an important role in the process of defense against bacteria, is almost always damaged in patients.
The course of the disease varies greatly. Generally, anemia evolves in spurts or "hemolytic crises", which are favored or triggered by infections. The painful vaso-occlusive crises occur at intervals varying, more or less marked. The trend is all the better that access to and quality of care are good.
To date, no one knows cure sickle cell disease, it is simply possible to relieve pain during a crisis, prevent serious infections at best. Painful crises are the leading cause of consultation or hospitalization. Analgesics (may not be sufficient, the pain is generally such as are used to morphine or morphine derivatives (opioids).
There may be mentioned as analgesics used in sickle cell disease, nonsteroidal anti-inflammatory drugs, acetaminophen, codeine, tramadol, buprenorphine, nalbuphine, the orphine, fentanyl, hydromorphone, oxycodone. In some cases, these treatments are not always enough to calm the pain. Often oxygen therapy is established during hospitalization, it consists of daily inhalation of an oxygen-enriched air to increase the oxygenation of organs and thus relieve pain. There is no specific treatment to treat anemia. When it worsens because of a splenic sequestration episode, a blood transfusion may be necessary. Drug therapy can be offered to patients with severe sickle cell disease, it is one of hydroxyurea (or hydroxyurea), a product used in leukemia. This molecule acts on ribonucleotide reductase. This is the key enzyme for the transformation of the four deoxyribonucleotides ribonucleotides essential to the synthesis of DNA. This molecule is capable of increasing in adults producing a hemoglobin normally present in the fetus and in minute quantities at birth (hemoglobin F). Forced production of the fetal hemoglobin F reduces the agglomeration of hemoglobin S. However, this molecule does not act on the lung or bone infections and do not put away secondary bone involvement. Furthermore, one hydroxyurea is not devoid of side effects, such an influence on male fertility. Currently, there is only one option to permanently treat the disease, bone marrow transplant, healthy marrow will produce healthy red blood cells. This procedure is however reserved only a small fraction of patients. This is an operation that requires an extremely heavy treatment and can lead to potentially fatal severe complications.
It is therefore clear that the treatments offered to people with sickle cell disease are far from sufficient. There is a significant medical need for new drugs with the least possible side effects since they are aimed at people physiologically vulnerable.
Polyunsaturated fatty acids of the Omega 3, 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 modulation of corresponding risk factors. In particular, they are known in the treatment of one hyperlipidemia, one of hypercholesterolemia and hypertension. The clinical trials conducted with formulations containing a high concentration of DHA ethyl ester on patients who had suffered a myocardial infarction showed their effectiveness in reducing mortality and sudden death in particular. These results were attributed in part to a stabilizing effect on cell membranes of ventricular cardiomyocytes, which prevent the appearance of malignant arrhythmia in the presence of ischemic myocytes in patients having had a myocardial or in experimental models which reproduce such conditions. Furthermore, low levels of DHA have been associated with, among other attention disorders (ADHD) and depression and it seems that taking DHA supplements to be effective in the fight against such diseases. Similarly, high levels of DHA would be correlated with a lower risk of developing dementia. DHA therefore play an important role in numerous pathologies.
In the case of sickle cell anemia, one lipid homeostasis is modified and erythrocyte DHA is reduced (Ren et al, Prostaglandins, leukotrienes and essential fatty acid. 72: 415-421, 2005), especially as the disease induces strong anemia. More recently, Ren et al, 2008 (Int J Vitam Nutr Res, 78 (3): 139-147). Shows that the omega-3 content is different in its distribution within the lipid bilayer of erythrocytes in sickle cell patients . The cause is increased peroxidation in these patients due to poor antioxidant capacity. A clinical study of 10 patients show interest a fish oil therapy in sickle cell disease by reducing the number of painful crises but without explaining the mechanism (Tomer et al., Thromb. Haemost. 85 (6) : 966-974, 2001). Very recently in a clinical pilot study (16 patients), the authors show that supplementation of six months in DHA + EPA (LOMG + 15mg / kg / d) in patients with sickle cell disease decreases the number of vaso-occlusive crises and hemolysis (Okpala et al, APMIS, 119 (7):. 442-448, 2011).
