ES2351909B1 - DEMONDS BASED ON DERIVATIZED PAMAM WITH SULFONILE RENTAL GROUPS. - Google Patents

Abstract

Dendrimers based on PAMAM derivatized with alkyl sulfonyl groups. # The present invention describes compounds derived from vinyl sulfones comprising a PAMAM core derivatized with alkylsulfonyl groups and the process for obtaining them. These compounds form complexes with DNA fragments that are useful for cell transfection, whereby the present invention also describes the use of these compounds as transfection agents.

Description

Dendrimers based on PAMAM derivatized with alkyl sulfonyl groups.

The present invention describes compounds derived from vinyl sulfones comprising a PAMAM core derivatized with alkylsulfonyl groups, as well as the process for obtaining these compounds and their use as transfection agents.

Prior art

The supposed capacity of gene therapy for the treatment of genetic diseases has led to a great research activity on the design of vectors for the administration of genes that are capable of compacting and protecting oligonucleotides such as DNA against degradation by DNases present in the serum. Non-viral vectors constitute an attractive alternative to the use of viruses, retroviruses and adenoviruses, since they solve problems of viral systems such as toxicity, immunogenicity and the problems of obtaining large-scale viral systems. However, virus-based systems have a high efficiency of transfection. Although non-viral systems per se have a lower transfection efficiency (due to the intra and extracellular barriers they encounter for cellular uptake), these non-viral vectors can be easily modified in their structure in a systematic way to allow the analysis of those factors involved in modulating their efficiency.

The design of cationic compounds, such as lipids, polymers, dendrimers and peptides that interact strongly and not covalently with the negatively charged skeleton of DNA, has been demonstrated as a useful strategy for the development of efficient synthetic transfection agents. Thus, highly branched dendrimers have demonstrated high potential as vectors for gene administration compared to classical polymer systems, due to their unique properties: a functionalizable multivalent surface, a well-defined three-dimensional architecture, as well as suitable molecular sizes and weights (O. Rolland, CO Turrin, AM Caminade and JP Majoral, New J. Chem., 2009, 33, 1809). Specifically, poly (amidoamine) based dendrimers (PAMAM) containing easily protonable amino terminal groups and internal tertiary amino groups are a frequent laboratory tool due to their simple chemical synthesis and availability (P. P. Kundu and

V. Sharma, Curr. Opin. Solid State Mater. Sci., 2008, 12, 89 M. Labieniec and C. Watala, Cent. Eur. J. Biol., 2009, 4, 434). The PAMAM-DNA complexes, called dendriplexes and constituted by the interaction between primary amino groups (D. Shcharbin, E. Pedziwiatr, J. Blasiak and M. Bryszewska, J. Control. Release, 2010, 141, 110), constitute supramolecular systems positively charged that have the ability to interact with the negatively charged cellular plasma membrane, allowing entry into the cell through an endosomal system, without producing significant cytotoxicity or immunogenicity. Once internalized, the buffer capacity of the non-protonated amino groups will allow them to act as a proton sponge in the acidic environment of the endosomes, thus facilitating the release of DNA from the dendiplex to the cell cytoplasm thanks to the inhibition of pH-dependent endosomal nucleases.

The influence of the dendrimer's generation on the efficiency of transfection (also called dendritic effect) was long identified and thus, medium-sized dendrimers (G5-G10) are considered the most efficient. On the other hand, three strategies have been developed to increase the transfection performance of PAMAMs: a) increase the flexibility of the branches, b) transform the dendrimer into ambifilic and c) make it biocompatible. The thermal degradation of high-generation PAMAMs results in "fractured" vectors with high flexibility and that are commercial (Superfect ™). In addition, surface modification through functionalization of the periphery of PAMAM has been extensively investigated to try to improve not only the formation of complexes with DNA, interaction with the plasma membrane and release of endosomes, but also to reduce cytotoxicity. and try to incorporate functional groups that produce the direction of the dendriplexes to a specific cell type (K. Jain, P. Kesharwani, U. Gupta and NK Jain, Int. J. Pharm., 2010, 394, 122). The conjugation of the primary amino groups with polyethylene glycol, amino acids, peptides, proteins, sugars, cyclodextrins, lipids and glucocorticoids has been described and has had variable results regarding the efficiency of transfection. Numerous works have demonstrated the advantages of incorporating lipophilic motifs to dendrimers to transform them into ambilic molecules capable of producing an increased transfection. Thus, lipid-modified PAMAMs have been constructed with topologies in which the hydrophilic dendritic portion was located in a part of the molecule while the hydrophobic portion, primarily alkyl chains of varying length, were located in the opposite part of the molecule (T Takahashi, C. Kojima, A. Harada and K. Kono, Bioconjug. Chem., 2007, 18, 1349). Alternatively, PAMAM dendrimers with a hydrophilic interior and a hydrophobic crown have been obtained by randomly joining alkyl tails at the periphery (J. L. Santos, H. Oliveira, D. Pandita, J. Rodrigues, A. P. Pêgo, P.

L. Granja and H. Tomás, J. Controlled Release, 2010, 144, 55.). In both cases, the in fl uence of parameters such as dendritic generation, the length of the alkyl chain and the degree of functionalization in the efficiency of transfection and cytotoxicity have been evaluated. Michael's addition of acrylate derivatives as well as acylation with activated fatty acid esters have been the synthetic tools used for the manufacture of these types of vectors

Description of the invention

The present invention describes a series of PAMAM-derived ambiflic compounds where some peripheral amino groups of these dendrimers have been linked to vinylsulfone derivatives containing hydrophobic chains. The transfection properties of the resulting compounds are also described, which depend on the chemical nature of the hydrophobic chain and the extent of surface modification of the dendrimers. Some of the PAMAM derived dendrimers prove to be useful vectors for transfection and have better properties than unmodified PAMAM or commercial transfection reagents such as LipofectaMINA 2000: a) greater transfection efficiency, b) lower cytotoxicity, c) capacity of maintaining transfection in the presence of serum and d) the possibility of transfecting different types of cell lines. It also describes the preparation of fluorescent probes to monitor the endocytosis pathways useful in transfection mediated by these dendrimers and obtain information on the internalization mechanisms employed by these vectors.

