IT201900018041A1 - POLYETHYLENIMMIN WITH N-ACYLATE HYDROXYPYRIDONIC FUNCTION, THEIR SYNTHESIS AND THERAPEUTIC USE - Google Patents

Description

DESCRIPTION

Attached to an INDUSTRIAL INVENTION patent application entitled:

POLYETHYLENIMMIN WITH N-ACYLATE HYDROXYPLRIDONIC FUNCTION, THEIR SYNTHESIS AND THERAPEUTIC USE

FIELD OF THE INVENTION

Iron chelating polymers and their use in the treatment or prevention of iron overload disorders affecting the mucosa: enteral pathologies and intestinal, genital or periodontal dysbiosis.

STATE OF THE ART

Iron is an essential but potentially toxic micronutrient. While iron deficiency leads to anemia, excess increases the risk of dysbiosis and infections, as well as promoting inflammatory diseases and predisposing to neoplastic forms.

The transition from ferrous (Fe <+2>) to ferric (Fe <+3>) and vice versa assist various biochemical events, but this redox facility makes iron potentially toxic and requires a coordinated transport and storage system to prevent free radical production (Andrews NC. N Engl J Med. 1999; 341 (26): 1986-95).

The host's free iron sequestration systems prevent microbial acquisition, a protective effect defined as "nutritional immunity" (Hood & Skaar. Nat Rev Micro.

20 12; 10 (S): 525 - 37). Enteral iron excess increases the effectiveness of pathogenic strains such as Salmonella (Kortman GA et al. PLoS ONE 2012; 7: e29968) (Dostal A et al. PLoS ONE 2014; 9: e93549) or aggravates intestinal inflammatory states (Seril DN et al. Dig. Dis.

Sci. 2002; 47: 1266-1278) (Carrier JC, Int. J. Colorectal Dis. 2006; 21: 381-387) (Uritski R et al. J. Nutr. 2004; 134: 2251-2255). Other research correlates luminal iron with the onset of colorectal cancer (CRC) (Ng et a Wien Med Wochenschr 2016; 166: 431-436) with direct and microbiota-mediated effects (Noor SO et al. BMC Gastroenterol. 2010; 10) : 134). This problem is particularly evident in the case of oral iron intake in the treatment of iron deficiency anemia (Mahalhal A et al. J Nutr Biochem. 2019; 67: 20-27).

Iron chelating polymers able to sequester iron selectively avoiding the depletion of systemic iron, contain hydroxamic, catecholic and pyridinonic type functionalities. Among the latter there are 3-hydroxy-4-pyrinonic unit polymers connected to the backbone with N-linker, eg. the acrylic resins of Feng M et al. (J. Med. Chem. 1993; 36, 2822-2821; J. Med. Chem. 1994; 37, 924-927; J. Dairy Sci. 1995; 78: 55-61) or the acrylic polymers of Saghaie L et al . (Res. Pharm. Sci., 2015; 10 (4): 364-377).

These iron chelators have the disadvantage of requiring a complex synthesis which requires the 3-benzyloxy-4-pyrinon-N-alkylene-acrylic monomer which, after radical polymerization, must be catalytically reduced to 3-hydroxy-4-pyrinon-N-alkylene - polyacrylate. Other metal sequestering polymers based on the direct condensation of carboxycatechols with polyamines are disclosed in WO2016057754. However, these catechol groups do not ensure a high selectivity on metals by the claimed chelating polymers.

In this sense we have thought of iron chelating polymers that are easy to obtain and at the same time powerful and selective on iron and high biocompatibility.

SUMMARY

The invention refers to polyethyleneimine type polymers functionalized with 3-hydroxy-4-pyrinone units by direct reaction with maltol or other pyrones and partially acylates having excellent characteristics of physiological compatibility.

We have in fact discovered that, while the functionalization of polyethylene imines with hydroxypyridone groups produces cytotoxic polymers due to the excessive cationic density, the acylation of part or all of such nitrogenous cationic centers greatly improves their tolerability without compromising their functionality and selectivity of the function. iron-sequestering agent. An inventive object is therefore to polymers derived from polyethylene imines functionalized with 3-hydroxy-4-pyridinone groups having a partially acylated structure, therefore substantially characterized by:

easy to obtain = direct synthesis without the need for preformed monomer power = high affinity for iron

selectivity = low affinity on other transition metals (except aluminum and gallium) high biocompatibility = non-cytotoxic.

