Stable crystalline chloride modifications dotap

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to enantiomerically pure (2S)- or (2R)-N,N,N-trimethyl-2,3-bis[[(9Z)-1-oxo-9-octadecenyl]oxy]-1-propanamide chloride (DOTAP chloride) which possess the transfection properties and can find application in medicine and pharmaceutics for intracellular transportation of pharmaceutically active compounds. The invention also refers to a pharmaceutical composition and the use of enantiomerically pure (2S)- or (2R)-DOTAP chloride as an ingredient for preparing drugs.

EFFECT: preparing the compounds which possess the transfection properties and can find application in medicine and pharmaceutics for intracellular transportation of pharmaceutically active compounds.

7 cl, 2 tbl, 6 dwg, 6 ex

 

The present invention relates to enantiomerically pure DOTAP chloride and crystalline modifications of racemic and enantiomerically pure DOTAP chloride, method of production thereof, and their use for the preparation of pharmaceutical compositions.

The DOTAP chloride, which is mentioned above and below in this invention, denotes N,N,N-trimethyl-2,3-bis[[(9Z)-1-oxo-9-octadecenyl]oxy]-1-propanamine chloride, also known as (Z,Z)-N,N,N-trimethyl-2,3-bis[(1-oxo-9-octadecenyl)oxy]-1-propanamide chloride or 1,2-tileorasi-3-trimethylammonium propachlor, and its hydrates.

C42H80ClNO4, Mm698,54

CAS number: 132172-61-3 and 477274-39-8 (racemate), 197974-73-58 (racemate, monohydrate) 428506-51-8 (2S form), 328250-28-8 (2R form)

Liposomes are synthetic multilayer vesicles (bubbles) (Autonomous spherical membrane)containing ambivilence substances, as a rule, natural lipids, in which hydrophilic substances can be encapsulated in the aqueous interior, and also lipophilic substances can be included in the internal part of the lipid membrane.

They are used in particular in cosmetics and medicine, especially in dermatology. In them, in particular, make vitamins, coenzymes, means on skin care and sunscreen. Typically, liposomes are applied topically.

One is about the liposomes are becoming increasingly important in pharmaceutical technology, because parenteral administration of liposomes allows you to achieve more specific delivery to a particular organ, than in the case of application of the active compounds in the freely dissolved form. If the introduced DNA, RNA or proteins, receive lipoplex.

Add oils and the use of high-pressure homogenizers can generate a so-called nanoparticles (nanoscale), which are popped from the liposomes. They are particles of approximately the same size as liposomes, but not with the aqueous phase, but instead contain an oil phase in the inner part. They are particularly suitable for the encapsulation of lipophilic substances.

Micro-emulsions are colloidal dispersed, single-phase system containing water, Lepidoptera and surface-active components. The size of the particles is 1-500 nm, and properties similar to the properties of liquids.

Especially in connection with the peptide active compounds, nucleotides, vaccines and other biological products, which generally have poor solubility, solubilizers effect is very important in the case of applications described above.

In addition, the splitting of the active compounds in the body can be reduced, and thus it is possible to obtain the effect of slow release.

Chloride DOT the DVD, which refers to the class of cationic lipids. Unlike naturally occurring phospholipids, they do not have zwitter-ionic nature. Liposomes containing cationic lipids, alone or in combination with phospholipids or other lepidopteran compounds are positively charged surface. This causes a high affinity to cells having a negatively charged surface from the outside, for example endothelial cells.

However, of particular importance is the ability of liposomes on the basis of DOTAP and other cationic liposomes and lipoplexes to penetrate into the cells and, thus, to transport the active compounds included in them, into cells (transfection).

All these properties make the DOTAP chloride is very interesting also for anticancer therapy. These properties give rise to the possibility of application of standard cytotoxic agents included in the cationic liposome DOTAP.

Transfection properties of DOTAP chloride and other salts DOTAP, such as, for example, acetate, bromide, dihydrophosphate, hydrosulfate, iodide, nelfinavir, methyl sulfate, triptoreline, sulfate or bisulfate and triflate, sufficiently known from the literature.

DOTAP dihydrophosphate and DOTAP mesilate in the literature are mentioned only in the form of a racemate. All other above salts are referred to as the racemate and as 2S-enantiomer, and, in addition, the chloride and ethyl sulfate referred to as 2R-enantiomer.