In a first aspect, the present invention relates to a compound of general formula (I):

where

G represents an aminated dendrimer which is selected from PAMAM, polyethyleneimines, polylysines or polypropyleneimine,

R1 is selected from C1-C20 alkyl, C2-C20 alkenyl, or the group -Y-R2

And you select between OOS,

R2 is selected from the group -CH2-CH2-NH-CO-R3 or a sterol,

R3 is selected from C1-C20 alkyl or C2-C20 alkenyl,

m is a value between 1 and 4,

n is a value between 1 and p, where p is the number of surface -NH2 groups of the aminated dendrimer.

The term "alkyl" refers, in the present invention, to radicals of hydrocarbon chains, linear or branched, having 1 to 20 carbon atoms, preferably 2 to 18, and which are attached to the rest of the molecule by a single bond, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, tert-butyl, sec-butyl, n-pentyl, n-hexyl etc. The alkyl groups may be optionally substituted by one or more substituents such as halogen, hydroxy, alkoxy, carboxyl, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto and alkylthio.

The term "alkenyl" refers to hydrocarbon chain radicals containing one or more double carbon carbon bonds and preferably having from 2 to 20 carbons, for example, vinyl, 1-propenyl, allyl, isoprenyl, 2-butenyl, 1,3-butadienyl etc. Alkenyl radicals may be optionally substituted by one or more substituents such as halo, hydroxy, alkoxy, carboxyl, cyano, carbonyl, acyl, alkoxycarbonyl, amino, nitro, mercapto and alkylthio.

The term "sterol" refers to spheroids with 27 to 29 carbon atoms. Its chemical structure derives from cyclopentaneperhydrophenanthrene or esterano, a 17-carbon molecule consisting of three hexagonal and one pentagonal rings. In sterols, a side chain of 8 or more carbon atoms in carbon 17 and an alcohol or hydroxyl group (-OH) in carbon 3 are added. Non-limiting examples of sterols are cholesterol, ergosterol, stigmasterol etc.

PAMAMs (polyamidoamines) are polymers of the dendritic type (three-dimensional hyper-branched macromolecules of arborescent construction with a precise chemical structure), which have a generational growth form and are formed by an alkyldiamine core with branches that are generated from tertiary amines Preferably the PAMAMs are second to sixth generation and more preferably, second generation being p = 16 and presenting the following structure:

In a preferred embodiment, m is 1.

In a more preferred embodiment, R1 is a C1-C18 alkyl and preferably R1 is selected from - (CH2) 2CH3, - (CH2) 10CH3 or - (CH2) 16CH3.

In another more preferred embodiment, R1 is a C1-C18 alkenyl and preferably R1 is (CH2) 7CH = CH (CH2) 7CH3. In another preferred embodiment, m is 2. In a more preferred embodiment, R1 is the group -O-R2. In another more preferred embodiment, R1 is the group -S-R2. In another more preferred embodiment, R2 is -CH2-CH2-NH-CO-R3, where R3 is a C1-C18 alkyl and preferably R3

is selected from - (CH2) 2CH3, - (CH2) 10CH3 or - (CH2) 16CH3.

In another more preferred embodiment, it is -CH2-CH2-NH-CO-R3, where R3 is a C1-C18 alkenyl and preferably R3 is (CH2) 7CH = CH (CH2) 7CH3.

In a more preferred embodiment, R2 is a sterol that is selected from the list comprising cholesterol, epicolesterol, stigmasterol, lanosterol, ergosterol and coprostenol and preferably cholesterol.

In another preferred embodiment, n is a value that is selected from 2 or 1.

In another preferred embodiment, the invention relates to a compound of general formula (II):

where G represents an aminated dendrimer that is selected from PAMAM, polyethyleneimines, polylysines and polypropylene

nimina,

R1 is selected from C1-C20 alkyl, C2-C20 alkenyl, or the group -Y-R2

And you select between OOS,

R2 is selected from the group -CH2-CH2-NH-CO-R3 or a sterol,

R3 selects between C1-C20 alkyl or C2-C20 alkenyl,

R4 is a fluorogenic group,

m is a value between 1 and 4,

n is a value between 1 and p, where p is the number of NH2 groups of surface of the aminated dendrimer,

q is a value that is selected between 1 and 2.

"Fluorogenic group" refers to a small chemical group that is part of a larger molecule, which can be excited by light to emit fluorescence. In some embodiments, the fluorophors produce fluorescence efficiently by excitation with wavelength light from about 200 nanometers to about 800 nanometers. The intensity and wavelength of the emitted radiation depend on the fluorophore and its chemical environment.

In a more preferred embodiment, q is 1.

In another preferred embodiment, R4 is rhodamine or a derivative thereof, preferably the derivative having the following structure:

Rhodamine is a fluorine that is used as a fluorescent marker in techniques such as fluorescence microscopy, flow cytometry or ELISA. Other non-limiting examples of fl urogenic groups that can be used in the present invention are: acridine, anthracens, allophycocyanin, BODIPY, cyanines, coumarins, fl uorescamine, fl uorescein, FAM (carboxy fl uorescein), HEX (hexachloro fl uorescein-6) JOE ', 5'-dichloro-2', 7'-dimethoxy fl uorescein), Oregon Green, phycocyanin, fi coeritirine, rhodamine, ROX (carboxy-X-rhodamine), TAMRA (carboxytetramethylrodamine), TET (tetrachloro-fl uorescein), Texas red, tetramethylrodamine and xanthinas.