Another object of the invention is the synthesis of said polymers by direct condensation of a 3-hydroxy-4-pyrone with a polyamine polymer, proceeded or followed by selective acylation and retrohydrolysis, concluded by the separation / purification step;

The invention refers to their use in the treatment of the effects caused by the local accumulation of iron, such as the prevention of colorectal cancer and inflammatory bowel diseases and the treatment of dysbiosis caused by the alteration of the microbiota in favor of iron-avid species. pathogenic nature in the enteral, vaginal and periodontal tracts.

These and other inventive details are best illustrated in the following description.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 shows the IR spectrum of functionalized succinylated polyethylenimine (PFS) Fig. 2 shows the IR spectrum of functionalized acetylated polyethylenimine (PFA)

Fig. 3 illustrates the sequestration capacity of different cationic metals by PFS and PFA, highlighting their high selectivity for the sequestration of iron (III).

DETAILED DESCRIPTION

The invention relates to new polymers derived from polyethylene imines in which part of the primary amino groups present is functionalized with 3-hydroxy-4-pyrinone units, while the other part of the same primary or secondary amino groups is converted into amide groups by means of acylation reaction.

The polymer according to the invention has a structure as described in formula (I):

where is it:

R is hydrogen or a C1-C34 alkyl chain, preferably C1-C18, of the saturated or unsaturated type, linear or branched, optionally functionalized with hydroxyl, carboxylic groups, etc; R <1> is hydrogen or a methyl or ethyl group;

R <2> is hydrogen, methyl, hydroxymethyl or C1-C10 alkoxymethyl;

as well as their salts, hydrates and solvates; said polymers also characterized by a minimum molecular weight of 1 KDa

NB Note that formula (I) is purely indicative as the starting polyethylene imines (PEI) possess random structures including a diversity of connections between 1st, 2nd and 3rd nitrogen deriving from the polymerization of aziridine.

Preferred polymers of formula (1) are derived from maltol or ethyl maltol, therefore with R <1> = methyl or ethyl and R <2> = H and acylates such as to provide

Examples of starting polyethylenimines are Lupasol ™ (BASF); Epomin ™ (Nippon Shokubai); Dow-xx PEI (Dow Corp) or low weight polyethylenimines are Lupasol ™ FG, G 20 wfr, PR 8515 anhydrous, or Lupasol ™ FC, G 20. G 35 in sol. aqueous 50%.

Polyethylenimines (PEI) also include cross-linked polyethyleneimines, polyalkyleneimines and polyamidoamines.

Cross-linked polyethylenimines are obtained from polyethylenimines by treatment with cross-linking agents, e.g. epichlorohydrin, diisocyanates (e.g. hexamethylene diisocyanate), dialoalkanes (e.g. 1,2-dichloroethane), diepoxides (e.g. polyethylene glycol bisepoxide), polyalkylene glycols from 2 to 100 ethylene oxide (EO) or propylene oxide (PO) units, alkylene carbonates (e.g. ethylene carbonate), bischloroformates (e.g. 2,2-dimethylpropylene bischloroformed), etc.

An example of cross-linked polyethyleneimine is colestipol, a solid resin originally developed by Pharmacia-Upjohn (now Pfizer) for the treatment of hypercholesterolemia due to enteric bile acid sequestration.

Polyalkyleneimines are obtained in a similar way to polyethylenimines but, instead of aziridine, they come from homologs such as azetidine, propyleneimines (2-methylaziridine), 1-or 2-butyleneimines (2-ethylaziridine), possibly pre- or post-cross-linked .

In one embodiment, the synthesis of the inventive polymers consists of the following steps:

a) condensation of a polyethyleneimine (after neutralization of the protonant salts if present) with a 3-hydroxy-4-pyrone, eg. maltol or ethylmaltol, by heating in a suitable solvent to give a polyethyleneimine-hydroxypyridone;

b) depleted acylation of the polyethyleneimine-hydroxypyridone in the presence of acylating agent in stoichiometric quantity or in excess to obtain an acetylated polyethyleneimineacyloxypyridone-N-acetylated;

c) selective hydrolysis with acids or bases of the acetylated polyethyleneimine-acyloxypyridone-N-acetylated to restore an acetylated polyethylenimine-hydroxypyridone-N-acetylated;

d) separation / purification of the polyethyleneimine-hydroxypyridone-N-acetylated, after possible neutralization, from the starting reagents.

In another embodiment, the synthesis of the inventive polymers consists of the following steps: e) condensation as described in (a);

f) selective acylation of polyethyleneimine-hydroxypyridone in the presence of an acylating agent in default to give an acetylated polyethyleneimine-hydroxypyridone-N-acetylated;

g) separation / purification as described in (d).

