Esters of l-carnitine or alkanoyl l-carnitines useful as cationic lipids for the intracellular delivery of pharmacologically active compounds.

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DESCRIPTION

L-carnitine or alkanoyl L-carnitine esters useful as cationic lipids for intracellular administration of pharmacologically active compounds.

The invention described in the present document refers to a class of new esters of L-carnitine and acyl L-carnitines and their use as suitable cationic lipids to favor the intracellular administration of pharmacologically active constituents, facilitating their transmembrane transport, or to promote their interaction with specific cell membrane sites (receptors).

The invention described herein also relates to better known L-carnitine and acyl L-carnitine esters, useful for the same purposes as the aforementioned new constituents.

What is meant herein by the term "intracellular administration" is the cellular transfection with polynucleotides or plasmids of natural or modified origin, endowed with therapeutic activity (gene delivery) or the introduction of immunogenic drugs or peptides into cells.

Many of the pharmacologically active substances, such as, for example, polypeptides and proteins or drugs in general need to penetrate cells to exert their effects influencing cellular functions at the molecular or subcellular level. For these molecules, the cell membrane constitutes a selectively impermeable barrier. The cell membrane, in fact, performs a protective function, preventing the entry of potentially toxic substances, but also the passage of constituents with therapeutic activity. The complex composition of the cell membrane includes phospholipids, glycolipids, and proteins; its function is influenced by cytoplasmic components such as Ca⁺⁺ and other ions, ATP, microfilaments, microtubules, enzymes and proteins that bind Ca⁺⁺. The interaction between the cytoplasmic and structural components of cells and the response to external signals are responsible for the selectivity shown by and between the various different cell types. The barrier effect of membranes can be overcome by combining substances in complexes with lipid formulations that reproduce the composition of naturally-occurring membrane lipids. These lipids are capable of fusing with membranes and releasing substances combined with them into cells. Lipid complexes are capable not only of facilitating intracellular transfer through their fusion with membranes, but they also reduce the repulsion of charges between the membrane and the molecule that has to penetrate the cell. Amphipathic lipids, such as membrane phospholipids, form lipid vesicles or liposomes in aqueous systems.

Liposomes are vesicles in which an aqueous volume is completely enclosed by one or more membranes composed of lipid molecules, usually phospholipids. Phospholipids, consisting of a hydrophilic head and a pair of carbon chains (hydrophobic tail), are the main components of biological membranes. In aqueous solution, the hydrophobic tails self-associate to expel the water, while the hydrophilic heads interact with the medium, spontaneously forming populations of vesicles of varying diameters. Lipids are generally bipolar, neutral, or anionic. These vesicles can be used as carriers for drugs, small molecules, proteins, nucleotides, and plasmids.

In recent years, cationic liposomes, a class of positively charged vesicles made from synthetic lipids, have been widely used for the transfer of genetic material into cells. The negative charge of DNA can interact with the positive charges of cationic lipids, forming a stable DNA-liposome complex. The simplicity and versatility of this technology has made liposomes an important vehicle for delivering genes for gene therapy in human patients. Currently, most vectors used for gene therapy and approved by the NIH Recombinant Advisory Committee include synthetic and viral systems.

Viral infection involves a series of complex mechanisms to be able to attack a specific cell and transport the DNA to the nucleus. The rationale for the use of viral vectors for gene therapy is based on the possibility of replacing viral genes with genes encoding a therapeutic function, without eliminating the ability of the viral particle to infect cells. The limitations of viral therapy have to do with those viral elements that can be immunogenic, cytopathic and recombinogenic.

High hopes are pinned on the use of cationic lipids for gene therapy. These vectors have great potential compared to those of biological origin, since they are much safer, less toxic and are also capable of incorporating large genes. Compared with biological-type vectors, however, they have a low intracellular gene transcription yield. It should be noted, however, that the use of such transfection systems is in an early stage of investigation. Cationic lipids play a very important role in the formation of DNA-lipid complexes, in complex-cell interaction, in membrane fusion, in the release of DNA within the cell, and in transcription.

There are important examples of in vivo applications of cationic liposomes. The first clinical trial in gene therapy was carried out by introducing an expression vector containing the complexed HLA-B7 gene to human liposomes for the treatment of melanoma. Another important application relates to the treatment of pulmonary cystic fibrosis by means of administration through the pulmonary route or as a nasal spray of the expression vector SV-40C-FTR complexed to liposomes. Other clinical trials involving the use of liposomes in gene therapy for cancer are currently underway.

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Four constituent elements are generally identified in the structure of cationic lipids: the positively charged cationic head, the spacer, the anchor lipid, and the binding bond.

The cationic head is responsible for the interactions between cationic liposomes and DNA, between the DNA-liposome complex and the cell membrane and the other components of the cell. It consists of mono- or polycationic groups (depending on the number of charges) that can be variably substituted.

The spacer is the part of the molecule that separates the cationic head from the hydrophobic tail and is involved in ensuring optimal contact between the cationic head and the negative charges of the DNA phosphates.

The anchoring lipid is the non-polar hydrocarbon part of the molecule and determines the physical properties of the lipid double layer, such as its stiffness and its rate of exchange with the lipids of the membrane.

What is meant by "bonding bond" is the bond between the hydrocarbon chains and the rest of the molecule. This bond determines the chemical stability and biodegradability of cationic lipids.

In recent years the use of liposomes has increased steadily in the cosmetics sector. The success of liposomes in this field is due to the fact that these compounds are very well tolerated by the skin. They are used as vehicles for active components and as constituents that promote the absorption of the latter.

