Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Liposomes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/22—Hormones
  • A61K38/28—Insulins

Definitions

• the present invention relates to the field of pharmacotechnology.
• compositions allowing administration in a particularly suitable form of peptides, and in particular hormonal peptides. It is known, in fact, that the administration of peptides with therapeutic properties is limited to a few specific routes, taking into account the ease of hydrolysis of these compounds at the gastric level or at the intestinal level.
• parenteral route by sublingual route or by pernasal route.
• the parenteral route which is the most used requires frequent and permanent use of injections and tends to become an easement for the patient.
• the unilamellar liposome technique has also been proposed but it has the drawback of subjecting to an intense sonication, an aqueous solution of phospholipids in water, which involves significant risks of oxidation of this material as well as other alterations. sonication chemicals.
• the substance which it is desired to incorporate is defined as being a vegetable or mineral oil or any oily substance or else a biologically active substance, a contrast agent, a biological reagent, or even an ink, a lubricant or a surface treatment agent.
• the solvent of the first phase must be sufficiently volatile to be able to be easily removed and this limits the choice of such a solvent to lower alcohols, lower ketones, certain hydrocarbons.
• the solvent of the second phase is a liquid which does not dissolve the polymer intended to form the capsule and the substance contained in the core, but which is miscible with the solvent of the first phase.
• This complex production process leads to the formation of nanoparticles of a size less than 500 nm, the outer wall of which is a polymer formed by nanoprecipitation and whose shape is regularly spherical.
• the object of the present invention is significantly different by its embodiment and by the technical problem. It aims to achieve a mode of administration of drugs, particularly suitable for the digestive or parenteral route and intended above all to allow the administration by these two routes, in a protected form, of drugs which, without it, would not pass not the intestinal barrier, either due to enzymatic degradation or due to insufficient absorption.
• a - This problem has therefore been resolved by producing liposomes, that is to say spherical vesicles with a diameter ranging from one tenth to ten micrometers, formed of several walls composed of a double layer of alternating phospholipids with one or more aqueous compartments.
• vesicles have the property of serving as vehicles for water-soluble or dispersible active principles which are housed in the aqueous compartment, lipophilic active principles which are inserted into the walls of lipids or amphiphilic active principles which are localized in both in the aqueous compartments by the hydrophilic part and in the lipid walls by their lipophilic residue.
• the process for preparing this new form of administration is characterized in that a mixture of phospholipids and cholesterol is dissolved in a chlorinated organic solvent and then the solvent is evaporated under reduced pressure, which is dispersed the dry residue in an ultrasonic tank in an aqueous solution or dispersion of the active ingredient to be incorporated until the total dispersion of the lipid phase in the aqueous phase is obtained.
• This suspension can then be diluted by liposomes which do not contain any active principle and whose aqueous phase contains only a solution of a buffering agent. We can therefore adjust the concentration of active ingredient at will.
• the active principle which is not attached to the liposomes is then separated by filtration, in particular on a polyacrylamide gel or on a dextran gel, eluting with the aid of a buffering agent.
• the liposomal suspensions obtained after gel filtration are stored at low temperature and protected from light.
• the method according to the invention therefore comprises two important stages: the formation of multilamellar liposomes from phosphatidyl choline and the separation of the active principle which is not fixed by gel filtration.
• the process is carried out by lyophilization and thus dry forms are obtained, no longer in suspension.
• the phospholipids used are phospholipids of natural origin such as egg phosphatidyl choline or soy phosphatidyl choline containing at least 9BX of phosphatidyl choline.
• lipid vesicles formed are kept in suspension in an aqueous solution of sodium citrate, the pH of which varies according to the concentration from 2.5 to 4.5 and preferably from 2.8 to 3.0.
• the lipid constituents are dissolved in an inert solvent such as a chlorinated solvent chosen from the group formed by chloroform, methylene chloride, dichlorethane, tetrabromoethane and hexafluoroacetone. Preference is given to the lowest boiling point solvents which thus allow easier and faster evaporation.
• Methylene chloride constitutes the most effective solvent according to the invention.
• the lipid solution can also be added with a nonionic surfactant such as a Tween, a Span, a Palmitostearate of sugar or a copolymer of propylene oxide and ethylene oxide.
• a nonionic surfactant such as a Tween, a Span, a Palmitostearate of sugar or a copolymer of propylene oxide and ethylene oxide.