In some in vitro studies other salts, such as, for example, DOTAP methyl sulfate, achieved better speeds transfection than DOTAP chloride. However, when using in vivo exchange of anions on the surface of the liposomes occurs in a living organism, which means that the advantages of other salts in this situation does not occur. Therefore, especially for medical use in humans, in particular for parenteral use, preferred salts DOTAP with physiologically acceptable anions, such as, for example, the corresponding chloride or acetate.

Medical, in particular parenteral application places the highest demands on the quality and purity of the active compounds and excipients. Therefore, there are very strict rules from regulatory authorities regarding the production, reproducibility of production and profile products for these compounds. In the case of substances used parenterally, in addition, it is necessary to prevent and to strictly control microbiological contamination of pathogenic microorganisms and endotoxins.

The DOTAP chloride and other salts DOTAP extremely unstable and therefore themselves are difficult to obtain in a reasonable clean so that they were suitable for use for the production of medicinal F. RMI.

Like all lipids containing radicals of oleic acid, such as, for example, natural phospholipids DOPC and DOPE, all salt DOTAP very sensitive to oxidation. However, the oxidation products of derivatives of unsaturated fatty acids, as a rule, are characterized by high toxicity.

In this case we require suitable methods of preparation and purification. Acetate DOTAP, for example, is a high-boiling oil, and therefore its industrial obtaining the right quality connected with great difficulties.

Standard ways to overcome instability problems, such as, for example, the addition of antioxidants in the form of ascorbic acid or restored L-glutathione, a very limited total convenience and ease of use of DOTAP chloride, so as not to exclude the interaction with the active compounds, which will be introduced later. The complete exclusion of oxygen during the production, storage and application virtually impossible or can only be carried out with very great difficulty.

The DOTAP chloride commercially available only in the form of a solution in chloroform or in the form of an amorphous solid.

In addition to its susceptibility to oxidation amorphous DOTAP chloride is extremely hygroscopic and dissolves for a very short time under normal atmosphere is Noah humidity with the formation of the oil film. This greatly complicates the manipulation with this connection.

Thus, the manufacturer of amorphous DOTAP chloride is usually recommends that you keep him under protective gas at -20°C and guarantees a shelf life, represents only about 6 months.

The literature points to various synthetic way only to obtain amorphous racemic DOTAP chloride:

Eibel and Unger, DE 4013632 A1, disclose the synthesis of DOTAP chloride of bromide DOTAP by ion exchange in the solvent system chloroform/methanol/aqueous solution of Hcl, after which purification by chromatography. Bromide DOTAP get in advance in situ from 1-bromo-2,3-dioleoylglycerol.

Leventis and Silvius, Biochim. Biophys. Acta, 1023 (1990) 124-132, reported the synthesis of DOTAP chloride of potassium iodide DOTAP by ion exchange in the two-phase solvent system/NaCl solution. Iodide DOTAP receive in advance by methylation of the corresponding dimethylaminomethylene using methyliodide.

Nantz et al., Biochim. Biophys. Acta, 1299 (1996) 281-283, J. Med. Chem. 40 (1997) 4069-4078, describe the synthesis of DOTAP chloride by nonaqueous ion-exchange chromatography. The desired compound is obtained by evaporation of the eluate.

Feigner et al., US 5,264,618, conduct methylation of the corresponding dimethylaminomethylene directly to DOTAP chloride with methyl chloride. They obviously get yellow wax crystallization from acetonitrile at -20°C. However, DOTAP chloride, practically insoluble in acetonitrile at room temperature. Attempts to reproduce this so-called crystallization allowed to receive only amorphous material solidification oily substance obtained from the hot solution by cooling. The fact that this is not a crystallization, is also evident based on the fact that the authors, obviously, does not achieve the effect of cleaning and should be cleaned with substances chromatography.

Therefore, neither the synthetic route to obtain two enantiomeric DOTAP chlorides, or their characteristic properties are not known at the moment. Although Chemical Abstracts assigned rooms for the two enantiomers, publications describe only work with racemic DOTAP chloride.

In particular, if the connections are intended for parenteral use, receipt, which includes the treatment of ion exchange resins, is extremely problematic due to possible microbiological contamination, as appropriate resins are an ideal breeding ground for bacteria, and even after their destruction is still the risk of contamination by endotoxins.