However, the authors do not seek to demonstrate the activity is driven primarily by the EPA or only DHA alone, or is it a combination of both which is pharmacologically active.
A study in man (Terano et al, Atherosclerosis, 46 (3):. 321-331, 1983) has shown that the EPA decision for 4 weeks in 8 healthy volunteers resulted in a reduction of viscosity blood and an increase in the deformability of red blood cells. The same authors reported a positive correlation between the content of EPA in red blood cell membranes and the deformability thereof.
Similarly, in another study (Ide et al, Int J Mol Med 11 (6):.... 729-732, 2003) conducted in patients with anemia due to chronic hepatitis C, the authors wanted to verify whether a contribution in EPA (1800 mg) for two months could be beneficial. It has thus been shown that the mean hemoglobin level in these patients was significantly increased after one month of treatment in all patients, and this increase was due to a reduction in loss in erythrocytes.
In view of these studies, it would seem that an intake of EPA is responsible for the demonstrated activity in patients with sickle cell disease.
Now, the inventors do the opposite hypothesis and believe it is DHA which plays an important role in this disease. An intake of DHA could increase the red blood cell DHA levels in people with sickle cell disease and thus reduce the adulteration of these red blood cells, these cells being the true hub of sickle cell disease, but also the alteration of other cells involved in pathophysiology of sickle cell disease such as endothelial cells, platelets, mononuclear cells.
Vitamins or provitamins in group B the benefits associated with their function. In particular, nicotinyl alcohol is the alcohol derived from nicotinic acid (vitamin B3).It is rapidly converted into nicotinic acid in the human body. Nicotinic acid, also called niacin, is a water soluble B vitamin that can be synthesized from tryptophan.Vitamin B3 plays an important role in the release of energy from food, but also in reducing the cholesterol. However, the effective therapeutic doses to hypocholesterolemic and hypolipidemic purposes are higher than the quantities synthesized by the body and an oral supplement proves to be necessary in a referred hypocholesterolemic and / or hypotriglycéridémiante. The vitamin B3 deficiencies still exist in some countries of Asia and Africa, ie in areas where sickle cell disease is rampant strongly. Vitamin B3 deficiency leading to a general fatigue, vitamin B3 could be a real benefit in the anemic people and thus already tiring faster.
Panthenol is the alcohol derivative of pantothenic acid, more known as vitamin B5. In the body, panthenol is transformed into pantothenic acid which becomes an important part of compound "coenzyme A", which is of particular interest in cellular metabolism. Indeed, it takes part in the metabolism of lipids, carbohydrates and proteins. Panthenol also participates in the formation of 1 acetylcholine and adrenal steroids. It is also involved in the detoxification of foreign bodies and in resistance to infection which is particularly interesting in people with sickle cell disease.
Inositol vitamin B7 or mobilizes fats by preventing their accumulation. It also has an anxiolytic effect. It tones the nervous system and liver. It also reduces cholesterol in the blood. It is involved in the increase in serotonin activity, control of intracellular calcium concentration, maintenance of cell membrane potential and cytoskeleton assembly. The inositol deficiency can cause muscle pain and eye diseases. Consequently a contribution in inositol can only be favorable to sickle.
The isosorbide, in particular isosorbide mononitrate is a powerful peripheral vasodilator. It also has diuretic properties easing the work of the kidneys, this member being a prime target during vaso-occlusive crisis, a contribution of isosorbide can also be beneficial in sickle cell disease.
It is important to note that vitamins B3 and B5 are involved in the production of red blood cells. The contribution of either of these vitamins, in people with sickle cell disease is therefore the preferred alcohols of this invention.
Surprisingly, the inventors have found that administration of DHA ester with an alcohol allowed a significant increase in DHA level within red blood cells.
The present invention therefore relates to a docosahexaenoic acid ester with an alcohol selected from the group consisting of:
- Nicotinyl the following formula:

panthenol of the following formula

inositol following formula

isosorbide of the following formula:

isosorbide mononitrate of the following formula:

or one of its pharmaceutically acceptable salts, enantiomers, diastereomers, or mixture thereof, including racemic mixtures, for use as a medicament for prophylactic and / or curative treatment of sickle cell disease.
Advantageously, the ester of the invention e docosahexaeneoate panthenyl, "DHA ester of panthenol" of the following formula:

for its use as medicament for the prophylactic and / or curative treatment of sickle cell disease.
In a particular embodiment of the invention, DHA ester with an alcohol selected from the group consisting of nicotinyl alcohol, panthenol, inositol, isosorbide or isosorbide mononitrate is used as a medicament for preventing and / or relieve the vaso-occlusive crises in a patient with sickle cell anemia.
In another particular embodiment of the invention, DHA ester with an alcohol selected from the group consisting of nicotinyl alcohol, panthenol, inositol, isosorbide or isosorbide mononitrate is used as a medicament for preventing and / or treating anemia in a patient with sickle cell anemia.
In the present invention, the term sickle cell anemia, all genetic forms of sickle cell disease, both homozygous sickle cell composite qu'hétérozygote.
In the present invention, is meant prophylactic treatment, a treatment aimed to prevent the emergence or spread of the disease. By means cure, a treatment that aims to cure, minimize or alleviate the symptoms.
In the present invention, is intended to denote "enantiomers" optical isomers of the compounds that have identical molecular formulas but that differ in their spatial configuration and which are images in a non-superimposable mirror. The term "diastereomers" optical isomers which are not mirror images of one another. Within the meaning of the present invention, a "racemic mixture" is a mixture of equal proportions of the laevorotatory and dextrorotatory enantiomers of a chiral molecule.
In the present invention is meant by "pharmaceutically acceptable" or "pharmaceutically acceptable" which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable and that is acceptable for veterinary use as well as human pharmaceutical use.
Is intended to denote "pharmaceutically acceptable salts" of a compound of the salts that are pharmaceutically acceptable, as defined herein, and that possess the desired pharmacological activity of the parent compound. Such salts include:
- The acid addition salts formed with inorganic 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, hydoxynaphtoïque acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic, muconic acid, 2-naphthalenesulfonic acid, propionic acid, salicylic acid, succinic acid, dibenzoyl-L-tartaric acid, tartaric acid, p-toluenesulfonic acid, trimethylacetic acid, trifluoroacetic acid and the like; or
- Salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an ion of alkaline earth metal or an aluminum ion; or 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 the salts formed from hydrochloric acid, trifluoroacetic acid, dibenzoyl-L-tartaric acid and phosphoric acid
It should be understood that all references to pharmaceutically acceptable salts include the forms of added solvents (solvates) or crystal forms (polymorphs) as defined herein, of the same acid addition salt.
The present invention further relates to a pharmaceutical composition comprising the DHA ester with an alcohol selected from the group consisting of nicotinyl alcohol, panthenol, inositol, isosorbide or isosorbide mononitrate, and at least one pharmaceutically acceptable excipient for its use as 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 the vaso-occlusive crises in a patient with sickle cell anemia.
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 anemia.
The pharmaceutical composition according to the present invention may be administered by oral or other route of pharmaceutical administration.
The pharmaceutical compositions according to the present invention may be formulated for administration to mammals, including man. These compositions are prepared so as to be administered by oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration. In this case, the active ingredient may be administered in unit forms of administration, mixed with conventional pharmaceutical carriers, to animals or humans. The appropriate unit administration forms include oral forms such as tablets, capsules, powders, granules and oral solutions or suspensions, sublingual and buccal administration, forms for subcutaneous administration , topical, intramuscular, intravenous, intranasal or intraocular forms for rectal administration.
When preparing a solid composition in tablet form, one mixes the main active ingredient with a pharmaceutical vehicle such as gelatin, starch, lactose, magnesium stearate, talc, gum arabic, silica or the like. Sucrose tablets can be coated or other suitable materials or they can be treated so that they have a prolonged or delayed activity and release a continuously a predetermined amount of active ingredient.