• the content of surface active agent it is possible to incorporate in the lipid solution * varies in wide limits and particularly from 1 to 20% by weight of phospholipids. A content ranging from 5 to 15% is particularly envisaged.
• Filtration on polyacrylamide or dextran gel is carried out on a column packed with such a gel, formed in particular with a polymer sold under the brand SEPHAROSE and in particular SEPHAROSE 6B.
• the filtration eluent is a mixture of the starting citrate buffer supplemented with sodium chloride.
• the liposomal populations obtained are not homogeneous and, whatever the method of preparation, there are two different sizes of vesicles, one ranging from 120 to 180 nm and the other ranging from 750 to 1750 nm.
• the size of the vesicles measured with the Coulter N 4 MD counter shows good reproducibility.
• the liposomes according to the invention show a plurilamellar structure.
• the little absorbable or hydrolyzable active principles, of peptide nature which it is possible to incorporate into the liposomes according to the invention, are of very varied nature and can include small peptides, globulins, proteins, macromolecules, such as TRH, lysine vasopressin, desglycinamide lysine vasopressin, ACTH fragments, encephalins as well as longer chain peptides such as CCK, CCK analogs, natural or chemically modified insulins, proinsulin and insulins polymers, somatotropin, EGF, calcitonin, thyrocalcitonin, natriuretic factor, polylysine, FSH, LHRH, gonadorelines such as RH, buserelin, intestinal peptides (VIP, ProVIP, preproVIP ...) and in general, any peptide natural or of synthetic origin or genetic origin having a degree of solubility in water sufficient to ensure its incorporation into l has aque
• the suspension of neutral liposomes devoid of active principle is prepared under the same conditions by replacing for each of them, the buffered aqueous solution of active principle, by a buffer solution at approximately pH 3.0 and using a phospholipid concentration of 50 ⁇ mol / ml approximately.
• This suspension is mixed with the buffered aqueous suspension of active principle so that the final concentration of the mixture is reduced in a proportion ranging for example from 1 to 10.
• Filtration on acrylamide gel has the effect of eliminating the excess of active principle which has remained absorbed on the surface of the liposomes and whose presence would be useless due to the degradation in the digestive tract or possibly harmful when the active principle is a substance. very active.
• the active ingredient incorporated is a peptide hormone such as insulin and in particular porcine insulin or human insulin, calcitonin, oxytocin.
• the content of active principle and in particular of insulin makes it possible to observe that the active principle retained by the multilamellar vesicles before filtration, can vary within very large proportions.
• active principle retained by the multilamellar vesicles before filtration can vary within very large proportions.
• an insulin it is of the order of 1.5 to 2.5 IU per umol and after filtration of 0.03 to 0.10 U / ⁇ mol.
• the incorporation rate therefore ranges from 0.25 to 65% of active principle relative to the theoretical quantity introduced.
• the presence of a negative charge in the wall of the liposomes significantly improves the encapsulation of the peptide active principles inside the liposomes, when these have a basic structure.
• the raw materials used are soy phosphatidylcholine, with a molecular weight of approximately 800, containing at least 98% of phosphatidylcholine, lanolin cholesterol, with a molecular weight of 387, and diketyl phosphate, with a molecular weight 560. These three components are combined in a 7/2/1 molar ratio and added tocoferol acetate, used as an antioxidant in the proportion of one mole per 100 moles of phosphatidylcholine.
• the lipid vesicles are suspended in an aqueous solution of citrate buffer at pH 3.0 thus composed: 0.1 M disodium citrate solution containing 2 ° / 0 o of methylparaben: 399 ml, concentrated hydrochloric acid: 3.3 ml and distilled water qsq 1000 ml.
• the mother insulin solution is a 100 IU / ml solution of porcine insulin, also buffered at pH 3.0, thus prepared: porcine insulin: 192.3 mg, citrate buffer pH 3.0 q.s.p 50 ml.
• the lipid mixture was prepared with 200 mg of phosphatidylcholine, 28 mg of cholesterol, 20 mg of diketyl phosphate and 1.2 mg of tocoferol acetate.
• the lipid constituents are dissolved in 20 ml of dichloromethane, then the solvent is eliminated in a rotary evaporator (Buchi), in a liter flask, under reduced pressure (water pump) and at 37 ° C.