Therefore, the object of the present invention is to provide salts and hydrates of DOTAP chloride with high purity and with acceptable chemical and fusionsrestaurant. Another object of the present invention is to ensure that these salts of long periods of storage that will allow you to use them for the preparation of compounds of medicinal forms. Remains an important requirement concerning the reproducibility of the method of preparation of stable forms of salts and hydrates of DOTAP chloride, which may be carried out on an industrial scale.

Enantiomerically pure DOTAP chloride can be obtained from enantiomerically pure starting materials analogously to the processes described for the racemate

through (R or S)-1-chloro-2,3-dioleoylglycerol,

through (R or S)-1-LG-2,3-dioleoylglycerol and ion exchange (LG=leaving group) or after (R or S)-1-dimethylamino-2,3-dioleoylglycerol. Another way of obtaining that you can mention, is the separation of the racemate racemic DOTAP chloride.

Through experiments it was found, surprisingly, and racemic and also enantiomerically pure, crystalline DOTAP chloride can be obtained in a simple way with high chemical purity and excellent stability. The crystalline products obtained in this way have virtually unlimited stability at room temperature under protective gas. Accordingly, they are suitable as a component or as source material for photomediarecovery forms.

The present invention accordingly relates to enantiomerically pure DOTAP chloride and stable crystalline modifications of racemic and enantiomerically pure DOTAP chloride.

Stable crystalline modification may be found in crystalline and partially crystalline form. They have a purity equal to >98%, which has never been achieved to date, together with stability, component >98% relative to the original size, which has never been achieved to date, after storage for 12 months without access of air at 25°C and 60%relative atmospheric humidity (see about the data in Table 1. Crystalline modification of DOTAP chloride are characterized by a content of less than 1 equivalent of water or solvent for crystallization on the equivalent of DOTAP chloride.

Crystalline modification of racemic DOTAP chloride are, for example, in three different crystalline modifications (type I, type II and type III) and show a moderately sharp bands at the x-ray diffraction analysis of powders (see about the data Figure 1 - Figure 3 and Table 2). Selected values of 2 theta for different crystalline modifications are 12.6, 19.5, 20.2, 21.5 and 25.2 (type I); or 3.3, 4.9, 19.3, 20.0 and 23.5 (type II); or 2.8, 5.8, 20.0, 21.2 and 25.1 (type III).

Enantiomerically pure DOTAP chloride get Chris is aricescu form in the same way. Selected values of 2 theta for the detected crystalline modifications are 12.8, 19.4, 19.8, 20.2 and 21.5 (type IV, see about the data Figure 4).

The optically active enantiomers. Thus, chloride (2S)-DOTAP is characterized by an optical rotation, equal-2.12°, chloride (2R)-DOTAP is characterized by an optical rotation is +2.12° ([<x]Dat 20°C, 1% solution in dichloromethane).

The invention, furthermore, relates to a method of obtaining crystalline modifications of DOTAP chloride, which is characterized by the fact that the DOTAP chloride is crystallized from aprotic environment. Aprotic environment used for this purpose can be an aprotic solvents or mixtures thereof.

Aprotic medium may also contain proton solvents, such as, for example, water, in small quantities. In exceptional cases, under suitable conditions, may also be present to 25% by weight of the proton solvents. Crystallization of DOTAP chloride can be carried out according to the invention directly from the reaction solution without previous purification. Similarly crystalline DOTAP chloride can be obtained by recrystallization of amorphous, partially crystalline or crystalline material.

Suitable aprotic solvents include, in particular:

ethers, such as tetrahydrofuran, METI is tetrahydrofuran, dioxane, diethyl ether, DIPROPYLENE ether, diisopropyl ether and methyl tributyl ether;

ketones, such as acetone and 2-butanone, methylisobutylketone, methylisobutylketone,

NITRILES, such as acetonitrile, and

esters, such as ethyl formate methyl acetate, ethyl acetate, propyl, isopropylacetate, butyl acetate, isobutyl acetate, dimethylcarbonate, diethylcarbamyl and 1,3-dioxolan-2-it.

These solvents can in each case be used in pure form or in the form of a mixture, so it is possible to use a variety of aprotic solvents in the group in the form of a mixture, and the types of aprotic solvents in the form of a mixture with one another. As already mentioned above, additives proton solvents may be present in an aprotic solvent or used solvent mixtures.