The patent and scientific literature is abundant in references to the preparation and use of liposomes; there are, however, very few references describing the use of carnitine derivatives useful for gene delivery, while for drug delivery there are no documents available dealing with known techniques for the preparation of constituents that remotely resemble those in accordance with the invention described herein.

Patent application EP 0 279 887 describes the use of a carnitine derivative, that is, phosphatidyl carnitine, optionally in mixtures with other phospholipids and lipids (cholesterol, phosphatidyl choline, phosphatidyl serine), for the preparation of liposomes.

In the example provided in relation to liposome preparation, phosphatidyl carnitine liposomes are produced with incorporated propanolol, a drug known to be active as an antihypertensive, anti-anginal and antiarrhythmic agent. The carnitine derivative is used here because of the pronounced myocardial tropism of carnitine. This tropism makes it possible to prevent liposomes from being metabolized by the liver, rather than reaching the desired target site.

The presence of phosphatidyl carnitine also makes it possible to administer the liposomes orally, as they are resistant to intestinal lipases.

In J. Med. Chem. 1998 Jun 18; 41 (13): 2207-2215, a series of L-carnitine esters useful for gene delivery are described, but are not described or proposed as useful agents for drug delivery.

WO 96/39193 describes novel targeted pharmacological agents that are directed to enter the mitochondria through the carnitine-acylcarnitine translocase system, but they are not suggested to be useful agents for liposome preparation.

EP 559 625 B1 describes a series of L-carnitine and acyl L-carnitine esters endowed with selective muscle relaxant activity of the gastrointestinal tract.

In recent years, molecular biologists have identified numerous defects at the chromosomal level that cause inherited diseases in human patients.

An important sector of modern medicine is involved in the treatment of these genetically based inherited diseases through the use of gene therapy protocols.

As already mentioned, cationic liposomes are widely used for the intracellular administration of pharmacologically active constituents, facilitating transmembrane transport or promoting their interaction with specific cell membrane sites (receptors).

These vectors have great potential compared to those of biological origin, since they are much safer, less toxic and are also capable of incorporating large genes. Compared with biological-type vectors, however, they have a low intracellular gene transcription yield.

Furthermore, conventional cationic lipid-mediated gene transfer requires that plasmid DNA and cationic lipids be kept separate, and that their mixing be carried out immediately prior to gene transfer.

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Attempts to stabilize these polynucleotide complexes have so far failed to give encouraging results; in fact, they remain stable for only a short period.

In the field of gene therapy or gene delivery and drug delivery, there is therefore a clearly perceived need for stable and reproducible site-specific systems that are also active after a suitable period of time.

It has now been found that a class of cationic lipids powerfully active in promoting intracellular delivery of pharmacologically active constituents comprises the novel esters of L-carnitine and acyl L-carnitines.

These new constituents are stable and highly selective because they are site-specific in reaching the target organ.

This characteristic makes them particularly useful for the transport of active constituents directly to the site where they can exert their pharmacological activity.

Those constituted according to the invention described herein are constituted with general formula (I):

<img file="ES2234591T3_D0001.tif" />

in which:

n is an integer between 1 and 3;

R is hydrogen or alkanoyl, linear or branched, with 2-6 carbon atoms;

Ri and R₂, which may be the same or different, represent a saturated or unsaturated linear acyl chain, with 3-20 carbon atoms; Y

X is the anion of a pharmacologically acceptable acid.

Examples of R are acetyl, propionyl, butyryl, valeryl, and isovaleryl.

Examples of Ri and R2 are hexanoyl, undecanoyl, myristoyl, palmitoyl, or oleoyl.

Preferred examples of constituted according to the invention are:

- L-carnitine bromide ester with 2-hydroxyacetyl-1,3-dipalmitoyl glycerol (ST 770);

- ester of acetyl L-carnitine bromide with 2-hydroxyacetyl-1,3-dipalmitoyl glycerol (ST 771);

- propionyl L-carnitine bromide ester with 2-hydroxyacetyl-1,3-dipalmitoyl glycerol (ST 772);

- isobutyryl L-carnitine bromide ester with 2-hydroxyacetyl-1,3-dipalmitoyl glycerol (ST 773);

- isovaleryl L-carnitine bromide ester with 2-hydroxyacetyl-1,3-dipalmitoyl glycerol (ST 774);

- L-carnitine bromide ester with 1,3-dihexanoyl-2-hydroxyacetyl glycerol (ST 810);

- ester of acetyl L-carnitine bromide with 1,3-dihexanoyl-2-hydroxyacetyl glycerol (ST 809);

- ester of propionyl L-carnitine bromide with 1,3-dihexanoyl-2-hydroxyacetyl glycerol (ST 808).

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What is meant by a pharmacologically acceptable anion of an acid is any anion of an acid that does not increase unwanted toxic or side effects.

These acids are well known to pharmacologists and experts in pharmaceutical technology.

Examples of these anions, although not exhaustively listed, are: chloride; bromide; I last; aspartate; acid aspartate; citrate; acid citrate; tartrate; acid tartrate; phosphate; acid phosphate; fumarate, acid fumarate; glycerophosphate; glucose phosphate; lactate; maleate; acid maleate; mucato; orotate; oxalate; acid oxalate; sulfate; acid sulfate; trichloroacetate; trifluoroacetate; methane sulfonate; pamoate and acid pamoate.