• the residual film is dispersed at room temperature, by stirring in an ultrasonic tank (Bransonic 220V), for 30 min, in 10 ml of the buffered insulin solution at 50 IU / ml. This sonication is followed by a 2 hour rest period at room temperature.
• the suspension is then cooled and subjected to a new sonication by probe (Branson 450W), 6 minutes in an ice bath (to keep the temperature below 40 ° C), in the open air.
• the lipid constituents are dissolved in 20 ml of dichloromethane containing an amount of Tween 60 representing 10% by weight of the phospholipids.
• the buffered aqueous insulin solution is added with constant stirring to Ultraturrax e * in the open air, at a temperature increasing to 60 ° C in 20 minutes. After stopping the heating, stirring is continued for 40 minutes. It allows the total evaporation of the organic solvent and the obtaining of a suspension whose lipid concentration is 25 ⁇ mol / ml (ie 20 mg / ml).
• the lipid constituents are dissolved in 5 ml of propylene glycol, maintained at 60 ° C (until dissolution).
• the buffered insulin solution 50 ml is heated to 30 ° C and introduced into an ultrasonic bath (Bransonic 220 V), with gentle agitation with Ultraturrax (0SI).
• the lipid solution at 60 ° C. is injected at a low rate (5 ml in 1 to 2 minutes) into the insulin solution maintained with stirring and sonication. Agitation with Ultraturrax is continued 2 minutes after the injection and sonication, 30 minutes.
• the bath of sonication should be cooled with ice to keep the temperature below 40 ° C.
• the separation of the free insulin is carried out by filtration on Sepharose 6B gel (column 25 cm high and 1.6 cm in section).
• the eluent for filtration is the sodium citrate buffer supplemented with 0.6% sodium chloride. Its flow rate is 0.5 ml / min.
• the volume of each fraction collected is 3 ml.
• the lipid vesicles are filtered between the 5th and the 8th fraction and free insulin, from the 12th.
• the separation of the free insulin is carried out by tangential ultrafiltration, on acrylic Minitan (Millipore), using a PTMK 300 KD polysulfone filter.
• the volume of the lipid suspension, concentrated by removing the filtrate, is readjusted to 100 ml, during filtration, with the citrate preparation buffer.
• the free insulin solution is eliminated in the filtrate each time the lipid suspension passes through the membrane, and its different fractions can be collected using a side tube of the filter circuit.
• the insulin concentration of the different filtrate fractions is determined by assay (HPLC) and filtration is stopped when this concentration is less than 2 IU / ml, which corresponds to a total volume of filtrate collected of the order of 5 to 600 ml. (i.e. 5 to 6 times the initial volume of lipid suspension).
• the insulin suspensions obtained by the different methods can be stored at 4 ° C and protected from light, or lyophilized as such, after distribution by 2 ml aliquots in 15 ml bottles, for 24 hours.
• This assay was carried out by a high performance liquid chromatography (HPLC) method in an isocratic system, with a Kromasil C 4 , 5 ⁇ m, 100 A column, 25 cm in length, associated with a Kromasil C 4 , 5 ⁇ m precolumn, 100 A, 1.8 cm, intended to stop most of the phospholipids.
• the eluting solution is a mixture of 2 solvents A and B, in respective proportions 590 ml / 410 ml, filtered through Millipore 0.45 ⁇ m and added with 0.2% acetonitrile (v / v).
• Solvent A is composed of acetonitrile: 100 ml and buffer pH 3.3: qs 1000 ml and solvent B, acetonitrile: 500 ml and buffer pH 3.3: 500 ml.
• the pH 3.3 buffer is prepared by mixing ammonium sulphate: 39.6 g, 2N sulfuric acid: 0.7 ml (qs pH 3.3) and distilled water: qs 1000 ml.
• the injection volume of the sample to be assayed is fixed at 100 ⁇ l.
• the retention time of the insulin on the column is of the order of 11 min.
• This assay method was developed with the buffered aqueous insulin solution used for the preparation of lipid suspensions. It has good sensitivity (1.9 ⁇ g of detectable insulin, so 0.05 IU), as well as unsatisfactory speci fici ty and reproducibility.
• the results are expressed in units of hormone / ml of the sample to be analyzed.
• the phospholipid level of insulin suspensions can be determined by the Takayama enzymatic method, using reactive kits from the company BIOMERIEUX.