Supplements proton solvents of this type can, as a rule, consist of the following solvents:

alcohols, such as methanol, ethanol, n-propanol, isopropanol, n-butanol, Isobutanol, 2-butanol, tert-butanol, 3-methyl-1-butanol and ethylene glycol, methoxyethanol ethoxyethanol, or

water.

Supplements proton solvents can in turn be additives pure solvents or mixtures of these proton solvents.

Crystallization modifications of DOTAP chloride so definitely achieved by slowly cooling the solution to temperatures lower than 30°C. Crystal formation occurs spontaneously or, or adding the corresponding crystalline modification of DOTAP chloride.

Different crystal modifications of DOTAP chloride can pass each other. Transition can be achieved by thermal processing of the selected crystalline modifications at elevated temperature or by prolonged mixing their suspensions under conditions of crystallization.

The use of amorphous or partially crystalline DOTAP chloride as a starting material for recrystallization gives the opportunity to receive, through the described process, highly crystalline DOTAP chloride with purity, which has never been achieved to date, together with stability, which has never been achieved to date.

The invention also relates to the use of crystalline DOTAP chloride for the preparation of dosage forms, as crystalline DOTAP chlorides have excellent stability in solid form under these conditions and have a constant and very good quality for an almost unlimited time.

Alternatively, enantiomerically pure DOTAP chloride have different physical properties compared to the racemate, in particular in combination with chiral compounds, such as phospholipids or cholesteric is.

New properties discovered for enantiomerically pure DOTAP chloride may advantageously be used alone or in combination with suitable phospholipids, cholesterol and its derivatives, to provide new classes of liposomes, which, compared with conventional forms, on the one hand, represent a more compact packing of the lipids and, on the other hand, have a more homogeneous structure. Thus, liposomes prepared from pure enantiomers, are characterized by 5°C higher core temperature of the transition phase in comparison with liposomes containing racemic DOTAP chloride. It is a measure of the density of the packing. Therefore, liposomes containing the enantiomers of DOTAP chloride, are also characterized by the reduction of the leakage connection is included in them.

The consequence of this is the fact that liposomes charged pharmaceutically active compounds will release the active compounds slow way in interaction with the metabolism in the animal or human body. In particular, so sensitive active compounds can be successfully delivered more specific to the desired area or organ, where it is desirable action of the medicinal product.

The enantiomers of DOTAP chloride have, especially in combination with chiral lipids, such as phospholipids, cholesteric and their derivatives, the transfection properties, which differ depending on the cell line.

For the preparation of these new liposomes specialist, skilled in this field can be specific to choose the form, or a mixture of DOTAP chlorides, secured thereby, with the purpose of preparation of liposomes with certain new properties.

Therefore, the invention also relates to pharmaceutical compositions resulting from the application forms of DOTAP chloride indicated in the claims. The pharmaceutical compositions of this type may contain crystalline modification of (2R,S)-, (2S)- and (2R)-DOTAP chloride together with other pharmaceutical active compounds and known auxiliary substances commonly used in the production of pharmaceuticals, as well as one or more solvents.

These pharmaceutical compositions can, for example, be in the form of liposomes, lipoplexes, microemulsions and nanoparticles and include, for example, the active compound from the group of peptides, nucleotides, vaccines or cytostatic agents.

This description enables the technician, qualified in this field, comprehensively implement the invention. In addition, the following examples serve to better understand and to illustrate possible embodiments of the invention. So uh the examples and in no way should be regarded as such restrict the invention.

All temperatures referred to in the following examples are in degrees Celsius. Unless otherwise noted, data on the content provided as % by weight.

Examples to illustrate the inventions

Example 1

Stability

To determine the stability of the crystalline DOTAP chlorides, substances stored together with the comparative samples at 25°C and 60% relative humidity without air. The residual content of DOTAP chloride is measured at certain intervals and indicate in comparison with the original value.

The purity and content of DOTAP chloride determined by HPLC. For type I obtained the following values:

The definition of stability can be repeated at any desired time, the values specified in Table 1, are reproducible.