Another aspect of the invention relates to the use of a compound of formula (I) or of formula (II) as described above as a gene transfection agent.

Another aspect of the invention relates to a complex comprising a compound of formula (I) and a DNA fragment.

Another aspect of the invention relates to the use of the complex as described for the manufacture of a pharmaceutical composition.

Another aspect of the invention refers to a composition comprising a compound of formula (I), a compound of formula (II), a complex as defined above or any combination thereof, characterized in that the stoichiometric relationship between G and sulfonated groups have a value between 0.5 and 2. Preferably, this stoichiometric ratio G: sulfonated groups is 0.5. This composition contains both dendrimers that have been functionalized with vinyl sulfone groups and dendrimers that are not functionalized.

Another aspect of the invention relates to the use of a composition as described above, as a gene transfection agent.

A final aspect of the invention relates to a process for obtaining a compound of formula (I) comprising a Michael addition reaction of the compound of formula (III):

where m and R1 are defined as above, to an aminated dendrimer selected from PAMAM, polyethyleneimines, polylysines and polypropyleneimine and particularly to a second to sixth generation PAMAM dendrimer.

A suitable medium for the reaction described above is tetrahydrofuran / water although it is also possible to perform it in other reaction media known to any person skilled in the art, such as, but not limited to, acetone-water or acetonitrile-water.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

Description of the fi gures

Fig. 1. Electrophoretic mobility test. A.-Electrophoretic mobility test of PAMAM-G2 or some PAMAM-G2 derivatives bound to pDNA at N / P ratios between 0 (pEGFP-N3 only) and 10. The absence of the plasmid band in the wells is related to inhibition of electrophoretic mobility of the plasmid. B.-Minimum N / P ratio necessary to completely inhibit electrophoretic mobility in the assay. C.-Electrophoretic mobility test for PAMAM-G2 or PAMAM-G2 derivatives labeled with rhodamine.

Fig. 2. Experiments of protection of digestion with DNase I. A-Representative samples of agarose gels corresponding to plasmid pEGFP-N3 incubated in the absence (-) or presence (+) of DNase I as control. Samples of pEGFP-N3 that have been pre-complexed with PAMAM-G2 derivatives before treatment with DNase I (as described in the experimental section) have been run in parallel. B.-Quantification of the relative intensity (the untreated pEGFP-N3 has been assigned a value of 100) of the sum of the relaxed and overcoiled plasmid corresponding to the samples complexed with PAMAM-G2 derivatives and treated with DNase I. The results correspond to the mean ± SEM (n = 4). C.-Samples of rhodamine-labeled PAMAM-G2 derivatives have been similarly processed and the protection of digestion with DNase I has been measured by agarose electrophoresis. (a) Dendrimer-pDNA complexes, (b) labeled dendrimer alone. Since dendrimers are marked with fluorescence, gels have been photographed before (UV) and after staining with ethidium bromide (EtBr). Due to the interaction of the free labeled dendrimer with the SDS used in the preparation of the samples, the free labeled dendrimers have a negative charge during electrophoresis.

Fig. 3 In vitro efficiency of PAMAM-G2 derivatives in CHO-K1 cells. CHO-k1 cells were transfected with dendriplexes using plasmid pEGFP-N3 as described in the experimental section. For each condition, the DNA was mixed with the PAMAM-G2 derivative in an N / P ratio of 2.5 to 50. As a positive control (Lp), a transfection with LP2000 is performed. Fluorescence was measured 48 hours after transfection. The fluorescence / protein ratio for each compound is shown in the figures. The fluorescence value of eGFP in transfection with LP2000 has been 100% normalized in each experiment. The results are shown as mean ± SEM (n = 8).

Fig. 4 Cytotoxicity of complexes. The cytotoxicity of the complexes formed between the pDNA and the modified dendrimers has been tested in CHO-k1 cells 24 hours after transfection by measuring the percentage of cell viability using an MTT-based assay. Dendrimers with greater transfection efficiency have been tested. The test was performed using the most efficient N / P ratio for the transfection of each compound (6b-G2 (I): 10; 6c-G2 (I): 2.5; 6d-G2 (I): 10; 9b -G2 (I): 20; 9d-G2 (III): 10; 11d-G2 (III): 10). The results are shown as% viability, normalizing the value of 100% of untransfected cells.

Fig. 5 Factors that influence the transfection efficiency of PAMAM-G2 derivatives. A.-Effect of serum on the transfection medium. The LP2000 (as a positive control) or 6c-G2 (I) (ratio N / P 2.5-50) have been complexed with the plasmid pEGFP-N3 and subsequently used for the transfection of CHO-K1 cells in the absence or presence of 10 % fetal bovine serum in the transfection medium. Fluorescence was measured 48 h after transfection. The fluorescence / protein ratio for each condition is shown in the graph. The fluorescence value of eGFP for transfection with LP2000 in the absence of fetal bovine serum has been normalized to 100%. The results are expressed as mean ± SEM (n = 7). B.-Cell type. CHO-k1, Neuro-2a or RAW 264.7 cells have been transfected using compound 6c-G2 (I) complexing with plasmid p EGFP-N3 as described in the experimental section. For each condition, the DNA was mixed with the PAMAM-G2 derivative at an N / P ratio of 2.5 to 50 ratios. For each cell line a transfection with LP2000 has been used as a positive control. The fluorescence has been determined 48 h after transfection. The fluorescence / protein ratio is shown for each condition. The fluorescence value due to eGFP has been 100% normalized in each cell line. The results are shown as mean ± SEM (n = 8).