The compounds of formula (I), in the form of liposomes, are useful agents both for the administration of plasmids or nucleotides of natural origin or modified useful in gene therapy, or that encode a peptide or protein useful as a vaccine, and for the general administration of drugs, such as, for example, anticancer drugs, antiviral agents, antibacterial agents, antifungals, antiprotozoa, drugs useful for the therapy of diseases of the cardiovascular system, or immunogenic peptides and other drugs useful in therapy.

The liposomes containing the compound of formula (I) are prepared by means of conventional techniques, well known to the person of ordinary skill in the art; see for example Allen TM, Drugs 56, 747-756 (1998). Liposomes according to the present invention can also be prepared using other components well known in the practice of liposome technology. In one embodiment of the present invention, the liposomes may contain adjuvant lipids, a term that is well understood in this art. Examples of adjuvant lipids are cholesterol, 1-palmitoyl-2-oleoyl phosphatidyl choline or dioleoyl phosphatidyl choline.

The liposomes according to the present invention are suitably presented in the form of compositions. In the embodiment pertaining to the administration of pharmacologically active constituents, the compositions are understood to be pharmaceutical, optionally comprising pharmaceutically acceptable carriers and / or excipients.

The compounds of formula (I), in the form of liposomes, may also be useful in the preparation of cosmetic compositions, which comprise the liposome per se as a cosmetic active agent and for the administration of substances with cosmetic activity, such as, for example, moisturizing agents, nutrients, substances for facial cleansing, anti-wrinkle agents, anti-cellulite agents and anti-stretch marks.

The liposomes comprising those of formula (I) can be administered transdermally, subcutaneously, intramuscularly, intravenously, parenterally or orally, or in the form of nasal or buccal sprays.

The process for the preparation of the compounds of formula (I) according to the invention is represented in the following reaction diagram, it is intended that this diagram applies to the entire general formula (I). The expert will be able to easily obtain all the groups represented by R, Ri and R₂, since all the necessary reagents are commercially available or described in the literature and the reaction conditions are generally applicable to the entire scope of the present invention, any modification being normally carried out if necessary within the common general knowledge.

CH.OK „2 CICO- &lt;CH.)„ CH,

CH.OH αι, χοιαίά.αι,

IT NaflH,

CO

CH.OCO (CH.) „CH, αςοσχοΗ.), ^,

CH-OH * ClOCCHJr

CH.OCO (CH.) „Clt,

CH.OCOfCH_s) ,. CH, | Π) í)

CO

I

CH.OCO (CH.) „CH,

CK.oco (CH.&gt; „Ca,

CH-OH

I

CH.0C0 (CH.),₄CH,

CK.OCO &lt;CH.)_ORCH,

CH-OCOCILBr

CK_OCO &lt;CH.) ,. CH, (2) 9)

ES 2 234 591 T3

<img file="ES2234591T3_D0002.tif" />

With reference to reaction diagram 1 above, the preparation of the compounds of formula (I) according to the invention is illustrated here below.

Example 1

Preparation of propionyl L-carnitine bromide ester with 2-hydroxyacetyl-1,3-dipalmitoyl glycerol (ST 772)

a) Preparation of 1,3-dihydroxypropan-2-one 1,3-dipalmitate (1)

Dihydroxyacetone (7 g, 0.078 mol) was dissolved in 300 ml of anhydrous chloroform at 0 ° C (external temperature) under anhydrous nitrogen flow.

Palmitoyl chloride (44 g, 0.16 mol) and anhydrous pyridine (15 ml) were added dropwise to the solution thus obtained.

The resulting mixture, the temperature of which was raised to room temperature, was kept under stirring for 24 hours.

The mixture was then extracted in the following order: with 300 ml of a 0.5% aqueous hydrochloric acid solution, 300 ml of a 5% aqueous sodium bicarbonate solution and finally with 300 ml of water.

The separated organic phase was dehydrated over anhydrous sodium sulfate, filtered on a cellulose filter and concentrated to dryness, obtaining the crude product (1).

The pure product (1) was obtained by crystallization from 500 ml of ethyl alcohol.

30.4 g of product (1) were obtained.

Yield: 73%

Mp = 80-81 ° C

NMR ^!H (CDCl₃): 0.9 (6H, t, CH₃CH₂-); 1.3 (48H, m, (CH₂)_n_₂₄); -OCOCH2-); 4.7 (4H, s, -OCCH₂-).

1.55 (4H, m, -OCOCH₂CH₂-); 2.4 (4H, t,

b) Preparation of 1,2,3-trihydroxypropane 1,3-dipalmitate (2)

Water (7.5 ml) was added to product (1) (5 g, 9 mmol) slowly, and it was dissolved in tetrahydrofuran (125 ml) and toluene (25 ml) with stirring.

The temperature of the milky white suspension obtained was brought to 5 ° C (external temperature) and sodium borohydride (500 mg; 13 mmol) was added in small fractions. The suspension was kept under stirring for 30 minutes at 5 ° C.

Glacial acetic acid was then slowly added until effervescence occurred due to decomposition of excess sodium borohydride, finally obtaining a solution.

Chloroform (100 ml) was added to the solution, obtaining the formation of a biphasic system.

The lower organic phase consisting of CHCl₃ it was separated, being extracted in the following order: with water (25 ml), sodium bicarbonate (25 ml of 10% aqueous solution) and water (25 ml).

The organic solution containing (2) was dehydrated over sodium sulfate, filtered and concentrated to dryness, obtaining a waxy product.

Product (2) was obtained by crystallization from acetone of the waxy crude product.

4.8 g of product (2) were obtained.

Yield: 94%.