The condensation reaction (a) takes place under heat, possibly assisted by microwaves, of the polyamine polymer with a 3-hydroxy-4-pyrone, with the reagents dissolved or suspended in water, mixtures of aqueous solvents or polar solvents.

The operations mentioned as "exhaustive acylation", "selective acylation", "selective hydrolysis" and "separation / purification" are carried out with known techniques, eg. described in the Examples. The inventive polymers can be administered topically, orally or enterally in formulations containing excipients and physiologically acceptable ingredients that act as a diluent, dispersant or vehicle of the substances of interest in the composition. The amount of the vehicle can range from 1% to 99%.

The amount of the inventive polymer in the medicinal composition may vary between 90% and 0.01% by weight, preferably between 10% and 1% by weight of the composition, dissolved or dispersed in a physiologically acceptable vehicle.

The term "medicinal composition" includes a drug, a medical device, a cosmetic with a soothing effect or a functional supplement.

The expression "physiologically acceptable" refers to medicinal compositions that do not produce an adverse reaction, allergic or other inconvenience when administered to animals, even better in humans. The preparation of a pharmaceutical composition containing at least one inventive polymer and optionally, one or more antibiotics will take place by means of practices known to those skilled in the art in the light of the present description.

Particularly useful are the gastro-resistant forms of the inventive polymers, such as for example those mentioned in the patents WO2005063256, EP 1547601, EP1312368 and W09826767.

The inventive polymers can find useful applications in the medical field in the treatment of the effects caused by the local accumulation of iron on the enteral and non-enteral mucous membranes, such as the prevention of colorectal cancer and inflammatory bowel diseases and the treatment of dysbiosis caused by the alteration of the microbiota a favor of pathogenic iron-avid species in the enteral, vaginal and periodontal tracts.

The treatment of enteral dysbiosis includes: subjects under oral iron therapy for the treatment of iron deficiency anemia; supportive therapy in enteral infections, post-antibiotic intervention to prevent enteritis or treatment in recurrent diarrhea.

Colorectal cancer prevention (CRC) is seen as a preventive regimen in individuals predisposed to CRC, eg. consumers of high or processed red meat; preventive treatment after cancer therapy and surgery to reduce relapse and mortality.

Treatment of inflammatory bowel disease (IBD), ulcerative colitis and Chron's disease includes: maintenance therapy in quiescent or mild IBD, preferably as a support in IBD drug protocols

NB The term "dysbiosis" is not a standardized medical term. Similar concepts are also described as microbial imbalance "," bacterial imbalance ", or" increased levels of harmful bacteria and reduced levels of beneficial bacteria. "The forms of dysbiosis treatable with the polymers according to the invention may relate to the microbial populations of various parts of the body in addition to the gastrointestinal tract, i.e. the genital area (female) and the mouth.

The compositions containing the inventive polymers can be administered for oral or enteral use in the case of treatment of intestinal dysbiosis; for intravaginal or intraurethral use in the case of female urogenital dysbiosis; buccally with final rinsing in the case of dysbiosis involving the oral cavity.

The composition of the present invention comprising the cited polymers may take various forms such that the compositions according to the present invention maintain an acceptable benefit / risk profile.

Medical compositions for the gastrointestinal tract may be packaged with known pharmaceutical practices (see Remington: The Science and Practice of Pharmacy (20th ed.), Ed. A.R. Gennaro, Lippincott Williams & Wilkins, 2000; or Encyclopedia of Pharmaceutical Technology, ed. . J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York). Particularly useful are the gastro-resistant forms, for example those mentioned in: WO2005063256, EP1547601, EP1312368 and WO9826767. Oral compositions may contain physiologically acceptable ingredients, e.g. ADA / PDR: Guide to Dental Therapeutics, 4th Edition by Physicians' Desk Reference and American Dentai Association. It is understood that this therapeutic treatment can be associated, concomitantly or in an alternating phase, with other treatments and drugs prescribed for the disorders described above.

The subjects that can benefit from the present invention are human beings, but also animals such as dogs, cats, horses, cattle, etc.

A wide range of modifications and improvements to the present invention can be achieved by experts in the field, while falling within the scope defined by the same.