Fig. 6 Effect of transfection inhibitors on PAMAM-G2 derivatives. CHO-K1 cells have been pretreated with chlorpromazine (14 μM) or genistein (200 μM) for 30 minutes before transfection with the following dendriplexes in the most efficient N / P ratio (6b-G2 (I): 10; 6c- G2 (I): 2.5; 6d-G2 (I): 10; 9b-G2 (I): 20; 9d-G2 (III): 10; 11d-G2 (III): 10). As a control (Lp), a transfection with LP2000 has been performed. The fluorescence has been tested 48 h after transfection. The fluorescence / protein ratio for each compound is shown in the graph. The fluorescence value due to the eGFP in transfection with LP2000 has been normalized to 100%. The results are expressed as mean ± SEM (n = 6).

Fig. 7 Confocal Microscopy. CHO-K1 cells were seeded in microscope covers and transfected with dendriplexes based on the rhodamine-labeled compound 9d-G2 (III) and Cy5-labeled pEGFP-N3 at an N: P ratio of 10. 24 hours after transfection, the cells were fixed and analyzed by confocal microscopy to detect the eGFP protein in the transfected cells (green), the rhodamine-labeled compound 9d-G2 (III) (red) or the Cy5-labeled DNA pEGFP (blue). An overlay image is displayed as well as individual images for each independent channel. A and B.-CHO-k1 cells transfected at two different magnifications. C.-CHO-k1 cells preincubated with chlorpromazine before transfection.

Examples

The invention will now be illustrated by tests carried out by the inventors, which demonstrates the specificity and effectiveness of the compounds of the invention.

Synthesis of vinyl sulfone derivatives for the functionalization of PAMAM-G2

To obtain new PAMAM-G2 derivatives useful as transfection vectors with improved efficiency and low toxicity, a set of compounds having hydrophobic chains are selected for conjugation with the terminal amino groups of dendrimers. In order to assess the importance of the length of the hydrophobic chain in the transfection process, commercial reagents with alkyl chains of 4, 12 and 18 carbons are chosen. Thus, brominated derivatives 1a-c, oleic alcohol (cis-9-octadecenol) 2, butanoyl chloride 7a, lauric acid (dedecanoic acid) 7b, stearic acid (octadecanoic acid) 7c and oleic acid (cis-9 acid have been used -octadecenoic) 7d. The aza-Michael type addition of vinyl sulfone derivatives of these molecules is considered as a synthetic tool suitable for coupling with peripheral amino groups given the high yield and ease of this reaction that can be carried out by simply mixing the compounds in a suitable solvent without the formation of unwanted by-products.

The different functionality of compounds 1a-c, 2 and 7a-b (alkyl bromides, alcohols and carboxylic acids, respectively) allows simple strategies to be designed for the preparation of their corresponding vinyl sulfone derivatives and also for the introduction of structural variability in the spacer that joins the hydrophobic alkyl chain and the dendrimer nucleus, whose influence on transfection efficiency is also a parameter under evaluation. Thus, the nucleophilic displacement of the brominated derivatives 1a-c and the mesylated derivative 3 of the oleic alcohol 2 by the sulfur atom of the 2-thioethanol produces the hydroxyethyl thioethers 4a-d with high yield. Oxidation with peracetic acid and the subsequent dehydration of 5a-d hydroxysulfones by forming their mesylated derivatives easily leads to 6a-d vinyl sulfones in which the original functionality has been replaced by the vinyl sulfone group (Scheme 1). Alternatively, a strategy has been designed based on 7a-d fatty acids that has allowed us to introduce a longer hydrophobic alkyl chain spacer into vinyl sulfone. Thus, the reaction with 2-aminoethanol of the fatty acid chlorides, using commercial reagent 7a or obtained "in situ" by treatment with thionyl chloride in the case of 7b-d acids, yields the corresponding 2- hydroxylethyl amides 8a-d. The reaction of these compounds with divinyl sulfone (DVS) produces vinyl sulfone ether9a-d with moderate yields (Scheme 1).

Additionally, in the case of oleic acid, the molecule homologous to vinyl sulfone thioether 11d is also prepared to evaluate the influence of the spacer heteroatom on the efficiency of transfection. This compound is obtained by the reaction of oleic acid chloride with cystamine dihydrochloride which produces 10d disulfide in high yield. The reduction with Zn powder of this compound followed by the addition of Michael to DVS allows obtaining the thioether derivative 11d in a single step (Scheme 1).

(Scheme goes to next page)

ES 2 351 909 A1

Taking into account that cholesterol has frequently been used as a hydrophobic motive in non-viral transfection agents including PAMAM derivatives to introduce a lipophilic character in them, a vinyl sulfone derivative of this steroid is also prepared to synthesize the corresponding cholesterol -PAMAM-G2 conjugate and evaluate whether this structural modification allows optimization of transfection. The treatment of commercial cholesterol 12 with DVS produces the corresponding vinyl sulfone derivative 13 with a moderate yield (Scheme 1).