Table 1
(R,S)-DOTAP chloride crystal type IThe exposure time in months
0123612
Area - %100%100.0% 100.0%100.0%100.0%100.0%
weight%98,6%97%97,9%97,2%of 98.2%98,7%

Example 2

[Chart powder diffraction]

To characterize the structural properties (crystalline modifications) crystalline DOTAP chlorides record charts powder diffraction (diffraction spectra) of these substances.

Crystalline DOTAP chlorides give spectra with a moderately sharp stripes, which have a relatively good solution for lipids. The spectra indicate a high content of crystalline components. Under polarizable microscope not observed any amorphous fraction.

Examples of the spectra shown in figure 1 (type I), 2 (type II), 3 (type III) and 4 (type IV).

For comparison, the spectrum of commercially available amorphous sample is shown in Figure 5 (amorphous).

Table 2 lists selected values of 2 theta for different crystalline modifications of racemic and enantiomerically pure DOTAP chloride.

Table 2
Selected values of 2 theta
Type I
Type II
Type III
racemic12.6, 19.5, 20.2, 21.5 and 25.2
3.3, 4.9, 19.3, 20.0 and 23.5
2.8, 5.8, 20.0, 21.2 and 25.1
Type IVenantiomerically pure12.8, 19.4, 19.8, 20.2 and 21.5

Example 3

Main junction temperature phase

Measurements of differential scanning calorimetry (DSC) performed on multilayer liposomes in water. Liposomes prepared thin-film method of calculation of quantities of racemic or enantiomerically pure DOTAP chloride. The concentration of lipid in each case is 0.1 g/ml Then the appropriate number of these dispersions is injected in resealable aluminium crucibles and measured by the calorimeter 204 Phoenix (Netzsch, Selb, Germany). In each case, spend three sequential heating/cooling -50°C to +20°C at 1°C/min

For all three variants of DOTAP chloride found junction temperature phase below 0°C. For cooling cycles it in each case is from -23°C to -24°C. the Differences between racemic and enantiomerically pure DOTAP chloride in each case is obvious in cycles of heating. (2R)-DOTAP chloride and (2S)-DOTAP both are characterized by an endothermic transition phase at-12.5°C, the as a transition phase for the racemate is 17.5°C (see 6).

Example 4

Properties transfection the cells COS-7

Racemic and enantiomerically pure DOTAP chloride and racemic DOTAP the methyl sulfate was dispersed separately with the same amount of cholesterol in the environment transfection and treated with ultrasound. Liposomes and solution GFP plasmids are mixed and incubated for 15 minutes, a total of 6 holes: 2 µg plasmid/8 µg lipid. After incubation for 5 hours, the supernatant is extracted from cells by sucking at 37°C/5% CO2add 2 ml of new medium and the mixture incubated for an additional 20 hours. After this analysis shows FACS efficient transfection for all lipoplexes. A significant difference between velocities transfection for individual lipid mixtures obvious:

Complex cholesterol/(R)-DOTAP chloride:32,4%
Complex cholesterol/(S)-DOTAP chloride:11,0%
Complex cholesterol/(R,S)-DOTAP chloride:25,9%
Complex cholesterol/(R,S)-DOTAP the methyl sulfate:20,2%

Example 5

Obtaining racemic chloride (2R,S)-DOTAP [chloride (R.S)-1,2-dioleoyl-3-propyltrimethylammonium]

[Railroad is e (R,S)-3-(dimethylamino)-1,2-propane diol and subsequent methylation with methyl chloride described in US 5264618 (Feigner and others]

Raw materials

In the synthesis used the following connections:

N,N'-carbonyldiimidazole (firm Sigma-Aldrich, lot 1252812)

Oleic acid (RCA, lot OA 11.G.01.2007, 99.1%, according to GHUR, the peak area)

(R,S)-3-(dimethylamino)-1,2-propandiol (Merck EPROVA, lot MS-103-a), or 0.11% of water, and 99.4% (GC),

1,8-diazabicyclo[5.4.0]undec-7-ene (firm Sigma-Aldrich, lot 1076841), of 99.7% (GC), methylene chloride (Linde, lot 61448),

iodide of sodium (firm Sigma-Aldrich, lot 1336385), 0,27% water,

aluminum oxide (firm Sigma-Aldrich, lot 1336643),

acetonitrile (firm ICC lot 0000426130), 100% (GC), <0,015% water,

n-heptane (company Brenntag Schweizerhall, lot 0000278245), 96,4% (GC),

2-propanol (firm Thommen Furler, lot 070920211487), of 99.96% (GC), 0.03% is water.

acetone (firm Thommen Furler, lot 080609324212), of 99.98% (GC), 0,16% water.