ES 2 234 591 T3

Mp = 71-72 ° C

NMR ¹H (CDCl₃): 0.9 (6H, t, CH₃CH₂-); 1.3 (48H, m, (CH₂)_n_₂₄); -OCOCH₂-); 4.2 (5H, m, -CHCH₂OR-).

1.55 (4H, m, -OCOCH₂CH₂-); 2.4 (4H, t,

c) Preparation of 1,3-dipalmitoyl-2-bromoacetyl glycerol (3)

The product (2) (2.5 g, 4.4 mmol) was solubilized in anhydrous chloroform (50 ml) under stirring and at 0 ° C (external temperature).

Pyridine (0.42 ml) and 3 ml of a chloroform solution containing bromoacetyl chloride (0.43 ml, 5.2 mmol) were added dropwise to the solution thus obtained.

The reaction mixture was kept for 30 minutes at 0 ° C (external temperature) and for 30 minutes at room temperature.

The reaction mixture was then treated in the following order with: a 1% aqueous hydrochloric acid solution (approximately 50 ml), a 5% aqueous sodium bicarbonate solution (approximately 50 ml) and water.

The product (3) was purified by crystallization from acetone, after dehydrating the reaction mixture (with sodium sulfate) and concentrating to dryness.

2.5 g of product (3) were obtained.

Yield: 89%.

Mp = 46-47 ° C

NMR ¹H (CDCl₃): 0.9 (6H, t, CH₃CH₂-); 1.3 (48H, m, (CH2V24); 1.55 (4H, m, -OCOCH₂.CH₂-); 2.4 (4H, t, -OCOCH₂-); 3.9 (2H, s, -OCH₂COO-); 4.2-4.4 (5H, m, -CHCH₂OR-); 5.25 (1H, m, -CHCH₂OR-).

d) Preparation of L-propionyl carnitine bromide ester with 2-hydroxyacetyl-1,3-dipalmitoylglycerol (4)

The internal L-propionyl carnitine salt (0.95 g, 4.4 mmol) previously dried under vacuum at 40 ° C was suspended in anhydrous dimethylformamide (approximately 20 ml).

Product (3) (3 g, 4.7 mmol) was added to the suspension in small portions. The suspension was slowly heated to 38 ° C and kept under these conditions until a solution was obtained.

After 10 minutes the solution was brought to 0 ° C for 30 minutes. A precipitate was obtained, which was filtered and washed with ethyl ether and dissolved in chloroform (100 ml). The opalescent solution obtained (30 ml) was filtered over Celite and concentrated. To this last solution hexane (100 ml) was added, and the precipitate of the product (4) obtained was filtered and dried under vacuum at 35 ° C.

3.19 g of the title compound were obtained.

Yield: 80%.

Mp = 127-128 ° C

[to]²⁵D = -3.9 (C = 1% chloroform)

Elemental analysis of C₄₇H₈₈BrNOi₀

<td></td><td>C%</td><td>H%</td><td>N%</td><td>Br%</td>

<td>Calculated</td><td> 62,23</td><td> 9,78</td><td> 1,54</td><td> 8,81</td>

<td>Found</td><td> 62,73</td><td> 10,15</td><td> 0,79</td><td> 8,77</td>

NMR ¹H (CDCl₃): 0.9-0.95 (6H, t, CH₃CH₂CH₂-); 1.1-1.2 (3H, t, CH₃CH₂CO); 1.2-1.4 (24H, m, (CH₂)_n_₂₄); 1.51.6 (4H, m, -OCCH₂CH₂-); 2.3-2.4 (4H, t, -OCCH₂CH₂-); 2.4-2.45 (4H, dd, -CHCH₂OR-); 2.95 (2H, d, -CH₂COOCH₂COO-); 3.5 (9H, s, N (CH₃)₃); 4.2 (4H, m, -CH₂OCOCH₂-); 4.35 (2H, m, -CH₂N-); 4.65 (2H, dd, -OCH₂CO-); 5.25 (1H, m, -OCH₂CHCH₂OR-); 5.75 (1H, m, -CHCH₂N-).

Examples 2-7

The following constituents were prepared in the same way as in the preceding example:

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- L-carnitine bromide ester with 2-hydroxyacetyl-1,3-dipalmitoyl glycerol (ST 770);

- ester of acetyl L-carnitine bromide with 2-hydroxyacetyl-1,3-dipalmitoyl glycerol (ST 771);

- isobutyryl L-carnitine bromide ester with 2-hydroxyacetyl-1,3-dipalmitoyl glycerol (ST 773);

- isovaleryl L-carnitine bromide ester with 2-hydroxyacetyl-1,3-dipalmitoyl glycerol (ST 774);

- L-carnitine bromide ester with 1,3-dihexanoyl-2-hydroxyacetyl glycerol (ST 810);

- ester of acetyl L-carnitine bromide with 1,3-dihexanoyl-2-hydroxyacetyl glycerol (ST 809);

- ester of propionyl L-carnitine bromide with 1,3-dihexanoyl-2-hydroxyacetyl glycerol (ST 808).