• Insulin concentrations can thus be expressed in IU / ⁇ mol of phospholipids.
• the insulin concentrations of the formulations obtained by the three preparation methods were calculated. The results are as follows:
• METHOD I made it possible to fix an amount of insulin of the order of 1 IU / ml of suspension, ie 0.45 IU / ⁇ mol of phospholipid, which corresponds to a yield of the order of 22% of the initial amount of hormone used. Furthermore, the reproducibility of the results from one batch of preparation to another has been found to be satisfactory.
• METHOD II provided only a tiny insulin fixation and therefore cannot be retained. The hormone was probably degraded by the use of T een at high temperature.
• METHOD III had two advantages: it provided both a higher insulin fixation rate than the two previous methods, and a quantitative preparation of lipid suspension loaded with insulin, thanks to the rapid filtration mode adopted.
• the level of fixed insulin was 15 IU / ml, or 0.74 IU / ⁇ mol of phospholipid and a yield of the order of 37%.
• the rate of hormonal fixation has reached 25 IU / ml, that is to say 0.60 IU / ⁇ mol of phospholipid and a yield of 60% of the initial quantity.
• soy phosphatidylcholine instead of egg phosphadidylcholine. improve the storage of preparations by freeze-drying the lipid insulin suspensions.
• liposomes were composed of egg or soy phosphatidylcholine, cholesterol and cholesterol hemisuccinate in the molar ratios 7/2/1, 7/1/1 and 7/0/2.
• Cholesterol hemisuccinate therefore cannot be used as a loading lipoid.
• This loading lipoid was introduced at three concentrations, in the following phospholipid / cholesterol / lipoid molar ratios: 7/2 / 0.5, 7/2/1 and 7/2/2.
• Freeze-drying tests were carried out, for 24 hours, on 2 ml aliquots of suspension, in the presence and in the absence of mannitol. The determination of the fixed insulin level was carried out before and after lyophilization. In addition, the size of the liposomal vesicles was determined under the same conditions.
• the lipid constituents (soy phosphatidylcholine, cholesterol and diketyl phosphate) are dissolved in 5 ml of propylene glycol maintained at 60 ° C.
• the insulin solution buffered to pH 3.0 is heated to 30 ° C and introduced into an ultrasonic bath, with gentle stirring with Ultraturrax.
• the lipid solution at 60 ° C. is injected at low flow rate (5 ml in 1 to 2 minutes) into the insulin solution still under agitation and sonication. Agitation with Ultraturrax is continued 2 min after injection and sonication, 30 min.
• the sonication bath must be cooled by ice to keep the temperature below 40 ° C. After resting for one hour, a new sonication of 30 minutes is carried out in the tank always cooled by ice.
• the separation of the free insulin is carried out by tangential ultra-filtration, on acrylic Minitan (Millipore), using a PTMK 300 KD polysulfone filter.
• This filtration method makes it possible to treat a volume of 100 ml of liposomal suspension. Associated with the preparation method previously described (which itself applies to high volumes of suspensions), it makes it possible to successfully envisage an industrial preparation of liposomal suspensions.
• the lipid composition is expressed as a molar ratio of phosphatidylcholine, cholesterol and cholesterol hemisuccinate.
• the yield is expressed in% of the theoretical quantity of insulin fixed, ie 2 IU / ⁇ mol of phospholipid. TABLE II influence of the diketyl phosphate concentration
• the lipid composition of the 3 formulations was: phospholipid, cholesterol and diketyl phosphate, in a 7/2/1 molar ratio.
• Liposomes composed of non-lyophilized soy phosphatidylcholine 140+ 60nm 70% 1.83 0.75 37.5% 1600 + 340nm 30%
• liposomes composed of non-lyophilized soy phosphatidylcholine 200+ 80nm: 70% 1.65 0.60 30% 5000 + 1300nm: 30%
• Venous blood samples will be taken from all fasting animals, then 30 min, 1 hour, 2, 3, 4, 5, 12 and 24 hours after administration of the various preparations in the treated animals and in the controls used to determine spontaneous changes in blood sugar. These samples are taken while taking care to avoid any stress or anesthesia of the animals, likely to cause a disturbance of their glycemia. The best way is to treat animals with a permanent collection catheter.
• the preparation doses, in liquid form, are determined by HPLC beforehand.

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