A preparation in capsules is obtained by mixing the active ingredient with a diluent (optional step) and pouring the mixture obtained into soft or hard capsules.
A preparation in the form of a syrup or elixir may contain the active ingredient together with a sweetener, an antiseptic, a flavoring and an appropriate color.
The powders or granules dispersible in water may contain the active ingredient mixed with dispersing agents or wetting agents, or suspending agents, as well as with flavor correctors or sweeteners.
For rectal administration, use is made of suppositories which are prepared with binders melting at the rectal temperature, for example cocoa butter or polyethylene glycols.
For parenteral administration (intravenous, intramuscular etc.), intranasal or intraocular administration, aqueous suspensions, isotonic saline solutions or sterile and injectable solutions which contain dispersion agents and / or pharmacologically compatible wetting agents.
The active principle can also be formulated as microcapsules, optionally with one or more additive supports.
Advantageously, the pharmaceutical composition of the present invention is intended for oral or intravenous administration, more advantageously orally.
The dosages of the pharmaceutical compositions containing DHA ester with an alcohol selected from the group consisting of nicotinyl alcohol, panthenol, inositol, isosorbide or isosorbide mononitrate in the compositions of the invention are adjusted to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular composition in the method of administration. The selected dosage level therefore depends upon 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 . Accordingly the optimum dosage should be determined based on the relevant parameters considered by the expert in the field. Preferably, the ester of DHA is administered in pharmaceutically acceptable compositions where 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, such as 1 g, 2 g, or 4 g / day. It may be necessary to use higher doses (called loading dose) at the beginning of prophylactic and / or curative, and then reduce the dose (maintenance dose) during processing.
The pharmaceutical composition according to the present invention may further comprise at least one other active ingredient, such as an analgesic and / or 1 hydroxyurea leading to a complementary or possibly synergistic.
The invention will be better understood by reference to the following examples.
Example 1: Effect of DHA nicotmol on the fatty acid composition of plasma and red blood cells of dogs treated orally. The purpose of the first study was to assay the total DHA in the blood (plasma and red blood cells) from dogs receiving the DHA nicotinyl orally.
Two groups of 10 dogs are used:
Group 1: control group
Group 2: nicotinyl alcohol to DHA to 2g per day.
All animals received orally for 28 days or a placebo or DHA nicotinyl to 2g per day. Blood samples are taken at Jl (control), D7, D14, D21 and D28.
The plasma total lipid (500 L) and red blood cells ("500 mg, weighing the red blood cells is more accurate than the measurement of a volume) are extracted with 4 ml of hexane and isopropanol (2/1, v / v) under acidic (3M HC1, 500 L) in the presence of margaric acid as internal standard (100 ug). After agitation and centrifugation (2000 g, 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 brine (NaCl 9 ° a). The solvents are evaporated under nitrogen flux at 40 ° C.
Total lipids from the plasma and red blood cells are then saponified (1 mL of 0.5M NaOH in methanol, 70 ° C, 30 minutes) and then converted to methyl esters (1 ml, BF 3 14% in methanol, 70 ° C, 15 minutes). After hydrolysis (4 ml NaCl 9 ° a) they are extracted by 4 and 2 mL of pentane. The organic phases are washed with 2 mL of brine (NaCl 9¾Ό). The solvents are evaporated under nitrogen flux at 40 ° C. The methyl esters are taken up in 200 L of hexane to the plasma and red blood cells. The methyl esters of fatty acids extracted were analyzed by gas chromatography. The chromatograph (Agilent Technologies 6890N) is equipped with a split injector heated at 260 ° C (1:10 split), a capillary column (length 60 m, diameter 0.25 mm) with a stationary phase BPX70 (70% cyanopropylpolyphénylène siloxane; μιτι 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 1.5ml / min). The column temperature is initially 150 ° C and then it rises according to a temperature gradient of 3 ° C / min up to 220 ° C then 40 ° C / min to reach 260 ° C for 5 minutes. The time standard methyl esters retention identify the methyl esters of fatty acids extracted.
DHA is quantified relative to the internal standard (C17: 0) added in known amount to the sample before the extraction of total lipids. It is expressed in g / mL for plasma, in g / g for red blood cells. Values are presented as mean ± SD (n = 10 in general). The significant differences are shown by a Student test at the 5% threshold.
The results of plasma DHA levels in the dog are summarized in Table 1
Table 1: Evolution of plasma DHA levels during treatment with DHA nicotinyl to 2 g / J.