EXAMPLES

Example 1 - Synthesis of functionalized succinylated polyethylenimine (PFS)

In a microwave reactor 2.5g of a 50% solution in water of PEI 600-1000 KDa dissolved in 20 mL of water are placed. 0.5 g of maltol are added to the solution kept under stirring. Once the reactor is closed, the solution is heated with microwaves at 110 ° C for 1 hour. The TLC control of the crude shows the complete disappearance of the maltol (eluent The solution is transferred into a flask with 2.0 g of succinic anhydride and 1.4 mL of TEA. The solution is stirred for 12 h at room temperature. Then NaOH is added. 1M up to pH = 1 and stirring continued for 1 h. Finally 10% HCl is added up to pH = 5. The solution is purified by dialysis (MWCO 12 KDa) and then lyophilized. The IR spectrum: Cm- 1 = 3271 (NH), 1697 (C = 0), 1558 (C = 0) is illustrated in Fig. 1

Example 2 - Synthesis of acetylated functionalized polyethylenimine (PFA)

2.5g of a 50% solution in water of PEI 600-1000 KDalton dissolved in 20 mL of water are placed in a microwave reactor. 0.5 g of maltol are added to the solution kept under stirring. Once the reactor is closed, the solution is heated with microwaves at 110 ° C for 1 hour. The TLC control of the crude shows the complete disappearance of the maltol (eluent The solution transferred into a flask is added with 1.98 mL of acetic anhydride and 1.4 mL of TEA. The solution is stirred for 12 h at room temperature.

At the end, NaOH IN is added up to pH = 11 and stirring continued for 1 h. Finally 10% HCl was added until pH = 5. The solution is purified by dialysis (MWCO 12 KDa) and then lyophilized. The IR spectrum: is in Fig. 2.

Example 3 - Determination of the iron chelating capacity of PFS and PFA

A known quantity of functionalized succinylated polyethylenimine (PFS) or acetylated (PFA) obtained as in examples 1 and 2 is suspended in a 10mM solution of FeCl3 and left under stirring for 24 h at room temperature. The reddish polymer is filtered and the concentration of iron in the solution is determined by atomic absorption spectroscopy and subtracted from the amount of iron initially added to obtain the amount of complexed iron. The data are summarized in Table I.

Table I. Iron chelating capacity of PFS and PFA

Example 4 - PFS and PFA selectivity test for measuring the complexation capacity

Known quantities of PFS and PFA are suspended in a 0.01 M solution of

at 25 ° C for 24 hours. Each sample is filtered and the residual quantity of metal is calculated on the resulting mother liquors with atomic absorption spectroscopy. The value is used to calculate the complexation of each metal. The results are summarized in table and III respectively.

Table II - Complexation of metals by PFS

Table III - Complexation of metals by PFA

Example 5 - Evaluation of cytotoxicity

The model for the evaluation of cytotoxicity is carried out on primary fibroblasts kept in culture media containing the substances added to the fibroblasts in the GO phase, exposed to the test substances for 24 and 48 hours with replacement of the culture medium every 24 hours. At the end of the incubation, the MTT test is performed to evaluate the viability of the treated cells compared to the control culture. The MMT test uses the tetrazolium salt (Borenfreund 1985; Mossman 1983) which is prepared by adding 15 mg of MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5diphenyltetrazolium bromide) to 30 mL of culture medium - reduced to formanzan salt by mitochondrial succinate dehydrogenase and by cytoplasmic dehydrogenases of viable cells. After exposure the cells are rinsed with 200 µL of PBS, the washing solution is removed and 200 µL of MTT medium is added to each well. The cells are then incubated for 4 hours at 37 ° C at 5% CO2. The medium with MTT is removed, then 200 µL of a solvent solution consisting of 10% Triton X-100 and 0.1 N of HCl in isopropanol are added. The plate is stirred for 20-30 'and the absorbance of this homogeneous solution is read at 570 nm (background 690 nm). Results are expressed as% cell viability compared to the untreated control culture. Statistical analysis is carried out by means of a T-test in comparison with the control at 24 and 48 hours. Cell cultures treated with inventive polymers, the poly (amino) (hydroxypyridones) acylated PFS and PFA, with polymers or functionalized non-acylated (PF) are used, in addition to the comparison, with data reported in table IV.

Table IV - Cytotoxicity of fibroblasts treated with PFS, PFA and comparative polymer (PF) expressed as% variation on the control

Example 6 - Gel

A bright green coloring gel is prepared by dissolving sodium hyaluronate in water, then adding preservatives and suspending the inventive polymer, finally staining with diluted cupric chlorophyll and making up to volume with water.