A preferred embodiment of the invention described herein consists in the preparation of liposomes with anticancer drugs, and particularly liposomes that act as vehicles for camptothecins, for example those described in WO 97/31003. In a more preferred embodiment, the invention described herein provides liposomes for the administration of camptothecins of general formula (IV):

<img file="ES2234591T3_D0003.tif" />

where: R₇ is a group -C (R₁₁) = NO_(n)R₁₀, in which R₁₀ is hydrogen or a C alkyl group₁_C₅ or alkenyl C₁_C₅, straight or branched, or a C cycloalkyl group₃_C₁₀, or a cycloalkyl group (C₃_C₁₀) alkyl (C₁_C₅) linear or branched, or a C aryl₆ C |₄, or an aryl group (C₆_C₁₄) alkyl (C₁_C₅) straight or branched, or a heterocycle or a heterocycloalkyl group (C₁_C₅) linear or branched, said heterocycle group containing at least one heteroatom selected from nitrogen atoms, optionally substituted by an alkyl group (C₁_C₅), and / or oxygen and / or sulfur; said alkyl, alkenyl, cycloalkyl, aryl, aryl-alkyl, heterocycle and heterocycle-alkyl groups being optionally substituted with other groups selected from: halogen, hydroxyl, C-alkyl₁_C₅, C alkoxy₁_C₅, phenyl, cyano, nitro, -NR₁₂R₁₃, in which R₁₂ and R₁₃, which may be the same or different, are hydrogen, alkyl (C₁_C₅) linear or branched, the group -COOH or one of its pharmaceutically acceptable esters; or the -CONR group₁₄R₁₅, in which R₁₄ and R₁₅, which may be the same or different, are hydrogen, alkyl (C₁_C₅) linear or branched; or

R₁₀ is an aroyl residue C₆-C₁₀, optionally substituted by one or more groups selected from: halogen, hydroxyl, C alkyl₁_C₅ linear or branched, C alkoxy₁_C₅ linear or branched, phenyl, cyano, nitro, -NR₁₆R₁₇, in which R₁₆ and R₁₇, which may be the same or different, are hydrogen, C-alkyl₁_C₅ linear or branched;

R10 is a polyaminoalkyl residue; or

R10 is a glycosyl residue;

n is the number 0 or 1;

R₁₁ is hydrogen, C alkyl₁_C₅ linear or branched, C alkenyl₁_C₅ linear or branched, cycloalkyl C₃-C₁₀, cycloalkyl (C₃-C₁₀) alkyl (C₁_C₅) linear or branched, aryl C₆-C₁₄, aril (C₆-C₁₄) alkyl (C₁_C₅) linear or branched;

R₈ and R₉, which may be the same or different, are hydrogen, hydroxyl, C alkoxy₁_C₅ linear or branched;

their N₁-oxides, single isomers, particularly syn and anti isomers of the -C group (R₁₁) = NO_(n)R₁₀, their possible enantiomers, diastereoisomers and related mixtures, their pharmaceutically acceptable salts and their active metabolites.

ES 2 234 591 T3

The constituents of formula (IV) are described in European patent application No. 99830124.6, filed on March 9, 1999.

With regard to the compounds of formula (IV) in which n is 1 and R₁₀ is as defined above, with the exception of aroyl, these constituents can be prepared from 7-aldehyde camptothecin (formula IVa, R₁₁hydrogen) or 7-keto camptothecin (formula IVa, R₁₁ other than hydrogen).

<img file="ES2234591T3_D0004.tif" />

in which R₇ is the group -C (R₁₁) = O, and R₁₁ is as defined in formula (IV), R₈ and R₉ it is as defined in formula (IV). The compound of formula (IVa) is reacted with the compound of formula (Va) R₁₀O-NH₂, in which R₁₀is as above, to give constituents of formula (I), in which R₇ is the group -C (R₁₁) = NOR₁₀, R₁₀ it is defined as in formula (IV), except for aroyl.

The reaction can be carried out with conventional procedures known to those skilled in the art, the procedure consists of the normal formation of oximes. Preferably, the molar ratio of 7-aldehyde or 7-keto camptothecin to hydroxylamine should be in the range of 1: 3 to 3: 1. The relevant hydroxylamine salts can also be used. The reaction is carried out in the presence of a base, for example, an inorganic base such as potassium carbonate, or an organic base, such as triethylamine or diazabicyclononane, using polar solvents, preferably methanol or ethanol, and carrying out the reaction at a temperature range between room temperature and the boiling point temperature of the solvent, optionally in the presence of dehydrating agents, for example sodium or magnesium sulfate, molecular sieves. If necessary, the reaction can also be carried out in the presence of a catalyst, for example a Lewis acid.

Alternatively, the aforementioned constituents can be prepared from the 7-aldehyde camptothecin oxime (obtained as described in Sawada et al., Chem. Phar. Bull. 39, 2574 (1991)), or the 7-ketone or from of the corresponding 7-acylcamptothecin by reaction with a halide R₁₀-X, where X is preferably iodine, in a polar solvent, for example tetrahydrofuran or alcohols, and in the presence of a base, for example sodium hydride or potassium carbonate.

As regards the constituents of formula (IV) in which n is 1 and R10 is aroyl, as defined in formula (IV), these constituents can be prepared from 7-oxime camptothecin, the preparation of which is described in the previous paragraph, with acyl chlorides R10-COCl, in polar solvents, and in the presence of a base, preferably pyridine, or directly in pyridine, as described by Cho et al., J. Org. Chem. 62, 2230 (1997).

Regarding those constituted of formula (IV) in which n is 0 and R₁₀ is as defined above, with the exception of aroyl, the constituents can be prepared from 7-aldehyde camptothecin (formula IVa, R11 hydrogen) or 7-keto camptothecin (formula IVa, R₁₁ other than hydrogen).