DHA levels are expressed in g / mL, Avg: Average value; SD: standard deviation; G 1: Group 1; G2: group 2. The differences between the 2 groups were statistically significant regardless of the processing time.
Plasma DHA levels are similar between the two groups at the beginning of experimentation. However, throughout the duration of treatment, plasma DHA content is higher in the group "nicotinyl DHA" compared to the control group.
Table 2 shows the DHA level of red blood cells Table 2: DHA evolution rate of red blood cells during the treatment with DHA nicotinyl to 2 g / J.
DHA J-l J7 J14 J21 J28
Moy SD Moy SD Moy SD Moy SD Moy SD
G 1 1,9 1,1 2,1 1,2 1,9 1,0 1,9 0,7 1,8 0,7
G 2 1.7 1.0 4.7 2.3 7.1 1.7 8.3 3.2 8.7 2.1 The DHA levels are expressed in g / mL, Avg: Average value; SD: standard deviation; G 1: Group 1; G2: group 2. The differences between the 2 groups were statistically significant regardless of the processing time.
DHA rate of red blood cells are equivalent between the two groups at the beginning of experimentation. However, throughout the duration of treatment, the DHA content of red blood cells is higher in the group "nicotinyl DHA" compared to the control group.
Thus, in dogs the treatment effect by the DHA nicotinyl is significant at all times of the treatment, nicotinyl alcohol to DHA induces an increase in plasma levels of DHA but also induces an increase in the DHA levels of red blood cells.
Example 2: Plasma and DHA incorporation in red blood cells of rats orally DHA panthenol. The purpose of this study was to assay the total DHA in the blood (plasma and red blood cells) of rats receiving DHA panthenol by oral gavage for 7 days.
Three groups of 4 rats (2 males and 2 females) were used:
Group 1: vehicle group (olive oil)
Group 2: panthenol DHA 300 mg / kg per day.
Group 3: panthenol DHA to 1000 mg / kg per day.
Total lipids of plasma (500 L) and red blood cells are extracted with a mixture of hexane and isopropanol (3/2, v / v) under acidic (HC1 3M, 1 mL) in the presence of acid margaric as internal standard. Total lipids from the plasma and red blood cells are then saponified (1 mL of 0.5M NaOH in methanol, 70 ° C, 30 minutes) and then converted to methyl esters (1 ml, BF 3 14% in methanol, 70 ° C, 15 minutes). The methyl esters of fatty acids are extracted with pentane and analyzed by gas chromatography. The chromatograph (Agilent Technologies 6890N) is equipped with a split injector heated at 250 ° C (1:10 split), a capillary column (length 60 m, diameter 0.25 mm) with a stationary phase BPX70 (70% cyanopropylpolyphénylène siloxane; thickness 0.25 μιτι). The carrier gas is helium. The column temperature is initially 150 ° C and then it rises according to a temperature gradient of 3 ° C / min up to 220 ° C and remains at 220 ° C for 10 minutes. The time standard methyl esters retention identify the methyl esters of fatty acids extracted.
DHA is quantified relative to the internal standard (C17: 0) added in known amount to the sample before the 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.
The results of plasma DHA levels in red blood cells and in the rat are shown in Figure 1.
1 shows the plasma DHA levels (upper panels) in male rats (panels at left) and in female animals (right panels) and DHA levels in red blood cells (lower panels) in the (GI) group, in rats receiving panthenol DHA 300 mg / kg / d (G2) and rats receiving 1000 mg / kg / d of DHA panthenol (G3).
In male rats, the amount of DHA found in red blood cells and plasma depends on the dose of panthenol DHA than animals received. In female rats the amount of DHA found in red blood cells and the plasma is increased with the highest dose of DHA of panthenol.
Thus panthenol DHA helps to liberate DHA in plasma but especially to incorporate DHA in red blood cells in rats.
EXAMPLE 3 DHA concentration in human red blood cells after DHA panthenol absorption.
The objective of this clinical study was to determine the concentrations of total DHA in red blood cells in volunteers receiving orally once daily DHA panthenol for 28 days. DHA panthenol three doses were tested in this study, 1, 2 and 4 g / day. Twelve subjects were included in this study, three subjects received placebo (without panthenol DHA) and 9 received DHA panthenol.
Blood samples were taken before the administration of DHA panthenol (corresponding to the base level) and on days 4, 7, 10, 14, 15, 19, 22, 25 and 29 to determine the concentrations of DHA in red blood cells. Two blood samples of 4 raL each were performed in tubes containing EDTA. The tubes are centrifuged at 3000 g for 15 minutes at room temperature within 30 minutes of venipuncture. Red cells were stored at 4 ° C and sent under refrigerated conditions (2 ° C to 8 ° C) in the laboratory that carried out the analyzes
Lipids were extracted from samples of red blood cells (~ 500 mg) with a mixture of hexane / isopropanol (3/2 v / v) in an acidic medium in the presence of margaric acid as internal standard (100] ig) . The extracted total lipids are saponified and converted into methyl esters. After extraction with pentane, methyl esters of fatty acids extracted were analyzed by gas chromatography. The chromatograph (Agilent Technologies 6890N) is equipped with a split injector heated at 250 ° C, a capillary column (length 60 m, diameter 0.25 mm). The carrier gas is helium (constant flow 1.5ml / min). The column temperature is initially 150 ° C and then it rises according to a temperature gradient of 3 ° C / min up to 220 ° C and remains at 220 ° C for 10 minutes. The flame ionization detector heated to 250 ° C (hydrogen: 40 ml / min, air: 450 ml / min). The time standard methyl esters retention identify the methyl esters of fatty acids extracted.
DHA is quantified relative to the internal standard (C17: 0) added in known amount to the sample before the extraction of total lipids. Values are presented as mean ± standard deviation.
The results of the DHA levels in the human red blood cells after administration of different doses of DHA panthenol (or placebo) for 28 days are shown in Figure 2. Figure 2 shows the rate of DHA at endpoint, calculated percentage of fatty acid in the human red blood cells based on panthenol doses of DHA administered.Whatever panthenol dose administered DHA, the DHA levels in red blood cells is increased compared to placebo. At 28 days of treatment, a dose-dependent effect is demonstrated, the maximum effect seems soon reached 2 g / day although the variability is lower with a dose of 4 g / day. Basic DHA levels (calculated as a percentage of fatty acids) in human red blood cells in the absence of treatment that is found in the literature are of the order of 4.8% (Payet et al. British Journal of Nutrition, 91: 789-796, 2004; Weill et al Annals of Nutrition & Metabolism, 46: 182-191, 2002), very close to our values found in the placebo group (4.9%).. In our treatment groups, the levels of DHA reach 6.6% to 1 g / day of DHA and panthenol 7.8% for group 2 and 4 g / day of DHA panthenol. These differences clearly indicate a content of DHA enrichment of human red blood cells by the panthenol DHA intake