Chemical synthesis of PAMAM-G2 ambylic dendrimers as novirale vectors

The synthesis of the transfection agents was carried out by adding a THF solution of the vinyl sulfone derivatives (6a-d, 9a-d, 11d and 13), to an aqueous solution of PAMAM-G2. To evaluate the effect of the degree of functionalization and to determine the minimum stoichiometric value that allows obtaining the most effective transfection agents, three vinyl sulfone molar ratios are used: PAMAM-G2 (I = 0.5, II = 1.0 and III = 2) obtaining the corresponding conjugates [6a-d-G2 (I-III), 9a-d-G2 (I-III) 11d-G2 (I-III) and 13-G2 (I-III)] after solvent removal (Scheme 2). To carry out studies of cellular uptake of dendrimers and their subcellular localization, during the transfection process with dendrimers based on PAMAM-G2, the synthesis of fluorescent PAMAM-G2 derivatives is carried out for use in fluorescent confocal microscopy. The rhodamine vinyl sulfone derivative 14 (Scheme 2), allows to incorporate fluorescence in a simple way in the original PAMAM-G2 molecule to give the PAMAM-G2-Rho dendrimer which is subsequently functionalized to incorporate the hydrophobic motifs that have demonstrated a better behavior in Transfection studies done previously.

(Scheme goes to next page)

ES 2 351 909 A1

Transfection capabilities of functionalized derivatives of PAMAM-G2

The ability of the new PAMAM derivatives to neutralize, bind and compact plasmid DNA (pDNA) has been studied. Plasmid pEGFP-N3 [encoding green fluorescent protein (eGFP)] is chosen for complex preparation and transfection assays. The PAMAM conjugates are mixed with the pDNA at different N / P ratios (0.5 to 50) to give rise to the formation of the corresponding dendriplexes. The electrophoretic mobility test on gels is performed as a measure that reflects the ability of PAMAM-G2 derivatives to compact and protect the plasmid. The formation of the complexes causes a lower electrophoretic mobility with respect to the naked pDNA. Additionally, staining with ethidium bromide after electrophoresis is used to evaluate the effectiveness of the protection of pDNA since when the pDNA is efficiently protected in dendriplexes it is not accessible to the ethidium bromide molecule to intercalate. The electrophoretic mobility experiments in gels revealed that the majority of PAMAM-G2 derivatives bound to pDNA at an N / P ratio of 0.5-1 (Fig. 1 and ESI Fig. 1).

The ability to protect the pDNA from degradation by endonucleases is evaluated below as this is a key process to achieve high transfection, since unprotected pDNA is rapidly degraded by nucleases present in the culture medium. The results obtained with the derivatives of PAMAM-G2 (Fig. 2 and ESI Fig. 2) show that they have the ability to protect the pDNA from degradation by endonucleases while the unmodified PAMAM-G2 completely fails to protect the pDNA of digestion

In the next stage, the cellular uptake of the new dendriplexes is tested in the CHO-k1 cell line, a standard cell line that is normally used with transfection agents of different kinds. The plasmid pEGFP-N3 coding for the green fluorescent protein allows to evaluate the transfection efficiency since the fluorescence measured in the transfected cells 48 hours after the transfection (to allow the expression of the green fluorescent protein) is proportional to the efficiency of the same.

Cellular uptake is tested in the absence of serum at N / P ratios of 2.5-50 (Fig. 3). A value of 100% is assigned to the value obtained with the lipofectAMINE ™ 2000 (LP2000), a commercial reagent with high transfection efficiency in numerous cell types. The results obtained clearly indicate that while unmodified PAMAM-G2 has a very low transfection efficiency (less than 5% compared to LP2000). This fact is in agreement with previously published results since the compound: a) does not produce delays in pDNA gels (Fig. 1A) and b) does not protect against degradation with DNase I (Fig. 2A). This confirms the benefits of incorporating a hydrophobic domain to facilitate binding of the dendrimer to the hydrophobic nature bases of DNA. In fact, it is observed that the binding of a moderately hydrophobic domain such as rhodamine is sufficient to enable DNA binding to PAMAM-G2 (Fig. 1C) although it is not able to protect it from digestion with DNase I (Fig. 2C) .

From the transfection results obtained, several conclusions can be reached on the influence on the transfection efficiency of the chemical structure of the modified PAMAM-G2. Of the compounds tested, the minimum length of the alkyl chain that allows efficient transfection in CHO-k1 cells is 12 carbons (compounds 6b-G2 and 9b-G2). The derivative of PAMAM-G2 6b-G2 (I) has an outstanding transfection efficiency (p <0.001 compared to the value of LP2000) in an N / P ratio between 5 and 20 while the efficiency of compound 9bG2 (I) is lower but similar to that of LP2000 at an N / P ratio of 20 (Fig. 3). Both compounds have a high capacity to bind pDNA (Fig. 1), although as expected from their transfection efficiency, compound 9b-G2

(I) has a lower ability to protect DNA from degradation by DNase I (Fig. 2). The functionalized dendrimer 6c-G2 (I) carrying a C18 hydrophobic chain has the highest level of transfection in CHO-k1 cells (approximately 3.1 times more efficient than LP2000 (p <0.001)), accompanied by a high capacity to form Stable complexes with the pDNA, which allows complete protection against degradation (Fig. 2). However, compound 9c-G2 having an aliphatic chain of the same length has a low transfection efficiency, which correlates with the susceptibility to endonuclases digestion of the bound pDNA, in particular the band corresponding to the relaxed plasmid ( ESI Fig. 2). PAMAM-G2 derivatives containing 6dG2 (I), 9d-G2 (III) and 11d-G2 (III) oleic acid, which have a double bond in the aliphatic chain also have high levels of transfection compared to LP2000 (1.6 to 2.7 times greater than LP2000, p <0.01) (Fig. 3). These dendrimers also have high binding capacity to pDNA (Fig. 1 and ESI Fig. 1) and completely protect the pDNA from DNases (Fig. 2, and ESI Fig. 2). Overall, it can be said that although the inclusion of a hydrophobic domain is sufficient to obtain a strong DNA binding, an 18-carbon aliphatic chain is optimal for complete DNase protection and high transfection efficiency.