Synthesis of (2R,S)-DODAP [(R,S)-1,2-dioleoyl-3-dimethylaminopropane]

2,41 kg of N,N'-carbonyldiimidazole was dissolved at room temperature in 6,33 kg of dry acetonitrile. The resulting solution was heated to 25°C, and then in the solution for 60 min gave the pump 4.0 kg of oleic acid, while the temperature of the solution was maintained at a level of not higher than 35°C, varying the feed rate of oleic acid (control of emission of the gaseous carbon dioxide). After adding the reagent, the reaction mixture was stirred at 30°C for 90 min (until the completion of the allocation of carbon dioxide). Then to the mixture was added 11 g of 1,8-Diaz is bicyclo[5.4.0]undec-7-ene, with the subsequent addition of a solution of 0.83 kg of racemic (R,S)-3-(dimethylamino)-1,2-propane diol in 0,37 kg of dry acetonitrile and the mixture was stirred at 30°C for 21 hours, the resulting emulsion was cooled to 25°C. and the stirring was stopped, it was observed stratification of the mixture into two layers. The bottom layer was separated, was degirolami at 1 mbar and 25°C for 200 min and finally diluted 11.7 kg n-heptane. To the solution was added 1.5 kg of basic aluminum oxide and the suspension was stirred at 0°C for 3 hours, the Suspension was filtered and the filter residue was washed with 1.5 kg of n-heptane, pre-cooled to 0°C. the combined filtrates homogenized, it was received 15.9 kg of a solution containing 4,08 kg of pure (2-R,S)-DODAP in n-heptane (lot MW-116, the contents according to the analysis of 25.7%, output 88,9%).

The second game was synthesized in a similar manner, when it received 16.6 kg of a solution containing 3,52 kg of pure (2-R,S)-DODAP in n-heptane (lot MVA-117, the content according to the analysis of 21.2%, a yield of 88.6%).

Synthesis of chloride (2-R,S)-DOTAP

23,4 kg of a solution containing 3,52 kg of pure (2-R,S)-DODAP, in n-heptane (15.9 kg lot no MVA 116 and 7.5 kg of lot No. 117 MVA) was loaded into the reactor and drove n-heptane under reduced pressure and the temperature in the casing 60°C. after attaining a stable vacuum at 8 mbar temperature in the casing is reduced to 25°C. Then was added to 7.0 kg of 2-propanol and 3.1 kg of sodium iodide, the fact is the temperature value of the reaction mixture increased to 30°C and the nitrogen atmosphere was replaced with methyl chloride at a constant pressure of 1200 mbar. The reaction mixture was stirred under these conditions for 137 hours to complete the reaction of methylation (2-R,S)-DODAP, to receive chloride (2-R,S)-DOTAP (96%conversion). Consumption of methyl chloride was 1,39 kg of the Reaction mixture was diluted 35,8 kg of dry acetone at 25°C and slowly cooled to -12°C. the resulting suspension is kept at -12°C for 14 h, and then filtered through a pre-cooled filter (-15°C). The crude chloride (2-R,S)-DOTAP twice washed with 6.0 kg of chilled dry acetone (-18°C), then dissolved at 35°C in a closed filter in a mixture of 44.1 kg of dry acetone and 3.5 kg of 2-propanol. The resulting solution was transferred into a reactor and cooled to 0°C. Then the solution was slowly cooled to -12°C at the rate of 0.004°C per minute, i.e. for approximately 50 hours, the resulting suspension is kept at -12°C for another 16 h, and then filtered into a chilled filter-drier (-15°C). The filter residue was twice washed 0.8 kg of dry acetone (-18°C) and dried in vacuum. During drying the filter-dryer is heated to room temperature. Stage drying was completed with a stable vacuum 7,9 mbar. Output: 3.46 kg of racemic chloride (2-R,S)-DOTAP (lot # MW-118, the contents according to the analysis 100%, yield: 37.8% in the calculation of (R,S)-3-(dimethylamino)-1,2-propandiol, purity of 99.9% (IHVR, the peak area).