<img file="ES2234591T3_D0005.tif" />

ES 2 234 591 T3 in which R₇ is the group -C (Rn) = O, and Rn is as defined in formula (IV), R₈ and R₉ are as defined in formula (IV). The compound of formula (IVa) is reacted with the compound of formula (Vb) Ri₀-NH₂, in which R₁₀ is as defined above, to give constituents of formula (IV), in which R₇ is the group -C (Rn) = NOR₁₀, R_1st it is defined as in formula (IV), except for aroyl. The reaction can be carried out with conventional procedures known to experts in pharmaceutical technology, the procedure consists of the normal formation of imines. Preferably, the molar ratio of 7-aldehyde or 7-keto camptothecin to imine should be in the range of 1: 3 to 3: 1. The relevant amine salts can also be used. The reaction is carried out in the presence of a base, for example, an inorganic base such as potassium carbonate, or an organic base, such as triethylamine or diazabicyclononane, using polar solvents, preferably methanol or ethanol, and carrying out the reaction at a temperature range between room temperature and the boiling point temperature of the solvent, optionally in the presence of dehydrating agents, for example sodium or magnesium sulfate, molecular sieves. If necessary, the reaction can also be carried out in the presence of a catalyst, for example a Lewis acid as described, for example, by Moretti and Torre, Synthesis, 1970, 141; or by Kobayashi et al., Synlett, 1977, 115).

7-aldehyde camptothecin and 7-oxime camptothecin are described in European patent application EP 0056692 and in the aforementioned article by Sawada et al., Chem. Pharm. Bull. 39, 2574 (1991).

The N1 -oxides of the compounds of formula (IV) are prepared according to known heteroaromatic nitrogen oxidation procedures, preferably by oxidation with acetic or trifluoroacetic acid and hydrogen peroxide, or by reaction with organic peroxyacids (A. Albini and S Pietra, Heterocyclic N-oxides, CRC, 1991).

Regarding the variable importance of R10, present in the different reagents of formula V, these reagents are commercially available, or they can be prepared according to procedures common to those in the literature, to which a person skilled in the art can resort. sector, as a supplement to your own knowledge of the subject.

The pharmaceutically acceptable salts are obtained with conventional procedures described in the literature, and that do not need a further description.

Example 8

7-benzyloxyiminomethylcamptothecin (CPT172)

500 mg (1.33 mmol) of 7-formylcamptothecin was dissolved in 100 ml of ethanol. 15 ml of pyridine and 638 mg (4 mmol) of O-benzylhydroxylamine hydrochloride were added. The solution was refluxed for 5 hours. The solvent was evaporated in vacuo and the residue thus obtained was purified by flash chromatography on silica gel using a 4: 6 mixture of hexane / ethyl acetate as eluent.

Yield: 65%

Mp = 200-205 ° C dec.

The product obtained consists of an approximately 8: 2 mixture of the two syn and anti isomers (isomer A: Rf 0.32; isomer B, Rf 0.19, on Merck 60 F254 silica gel; eluent: hexane: ethyl acetate 3: 7).

HPLC: the analyzes were carried out in an apparatus equipped with a quaternary pump (HP 1050) with a Rheodyne injector (20 µ / l loop) and a diode-array detector (HP 1050) run by the HPLC ChemStation program. The spectrum was obtained between 200 and 600 nm and the chromatograms were taken at 360 and 400 nm.

A C18 reversed phase column (Rainin C18, 25 x 0.4 cm, Varian) was used with an RP18 pre-column. The analysis was carried out with a linear elution gradient, starting with 30:70 acetonitrile: water to 100% acetonitrile in 20 minutes, with a flow rate of 1 ml / min. The retention times were: 12.51 min for isomer B and 14.48 min for isomer A.

NMR ¹H (300 MHz; DMSO-d₆): or 0.88 (t, H₃-18A + H₃-18B), 1.87 8m, (H₂-19A + H₂-19B), 5.18 (s, H₂-5B), 5.21 (8s, H2-Ph B), 5.30 (R-Ph A), 5.40 (s, R-5A), 5.45 (s, R-17A + R- 17B ), 6.53 (s, -OH A + -OH B), 7.3-7.6 (m, Ar A + Ar B + H-14A + H-14B), 7.75 (m, H-11A + H-11B), 7.85-7.95 (m, H-10A + H-10B), 7.98 (dd, H-12B), 8.18-8.27 (m, H-12A + H -9B), 8.45 (s, CH = NB), 8.59 (dd, H-9A), 9.38 (s, CH = NA).

Masses m / z 481 (M + 100) 374 (30) 330 (70) 300 (30) 273 (20) 243 (20) 91 (34).

Example 9

7-butoxyiminomethylcamptothecin (CPT 184)

400 mg (1.06 mmol) of 7-formylcamptothecin was dissolved in 80 ml of ethanol. 12 ml of pyridine and 400 mg (3.18 mmol) of Ot-butylhydroxylamine hydrochloride were added. The solution was refluxed for 4 hours. The

ES 2 234 591 T3 solvent was evaporated in vacuo and the residue thus obtained was purified by flash chromatography on silica gel using a 4: 6 mixture of hexane / ethyl acetate as eluent.

322 mg (0.72 mmol) of yellow solid were obtained.

Yield: 68%

Mp = 250 ° C dec.

The product obtained consists of an approximately 8: 2 mixture of the two syn and anti isomers (isomer A: Rf 0.31; isomer B, Rf 0.24, on Merck 60 F254 silica gel; eluent: hexane: ethyl acetate 3: 7).

HPLC: the analyzes were carried out in an apparatus equipped with a quaternary pump (HP 1050) with a Rheodyne injector (20 µl loop) and a diode-array detector (HP 1050) run by the HPLC ChemStation program. The spectrum was obtained between 200 and 600 nm and the chromatograms were taken at 360 and 400 nm.