Regarding the extension of functionality due to hydrophobic chains, the comparison of dendrimers of series 6 and 9 indicates that the best transfection efficiency is related to the number of alkyl chains attached to each dendrimer. Series 6 compounds obtained using a stoichiometric vinyl sulfone: PAMAM-G2 ratio of 0.5 are very efficient transfection compounds while functionalized series 9 dendrimers require a higher stoichiometric vinisulfone ratio: PAMAM-G2 of 2 to transform into compounds of efficient transfection. The reason for this different behavior is not obvious although it follows that an advantage of the present invention is the possibility of using very low stoichiometric ratios vinyl sulfone: PAMAM: G2 that facilitate the water solubility of said transfection compounds.

On the other hand, the nature of the heteroatom in the ether bond, oxygen for compound 9d and sulfur for compound 11d, does not seem to have an influence on the transfection capacity of the compounds, similar in both (Fig. 3).

Electrokinetic properties of functionalized derivatives of PAMAM-G2

The determinations of “dynamic light scattering” and “ζ-potential” have been made for the best transfection agents in the optimum N / P ratio for each of them, in order to determine their electrokinetic properties (Table 1). As expected, the potential ζ values decreased as the complexes formed with the pDNA. However, a relationship between particle size and transfection efficiency is not observed, since the best 6c-G2 (I) and 9d-G2 (III) compounds are 524 ± 18 and 226 ± 3 nm in size, respectively.

TABLE 1

Hydrodynamic and Potential Diameter ζ for PAMAM-G2 derivatives and their DNA complexes

Cytotoxicity of functionalized derivatives of PAMAM-G2

The cytotoxicity of dendriplexes is assessed by determining the percentage of viable cells with respect to cells that have not been exposed to transfection agents using an MTT-based assay (Fig. 4). The assays are performed on CHO-k1 cells using the N / P ratio that provides greater transfection efficiency for each PAMAM-G2 derivative tested. Unmodified PAMAM-G2 in the presence of pDNA does not show, as expected, no toxicity, while complexes with LP2000 have a high toxicity since they reduce cell viability by 50% (p <0.001). For the PAMAM-G2 derivatives the most efficient compounds 6c-G2 (I) and 9d-G2 (III), a high cell viability was obtained.

Effect of the presence of serum in the medium on the efficiency of transfection

The effect of the presence of serum in the medium on the efficiency of transfection is evaluated for compound 6cG2 (I). This compound is chosen on the basis that it is the compound that best protects the degradation by DNases and is the one with the highest transfection efficiency at very low N / P ratios (Fig. 5A). In the presence of serum, transfection levels were similar to those obtained with LP2000 when tested at 5-10 N / P ratios. The increase in the N / P ratio in transfection in the presence of serum has also been previously described for liposome-based transfection reagents of a cationic nature.

Transfection capacity of functionalized derivatives of PAMAM-G2 in other cell lines

The transfection capacity of the PAMAM-G2 6c-G2 derivative (I) is tested for the transfection of neuroblastomas (Neuro-2a) and a macrophage-derived cell line (RAW 264.7) that usually has a low transfection efficiency by non-agents. - viral. A value of 100% is assigned to the transfection levels produced by the LP2000 in each cell line. Compound 6c-G2 (I) has the ability to transfect both cell lines in a manner similar to LP2000 (Fig. 5B).

Studies on the mechanism of cellular uptake and internalization of PAMAM-G2 derivatives

DNA complexes with non-viral transfection agents can enter cells through different endocytosis pathways. Therefore, in the case of new transfection agents it is important to determine the internalization route since it will determine intracellular processing and therefore influences the efficiency of transfection. Most previous studies suggest that the endocytosis pathway is the main cellular gateway of non-viral transfection agents. There are two endocytosis pathways, dependent and independent of clatrine, responsible for the entry of these DNA complexes. Depending on the type of cell, the nature of the transporter and the size of the particle, one way or another is chosen. Clatrin-dependent pathway is preferred for particles smaller than 200 nm in size. The independent clatrin pathway includes cellular entry from lipid rafts in caveolae or entry through a fl otillin-dependent route.

To determine the endocytosis pathway used by PAMAM-G2 derivatives, two endocytosis inhibitors are used: chlorpromazine as a clatrine-dependent and genistein-dependent pathway inhibitor as a clatrine-independent pathway inhibitor. Fig. 6 shows that chlorpromacin significantly increases (p <0.001) the transfection efficiency of LP2000, 6c-G2 (I) or 9d-G2 (III) while not modifying the transfection efficiency of compounds 6b -G2 (I), 6d-G2 (I), 9d-G2 (III) and 11d-G2 (III). Conversely, preincubation with genistein decreases the transfection efficiency of most compounds, including LP2000. Therefore, it can be concluded from these data that the functionalized PAMAM-G2 fundamentally use a clatrine-independent route.