To obtain enantiomerically pure CHL is reed (2R)-DOTAP [chloride (R)-l,2-dioleoyl-3-propyltrimethylammonium]

Raw materials

In the synthesis used the following connections:

N,N'-carbonyldiimidazole (firm Sigma-Aldrich, lot 1252812)

Oleic acid (firm Acme Synthetic Chemicals, lot 060528, 97.8 per cent according to

IHVR)

(R)-3-(dimethylamino)-1,2-propandiol (firm Daiso, lot RMA062151), 0,11% water, 99,6% (GC),

1,8-diazabicyclo[5.4.0]undec-7-ene (firm Sigma-Aldrich, lot 1076841), of 99.7% (GC),

methylene chloride (Linde, lot 61448),

iodide of sodium (firm Sigma-Aldrich, lot 1336385), 0,27% water,

acetonitrile (firm Sigma-Aldrich, lot C, 100% (GC), 0,005% water,

n-heptane (company Brenntag Schweizerhall, lot 0000278245), 96,4% (GC),

2-propanol (firm Thommen Furler, lot 070629176434), of 99.96% (GC), 0,016% water,

acetone (firm Thommen Furler, lot 061201101946), of 99.98% (GC), of 0.10% is water.

Synthesis of (2R)-DODAP [(R)-1,2-dioleoyl-3-dimethylaminopropane]

1.63 kg N,N'-carbonyldiimidazole was dissolved at room temperature in 4.3 kg of dry acetonitrile and the resulting solution was heated to 25°C. Then the solution for 60 min gave the pump 2.7 kg of oleic acid, while the temperature of the solution was maintained at a level of not higher than 35°C, varying the feed rate of oleic acid (control of emission of the gaseous carbon dioxide). After adding the reagent, the reaction mixture was stirred at 30°C for 105 min (until the completion of the allocation of carbon dioxide). Then to the mixture was added 7.5 g of 1,8-diazabicyclo[5.4.0]undec-7-ene, followed by the addition is of astora 0,56 kg enantiomerically pure (R)-3-(dimethylamino)-1,2-propane diol in 0,26 kg of dry acetonitrile and the mixture was stirred at 30°C for 19 hours The resulting emulsion was cooled to 10°C. and the stirring was stopped, it was observed stratification of the mixture into two layers. The bottom layer was separated, was degirolami at 1 mbar and 20°C for 30 min and finally diluted 9.7 kg n-heptane. The suspension was stirred at 0°C for 1.5 h and filtered, received 12,3 kg of a solution containing 2.66 kg of pure (2R)-DODAP in n-heptane (lot MBR-001 the content according to the analysis of 21.6%, output 88,2%).

Synthesis of chloride (2R)-DOTAP

12,2 kg of a solution containing 2.66 kg of pure (2R)-DODAP in n-heptane (lot MBR-001)were loaded into the reactor and drove n-heptane under reduced pressure and the temperature in the casing 60°C. after attaining a stable vacuum of 1 mbar, the temperature in the casing was lowered to 20°C. Then was added 3,26 kg of 2-propanol followed by the addition of 1.4 kg of sodium iodide, the temperature of the reaction mixture increased to 30°C and the nitrogen atmosphere was replaced with a methylene chloride at constant pressure 1250 mbar. The reaction mixture was stirred under these conditions for 330 hours to complete the reaction of methylation (2R)-DODAP, to receive chloride (2R)-DOTAP (conversion of 97%). Consumption of methyl chloride was 0,58 kg of the Reaction mixture was diluted 16,2 kg of dry acetone at 25°C and slowly cooled to -12°C. the resulting suspension is kept at -12°C for another 1 h, and then filtered through a pre-cooled filter (-12°C). Untreated the military chloride (2R)-DOTAP twice washed 3.2 kg of chilled dry acetone (-18°C), then was dissolved at 35°C in a closed filter in a mixture of 20.5 kg of dry acetone and 1.63 kg of 2-propanol. The resulting solution was transferred into a reactor and cooled to 25°C. Then the solution was slowly cooled to -12°C at the rate of 0.05°C per minute, i.e. within approximately 12 1/3 hours, the resulting suspension is kept at -12°C for 9 h, and then filtered through a cooled filter-dryer (-12°C). The residue on the filter is washed twice 3.2 kg of chilled dry acetone (-18°C) and dried in vacuum. During drying the filter-dryer is heated to room temperature. Stage drying was completed with a stable vacuum of 0.6 mbar. Output: 1,47 kg enantiomerically pure chloride (2R)-DOTAP (lot # MBR-002, the contents according to the analysis of 99.7%, output: 44,7% for (R)-3-(dimethylamino)-1,2-propandiol, purity of 99.9% (IHVR, the peak area).