Ingredients _ Quantity

Sodium hyaluronate 1.0 g

Benzyl alcohol 0.7 g

Phenoxyethanol 0.7 g

Polymer of formula (II) 1.0 g

Copper chlorophyll to taste

Demi water to taste at 100

Example 7 - Gastro-resistant tablet for colo-intestinal release

A polymer of formula (I) is in a quantity of about 3 g and is assembled with the following ingredients (expressed in centesimal composition): lactose: 80 mg, starch: 17 mg, magnesium stearate: 3 mg, microcrystalline cellulose 10 mg; covered with a 7 mg / mm2 film by spray of hydroxypropylmethylcellulose phthalate and ethylcellulose solution.

Example 8 - Skin spray

A polymer of formula (II) is processed in a quantity of about 1.7 g with the following ingredients expressed in centesimal composition: macrogol 2 g; isopropanol 10 g; water q.s. to 100 g. Example 9 - Simple capsules

A quantity of 4 kg of a substance of formula (II) is placed in hard gelatin capsules in such a way that each capsule contains 40 mg of active compound.

Example 10 - Solution

A solution of 1 kg of a substance of formula (II) in 601 of double distilled water is filtered in sterile, placed in ampoules, lyophilized under sterile conditions and aseptically sealed. Each ampoule contains 20 mg of active compound

Example 11 - Bottles

A solution of 200 g of a substance of formula (II) and 5 g of disodium hydrogen phosphate buffered at pH 6.5 is dissolved in 3 1 of bi-distilled water containing a little diluted HCl, is sterile filtered, placed in injection bottles, lyophilized under sterile conditions and aseptically sealed. Each injection bottle contains 10 mg of active compound.

Example 12 - Suppositories

A mixture of 40 g of a substance of formula (II) is fused with 100 g of soy lecithin and 1400 g of cocoa butter, poured into molds and cooled to obtain suppositories with 40 mg of active.

Example 13 - Tablets

A mixture of 2 kg of a substance of formula (II), 4 kg of lactose, 1.2 kg of potato starch, 0.2 kg of talc and 0.1 kg of magnesium stearate is compressed to give tablets each containing 20 mg of active compound.

Example 14 - Coated tablets

The tablets prepared similarly to Example 13 are coated with sucrose, potato starch, talc, tragacanth gum and dye.

In practice it has been found that the present invention achieves the intended purposes. It is evident that the compositions and methods according to the invention are susceptible to variations within the inventive concept. The purpose of the invention is better defined by the claims, rather than by the examples reported above.

Claims (8)

CLAIMS 1. Polymers derived from polyethylene imine (PEI) functionalized with 3-hydroxy-4-pyridinone groups and partially acylated, having formula: where: R is hydrogen or a C1-C34 alkyl chain, preferably C1-C18, saturated or unsaturated, linear or branched, optionally functionalized with hydroxyl or carboxylic groups; R <1> is hydrogen or a methyl or ethyl group; R <2> is hydrogen, methyl, hydroxymethyl or C1-C 10 alkoxymethyl; as well as their salts, hydrates and solvates. 2. Polymer according to claim 1 wherein R = CH3, CH2CH2COOH, CH2OH, C6H4 (O) OH, O CH (OH) CH (OH) COOH; R <1> = CH3 or CH2CH2; and R <2> = H. Medical composition containing a polymer according to claim 1 or 2 in a therapeutically useful quantity for the treatment or prevention of an enteral pathology caused directly or indirectly by iron overload in the gastrointestinal tract. 4. Medical composition according to claim 3 wherein said enteral pathology is a dysbiosis in the gastrointestinal tract. 5. Medical composition according to claim 3 wherein said enteral pathology is an inflammatory bowel disease (IBD). 6. Medical composition according to claim 3 wherein said enteral pathology is colorectal cancer (CRC). 7. Composition according to claim 1 or 2 in an amount therapeutically useful for the treatment of dysbiosis in the female genital tract or in the periodontal cavity. 8. A polymer synthesis method according to claim 1 or 2 comprising: a) condensation of a polyethylenimine (after neutralization of the protonant salts if present) with a 3-hydroxy-4-pyrone, eg. maltol or ethylmaltol, by heating in a suitable solvent to give a polyethyleneimine-hydroxypyridone; b) depleted acylation of the polyethyleneimine-hydroxypyridone in the presence of acylating agent in stoichiometric quantity or in excess to obtain an acetylated polyethyleneimineacyloxypyridone-N-acetylated; c) selective hydrolysis with acids or bases of the acetylated polyethyleneimine-acyloxypyridone-N-acetylated to restore an acetylated polyethylenimine-hydroxypyridone-N-acetylated; d) separation / purification of the polyethyleneimine-hydroxypyridone-N-acetylated.