We Claims:-
1. An ester of decoseheraonoic acid with an alcohol selected from the group formed by:
- nicetinol having the following formula:
panthenol having the following formula:
- inositol having the following formula:
- isosorbide having the following formula:
and inosorbide mononitrate having the following formula:
or one of the pharmaocutically acceptable aalts ftherof, the anantiomers, diastereoisomers or a mixture thereof, including racemic mixtures, for use thereof as medicinal product for the prophylactic and/or curative tratment of drepanocytosis.

2. The ester according to claim1, having the folloeing formula:

3. The ester according to claim1 or 2, for use thereof as medicinal product intended to prevent and/or relieve vase-occlusive orises in patients suffer from drepanesytools.

4. The ester according to claim1 or 2, for use thereof as medicinal product intended to prevent and/or treat anaemia in patients suffering from drepanocytosis.

5. A pharmaceutical composition comprising an ester according to claims1 to 4 and an excipient acceptable from a pharmaceutical viewpeint for use thereof as medicinal product for the prophylactic and/or curative treatment of drepanoeytosis.

6. The pharmaseutioal composition according to claim 5, for use thereof as medicinal product intended to prevent and/or relieve vaso-occiusive orises in patients suffering from drepanocytosis.

7. The pharmasoutical composition according to claim5, for use thereof as medicinal product intended to prevent and/or treat anaemia in patients suffering from drepanceytosis.

8. The pharmaceutical composition according to any of one claims 5 to 7 for administration thereof via oral route.

9. The pharmaceutical composition according to any one of claims 5 to 8, further comprising at least one other active ingredient such as an analgesic and/or hydroxyures.