A C18 reversed phase column (Rainin C18, 25 x 0.4 cm, Varian) was used with an RP18 pre-column. The analysis was carried out with a linear elution gradient, starting with 30:70 acetonitrile: water to 100% acetonitrile in 20 minutes, with a flow rate of 1 ml / min. The retention times were: 12.92 min for isomer B and 14.61 min for isomer A.

NMR ^!H (300 MHz; DMSO-d₆): or 0.88 (t, H₃-18A + H₃-18B), 1.30 (s, t-but.B), 1.47 (s, t-but.A), 1.87 (m, H₂19A + H₂-19B), 5.18 (s, H2-5B), 5.37 (H2-5A), 5.42 (s, H2-17A + H2-17B), 6.54 (s, -OH A + -OH B), 7.35 (s, H-14A), 7.36 (s, H-14A), 7.69-7.83 (m, H-11A + H-11B), 7.85-7, 98 (m, H-10A + H-10B), 8.07 (dd, H-9B), 8.16-8.27 (m, H9A + H-12B), 8.40 (s, CH B) , 8.62 (dd, H-12A), 9.31 (s, CH A).

Masses m / z 448 (M + 28) 391 (40) 374 (100) 362 (40) 330 (34) 57 (17).

Preparation of liposomes

Those constituted according to the invention can be used to prepare multilamellar liposomes (MLV) and unilamellar liposomes (SUV), both in the form of dry powders and in the form of suspensions in aqueous solutions.

Those constituted according to the invention, prepared as described in Examples 1-7, are used to prepare the liposomes according to the following procedure. A suitable amount of the constituent is dissolved in chloroform; the solution is concentrated in vacuo to dryness on a rotary evaporator until a lipid film is obtained. The lipid film is dried under high vacuum until the last remaining traces of solvent have been removed and then dissolved in tert-butyl alcohol or with water. The solution thus obtained is lyophilized, obtaining a soft and dry powder.

The powders are hydrated with a suitable amount of aqueous solution, obtaining the liposome of the used constituent, which is then complexed with the polynucleotide or with the desired drug.

Another method of preparing liposomes consists in the adsorption of a lipid film, which consists of a compound according to the invention in a solvent, or in a suitable inert support such as sorbitol, mannitol or other pharmacologically acceptable carbohydrates. The mixture is dried under vacuum, obtaining a solid that can be hydrated easily and very quickly before use.

Preparations in the form of dry powders have the advantage of being stable for long periods, and are easy to use.

Furthermore, those constituted according to the invention can be used to prepare liposomes complexed with DNA or with the desired drug, in the form of dry powders, according to the following procedure. The compound according to the invention is dissolved in tert-butyl alcohol or with water; The solution thus obtained is mixed with the DNA or the desired drug and the mixture is lyophilized, obtaining the complex that we can define as proliposome-DNA or proliposome-drug, in the form of a soft and dry powder.

The powders thus obtained (proliposomes) can be used for the preparation of pharmaceutical compositions that can be administered by aerosols, or which, alternatively, when reconstituted with water, or with a suitable buffer solution, can be administered parenterally or orally.

Liposomes complexed with DNA or with drugs, in solid form, can also be obtained with the adsorption process of the lipid film on an inert support such as sorbitol, mannitol or other carbohydrates by means of the procedure described above.

ES 2 234 591 T3

Liposome formation test

Liposome formation was tested by a colorimetric procedure, using a water-soluble dye, according to the following procedure. An aqueous solution of the water soluble dye Arsenazo III (Mw = 776.37; 2.3 mg / ml) was obtained.

This solution was used to hydrate the lipid films from the above-mentioned preparation instead of the water.

An aliquot of the suspension containing the liposome encapsulating the dye was diluted 100 times with water.

2 ml of the liposome suspension was used to obtain the first optical density reading at 660 nm; the reading was obtained relative to an equal mixture defined as blank. 200 µl of a CaCl solution were added₂ (15 mg / ml; 100 mM) to the first sample, and the optical density was measured at 660 nm against the blank, to which 200 µl of water was added. The absorbance value obtained was indicated as reading 2. We continue adding 100 µl of a Triton X-100 solution (5% v / v; final concentration of 0.26%) to the sample and 200 µl of water to the blank; the optical density read at 660 nm gave the value of the optical density defined as reading 3. To calculate the percentage of encapsulated dye, the following formula was used:

% Encapsulated Dye = [Read 3 - Read 2] x 100 / Read 3

The percentage of encapsulated dye provides a measure of liposome formation and is on average approximately 40%: the measurement of liposome size was performed using Laser Light Scattering with a positive result.

Examples of liposome preparation

Example 8

Preparation of taxol-liposomes SUVST 772 (1:70)

20 mg (0.0234 mmol) of taxol and 1485 mg (1.638 mmol) of ST 772 were dissolved in 20 ml of chloroform.

The solution was concentrated until a lipid film was obtained on the surface of the glass flask.

After removal of the last traces of chloroform with a high vacuum pump, 20 ml of tert-butyl alcohol was added to the lipid film. To obtain a clear solution, it had to be heated to 60 ° C. The solution was immediately frozen at -70 ° C with liquid nitrogen and lyophilized for 24 hours.

To obtain the final SUV liposome suspension, the lyophilized product, hydrated with a PBS solution (20 ml), was sonicated for 20 minutes at 0 ° C.

Filtration was then carried out on a 400 nm filter to remove traces of titanium released by the sonicator probe.