Finally, the process of internalization of the complexes has been observed by confocal microscopy using Cy5-labeled pDNA and the 6c-G2 (III) -Rho and 9d-G2 (III) -Rho labeled with rhodamine. These compounds were prepared using the vinyl sulfone chemistry indicated in scheme 2 which involves the functionalization of PAMAM-G2-Rho with the vinyl sulfones of stearic and oleic acid 6c and 9d, respectively. For the compound 9d-G2 (III) -Rho the corresponding confocal images are shown. Cells that are transfected are identified by the expression of eGFP since the experiments are performed using plasmid pEGFP-N3. Images were taken 24 h after transfection to allow eGFP expression. Fig. 7A, which corresponds to visible differential contrast images (Nomarki) and fluorescence, shows how fluorescence is localized in all cells, transfected or not. In all of them, the red fluorescence corresponding to the marked dendrimers can be observed in the cytosol of the cells near the nucleus. Almost all the blue fluorescence that corresponds to the labeled DNA is associated with the red fluorescence of the dendrimers (purple particles in the superimposed fluorescence images). Only in transfected cells, blue free fluorescence is observed corresponding to the DNA released from the dendrimer and which can be observed inside the cell nucleus (Fig. 7B). There is no free blue fluorescence in the nuclei of untransfected cells. The results obtained confirm that the limiting step in the transfection mediated by hydrophobic derivatives of PAMAM-G2 is the release of pDNA from the endosomes to the nucleus, since while practically all cells are capable of capturing dendriplexes, only in a set of The pDNA is released from these internal deposits to reach the nucleus. Fig. 7C shows CHO-k1 cells that have been pre-incubated with chlorpromazine before transfection with the rhodamine-labeled dendrimer 9d-G2 (III) -Rho complexed with Cy5-labeled pEGFP-N3. In these cells the entry of dendrimers into the perinuclear space is very small and shows an apical distribution. However, the efficiency of transfection (the blue fluorescence associated with the pDNA released in the nucleus) is increased. These results, supported by the efficiency of transfection in the presence of endocytosis inhibitors, confirm that the independent clathrin route is the productive route of endocytosis used by these functionalized derivatives of PAMAM-G2.

Claims (28)

1. Compound of general formula (I): where G represents an aminated dendrimer that is selected from PAMAM, polyethyleneimines, polylysines or polypropylene. pilenimine, R1 is selected from C1-C20 alkyl, C2-C20 alkenyl, or the group -Y-R2 And you select between OOS, R2 is selected from the group -CH2-CH2-NH-CO-R3 or a sterol, R3 is selected from C1-C20 alkyl or C2-C20 alkenyl, m is a value between 1 and 4, n is a value between 1 and p, where p is the number of surface -NH2 groups of the aminated dendrimer.

2.

Compound according to claim 1 wherein the aminated dendrimer is a second generation PAMAM, p = 16.

3.

Compound according to claim 2 wherein m is 1.

Four.

Compound according to claim 3 wherein R1 is a C1-C18 alkyl.

5.

Compound according to claim 4 wherein R1 is selected from - (CH2) 2CH3, - (CH2) 10CH3 or - (CH2) 16CH3.

6.

Compound according to claim 2 wherein R1 is a C1-C18 alkenyl.

7.

Compound according to claim 6 wherein R1 is (CH2) 7CH = CH (CH2) 7CH3.

8.

Compound according to claim 2 wherein m is 2.

9.

Compound according to claim 8 wherein R1 is the group -O-R2.

10.

Compound according to claim 8 wherein R1 is the group -S-R2.

eleven.

Compound according to any of claims 9 or 10 wherein R2 is -CH2-CH2-NH-CO-R3, where R3 is a C1-C18 alkyl.

12. Compound according to claim 11 wherein R3 is selected from - (CH2) 2CH3, - (CH2) 10CH3 or - (CH2) 16CH3. 13. Compound according to any of claims 9 or 10 wherein R2 is -CH2-CH2-NH-CO-R3, where R3 is a C1-C18 alkenyl. 14. Compound according to claim 13 wherein R3 is (CH2) 7CH = CH (CH2) 7CH3. 15. A compound according to claim 9 wherein R2 is a sterol that is selected from the list comprising cholesterol, stigmasterol epicolesterol, lanosterol, ergosterol and coprostenol.

16.

Compound according to claim 15 wherein R2 is cholesterol.

17.

Compound according to any of the preceding claims wherein n is a value that is selected from 2 or

one. 18. Compound of general formula (II): where G represents an aminated dendrimer that is selected from PAMAM, polyethyleneimines, polylysines or polypropylene. pilenimine, R1 is selected from C1-C20 alkyl, C2-C20 alkenyl, or the group -Y-R2 And you select between OOS, R2 is selected from the group -CH2-CH2-NH-CO-R3 or a sterol, R3 is selected from C1-C20 alkyl or C2-C20 alkenyl, R4 is a fluorogenic group, m is a value between 1 and 4, n is a value between 1 and p, where p is the number of groups -NH2 of surface of the aminated dendrimer, q is a value that is selected between 1 and 2.

19.

Compound according to claim 18 wherein q is 1.

twenty.

Compound according to any of claims 19 or 20 wherein R4 is rhodamine or a derivative thereof. 21. A complex comprising a compound of formula (I) or a compound of formula (II) and a DNA fragment.

22. Use of the complex according to claim 21 for the manufacture of a pharmaceutical composition. 23. Composition comprising a compound of formula (I), a compound of formula (II), a complex according to claim 21 or any combination thereof, characterized in that the stoichiometric ratio between G and the sulphonated groups has a value between 0.5 and 2 .

24.

Composition according to claim 23 wherein the stoichiometric ratio G: sulfonated groups is 0.5.

25.

Use of a composition according to any of claims 23 or 24 as a gene transfection agent.

26.

Process for obtaining a compound of formula (I) comprising a Michael addition reaction of the compound of formula (III):

where: myR1 is defined as in claim 1, to an aminated dendrimer selected from PAMAM, polyethyleneimines, polylysines or polypropyleneimine.

27.

Method according to claim 26 wherein the aminated dendrimer is a second to sixth generation PAMAM dendrimer.

28.

Process according to any of claims 26 or 27 wherein the reaction occurs in a tetrahydrofuran / water medium.

SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 201001350 SPAIN Date of submission of the application: 14.10.2010 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: See Additional Sheet RELEVANT DOCUMENTS

Category

Documents cited Claims Affected

TO

SANTOS, J.L. et al. Functionalization of poly (amidoamine) dendrimers with hydrophobic chains for improved gene delivery in mesenchymal stem cells. Journal of Controlled Release. May 21, 2010, Vol. 144, No. 1, pages 55-64. Whole document. 1-28

TO

TAKAHASHI, T. et al. Synthesis of novel cationic lipids having polyamidoamine dendrons and their transfection activity. Bioconjugate Chem., 2003, Vol. 14, No. 4, pages 764-773. Whole document. 1-28

TO

JONES, S.P. et al. Synergistic effects in gene delivery-a structure-activity approach to the optimization of hybrid dendritic-lipidic transfection agents. Chemical Communications 2008, Vol. 39, pages 4700-4702. Whole document. 1-28

TO

JEVPRASESPHANT, R. et al. Engineering of dendrimer surfaces to enhance transepithelial transport and reduce cytotoxicity. Pharmaceutical Research 2003, Vol. 20, No. 10, pages 1543-1550. Whole document. 1-28

TO

GUILLOT, M. et al. Effects of structural modification on gene transfection and self-assembling properties of amphiphilic dendrimers. Org. Biomol Chem. 2006, Vol. 4, pages 766-769. Whole document. 1-28

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 29.12.2010

Examiner E. Albarrán Gómez Page 1/4

REPORT OF THE STATE OF THE TECHNIQUE Application number: 201001350 CLASSIFICATION OBJECT OF THE APPLICATION C08G83 / 00 (01.01.2006) C07C233 / 36 (01.01.2006) A61K47 / 48 (01.01.2006) A61K48 / 00 (01.01.2006) Minimum documentation sought (classification system followed by classification symbols) C08G, C07C, A61K Electronic databases consulted during the search (name of the database and, if possible, search terms used) INVENES, EPODOC, REGISTRY, HCAPLUS, WPI, PUBMED, MEDLINE, BIOSIS, EMBASE State of the Art Report Page 2/4  WRITTEN OPINION Application number: 201001350 Date of Written Opinion: 29.12.2010 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims Claims 1-28 IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims Claims 1-28 IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/4  WRITTEN OPINION Application number: 201001350 1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

SANTOS, J.L. et al. Functionalization of poly (amidoamine) dendrimers with hydrophobic chains for improved gene delivery in mesenchymal stem cells. Journal of Controlled Release. May 21, 2010, Vol. 144, No. 1, pages 55-64.

D02

TAKAHASHI, T. et al. Synthesis of novel cationic lipids having polyamidoamine dendrons and their transfection activity. Bioconjugate Chem., 2003, Vol. 14, No. 4, pages 764-773.

D03

JONES, S.P. et al. Synergistic effects in gene delivery-a structure-activity approach to the optimization of hybrid dendriticlipidic transfection agents. Chemical Communications 2008, Vol. 39, pages 4700-4702.

D04

JEVPRASESPHANT, R. et al. Engineering of dendrimer surfaces to enhance transepithelial transport and reduce cytotoxicity. Pharmaceutical Research 2003, Vol. 20, No. 10, pages 1543-1550.

D05

GUILLOT, M. et al. Effects of structural modification on gene transfection and self-assembling properties of amphiphilic dendrimers. Org. Biomol Chem. 2006, Vol. 4, pages 766-769.

2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The present invention relates to dendrimers comprising a PAMAM (G2) core derivatized with alkyl sulfonyl or cholesterol-sulphonyl groups, to its method of obtaining by Michael addition reactions between compounds derived from vinillsulfone and PAMAM-type dendrimers, to complexes comprising these dendrimers and a DNA fragment, for use in the preparation of a pharmaceutical composition and for the use of the compositions of these dendrimers as gene transfection agents. Document D01 refers to medium sized G5 PAMAM dendrimers with high amino group content both in the periphery and in the interior, conjugated with fatty acids: lauric acid (dodecanoic acid), myristic acid (tetradecanoic acid) and palmitic acid (hexadecanoic acid), in order to obtain PAMAM G5 dendrimers attached at the periphery to hydrophobic chains. These vectors showed neutralization, binding and compaction capacity of pDNA (plasmid DNA), as well as conferring protection against serum nucleases. In vitro experiments with mesenchymal stem cell cultures (with relevance in regenerative clinical medicine) revealed the remarkable ability of these vectors to internalize pDNA with low levels of toxicity, this effect being related to the CH2 content of the hydrophobic part. Gene expression was also enhanced using these vectors, the highest gene expression efficiency corresponds to the vector with the shortest hydrophobic chain (with lauric acid). Document D02 discloses a gene transfection study using PAMAM dendrimers of generations G1 to G4 functionalized with two chains of two dodecyl groups. The authors point out that while lipoplex formed with G1 had no transfection capacity, lipoplexes formed with G2, G3 and G4 have this activity, and that this activity increases with increasing dendrimer generation. The transfection efficiency of lipoplex formed with G3 and supplemented with dioleylphosphatidylethanolamine (DOPE) is superior to that of the widely used non-viral vector that carries cholesterol and known as DC-Chol. In D03 reference is made to PAMAM G1 and G2 dendrimers functionalized with 1 or 2 cholesterol molecules as hydrophobic motive for these non-viral transfection agents. Document D04 uses PAMAM dendrimers of generations G2, G3 and G4 conjugated with lauric chains. Document D05 discloses a study in which amphiphilic dendrimers formed by a lipophilic dendron and another hydrophilic dendron joined by a rigid diphenyletin core are used. No PAMAM-type dendrimers functionalized with alkyl sulfonyl or cholesterol-sulfonyl groups have been disclosed in the state of the art. Consequently, it is considered that the invention set forth in claims 1-28 of the present application is novel and involves inventive activity (Art. 61. and 8.1 LP11 / 1986). State of the Art Report Page 4/4