To obtain enantiomerically pure chloride (2S)-DOTAP [chloride (S)-l,2-dioleoyl-3-propyltrimethylammonium]

Chloride (2S)-DOTAP received, as described above for chloride (2R)-DOTAP, except that the starting material used enantiomerically pure (S)-3-(dimethylamino)-1,2-propandiol (firm Daiso, lot SMA062281), 0.14% of water, and 99.8% (GC), all other reagents and solvents were identical), received 1,67 kg enantiomerically pure chloride (2S)-DOTAP (lot # MBS-002, the contents according to the analysis 99,6%, output: 50,4% (based on (S)-3-(dimethylamino)-1,2-propandiol, purity 100% (IHVR, the peak area).

Example 6

Evaluation of transfection

Liposomes of racemic and enantiomerically pure DOTAP chloride and cholesterol (4:1, mol/mol) was obtained by the method of dry film and hydrating in HEPES buffer solution, extrusion and dilution to a concentration of 2 mg/ml Liposomes were added in equal volume of a solution plasmids GFP (of 1.56 µg/µl in buffer solution THOSE of 1.5 μg/well). Cell line CHO-S cells were then incubated with the mixture for 24 h (0,5×106cells/ml, 2 ml per well in 24-hole tablet with deep holes, incubation at 250 rpm and 37°C, relative humidity 90%, 5% CO2). For staining to identify the dead/live cells in suspension was added iodide of propecia. An estimate of the number of cells, transfection and viability was performed by flow cytometry.

The level of transfection (viable transfection cells) is shown below (mean±standard deviation, n=6):

Chloride (2R,S)-DOTAP/cholesterol:17,3%±5,5%
Chloride (2R)-D/cholesterol:51,3%±3,1%
Chloride (2S)-D/cholesterol:16,4%±2,2%

In this experiment, the level of transfection when using the chloride (2R)-DOTAP unexpectedly significantly higher compared with the level of transfection using (2S)-enantiomer or the racemate. These data strongly suggest that the level of transfection when using a racemate not be considered as the sum of the levels of transfection pure enantiomers, and, therefore, it is not predictable.

1. Enantiomerically pure (2S)- or (2R)-N,N,N-trimethyl-2,3-bis[[(9Z)-1-oxo-9-octadecenyl]oxy]-1-propanamine chloride (DOTAP chloride).

2. Enantiomerically pure (2S)-DOTAP chloride according to claim 1.

3. Enantiomerically pure (2R)-DOTAP chloride according to claim 1.

4. The use of enantiomerically pure (2S)- or (2R)-DOTAP chloride as a component for the production of medicines.

5. Pharmaceutical composition comprising enantiomerically pure (2S)-or (2R)-DOTAP chloride together with a pharmaceutical active compounds, excipients or solvents.

6. The pharmaceutical composition according to claim 5, containing enantiomerically pure (2S)- or (2R)-DOTAP chloride, characterized in that the pharmaceutical active compound is an active compound selected from the group of peptides, nucleotides, vaccines and cytostatic agents.

7. The pharmaceutical composition according to claim 5, containing enantiomerically pure (2S)- or (2R)-DOTAP chloride, characterized in that it has the liposomes, lipoplexes, nanoparticles or micro-emulsions.



 

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113 cl, 25 dwg, 4 tbl, 30 ex

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3 cl, 2 ex, 2 tbl, 2 dwg

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14 cl, 2 dwg, 3 tbl, 4 ex

Colloidal solution // 2449776

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3 cl, 1 ex, 3 tbl

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1 tbl, 3 ex

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1 tbl, 3 ex

FIELD: medicine, pharmaceutics.

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3 ex, 1 tbl

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46 cl, 6 ex, 2 tbl, 3 dwg

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22 cl, 41 ex, 19 tbl, 14 dwg

FIELD: medicine, pharmaceutics.

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10 cl, 51 ex

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