Test of physical stability of the preparation

The physical stability of the preparation was tested by means of turbidimetry with the recording of a TDC (Time Drive Curve) at 800 nm, at 20 ° C, for 6 hours.

A constant turbidity trend was recorded, indicative of the stability of the preparation, without precipitation phenomena.

MLV liposomes were obtained.

Gene administration

Preparation of the liposome-DNA complex

The liposome and plasmid DNA were separately adequately diluted in PBS. The DNA was then added to the liposome and the liposome-DNA complex was left for approximately 30 minutes at 4 ° C to facilitate the formation of a stable liposome-DNA interaction.

In in vitro experiments, 1,2-dioleoyloxy-3-trimethylammonium propane (DOTAP) was used as the reference cationic lipid; 2.5 pg of plasmid DNA were used per 2 x 10⁵ HeLa cells, the liposome concentration was 9 juM.

ES 2 234 591 T3

In in vivo experiments, both DOTAP and [2,3- (dioleoyl) propyl] trimethylammonium (DOTMA) were used as reference cationic lipids.

The molar ratios defined in the results refer to nmol concentrations of the respective cationic lipids per mg of DNA.

In in vivo transfection experiments, 25 pg of plasmid DNA was used per animal.

The plasmid pCMVluc used in these experiments contained the cDNA of the luciferase gene under the transcriptional control of the cytomegalovirus (CMV) promoter.

Quantitative determination of luciferase activity

Luciferase protein activity was determined in cells and tissues using the Boehringer Mannheim kit (cat No. 1669 893).

Cells were washed three times in PBS and then removed from the plate with a scraper in lysis buffer (100 mM potassium phosphate, pH 7.8; dithiothreitol-1 mM DTT) and subjected to three consecutive freeze-thaw cycles. . After centrifugation in 1.5 ml Eppendorf tubes, the supernatant was used for the luminescence test no more than 5 hours after extraction of the proteins. Luminescence emission measurements were made using a luminometer at 562 nm. After first freezing in liquid N2 followed by fine grinding to a powder, the tissues were resuspended in lysis buffer and incubated for 10-15 min on ice.

The samples were then centrifuged in 2 ml Eppendorf tubes and the supernatant tested for luciferase activity.

Two-dimensional analysis

Cellular DNA was extracted according to the alkaline lysis procedure described by Sanbrook, Fritsch and Maniatis in Molecular Cloning, 1989.

pg of DNA extracted from cells was pre-absorbed on nylon filters (Boehringer) using Biorad's two-dimensional apparatus. The filters were then prehybridized for 4 hours at 65 ° C with a solution containing 0.5 M sodium phosphate (NaPi), 1 mM EDTA, 7% SDS. The probe marked with³²P (alpha) was prepared using the DNA plasmid pCMVluc as a template and the Amersham kit randomly primed. The filter was hybridized in the same prehybridization buffer for 12 hours at 42 ° C using 1 x 10⁶ CPM / ml. The filter was then subjected to 3 washes of 10 minutes at 65 ° C in buffer containing 40 mM NaPi, 1% SDS. Autoradiographic analysis was carried out with the aid of a phosphor camera using ^ -activation activated phosphor screens that are read and quantified by means of a photomultiplier system in conjunction with an image analysis program. The two-dimensional densitometry was done using an IP-LabGel image analysis program.

DNA plasmid transfection tests

A series of DNA plasmid transfection tests were carried out both in vitro and in vivo.

Either DOTAP and DOTMA were used as reference cationic lipids, the transfectability of which has been extensively characterized and described by Abkenet et al., Proc. Natl. Acad. Sci., USA 1993, 90, 6518. In in vivo experiments, several different molar ratios of cationic lipids to DNA plasmids were analyzed in order to determine the activity of cationic lipids and the respective concentrations most effective for gene transfection. The transfection capacity of the various liposomes was evaluated both in vitro and in vivo using the luciferase gene transporter contained in the plasmid pCMVluc, whose activity, in terms of relative luminescence units (RLU) (previously described) contributes to a easy quantification.

Alternatively, the transfection efficiency of a series of cationic liposomes was evaluated by means of densitometric analysis (phosphor chamber) of DNA samples extracted from transfected cells pre-absorbed on nitrocellulose filters (two-dimensional) and hybridized with plasmid DNA markers. ³²P marked, as previously described.

In vivo transfection tests

Dependence of in vivo transfection efficiency on the ST 983 liposome: DNA molar ratio

In this experiment, the dependence of the efficiency of liver, lung and heart transfection on the ST 983 liposome: DNA molar ratio was evaluated. The following liposome nm ratios per pg DNA were tested: 12: 1, 24: 1, 36: 1 and 48: 1.

ES 2 234 591 T3

Groups of 6 Balb / c mice weighing approximately 20 g were treated intravenously with the aforementioned amounts of liposome-DNA complex in 200 µg volumes of PBS and sacrificed 24 hours after complex administration.

Luciferase activity extracted from lung, heart and liver tissue revealed a predominantly pulmonary luciferase distribution at all molar ratios analyzed. In fact, approximately 99% of the total luciferase extracted from the three tissues was located in the lungs. The liposome: DNA molar ratio of 12: 1 proved to be the best.

Transfection of DNA plasmids into HeLa cells with ST 772 liposomes

Figures 1 and 2 show the efficiency of transfection of the ST 772 liposome DNA plasmid into HeLa cells. For this purpose, densitometric analysis of DNA extracted from cells transfected with ST 272 and with DOTAP as reference cationic lipid was carried out. The results of the blot analysis reveal amounts of plasmid DNA of the same order of magnitude as those obtained with DOTAP.