WO2011111084A1 - Pharmaceutical composition based on glycyrrhizin and eg56 polymer for the preparation of anti-inflammatory products - Google Patents

Abstract

Pharmaceutical composition containing the thermo-sensitive polymer produced and marketed under the tradename EG56, corresponding to Bis-Methoxy PEG-13 PEG-438/PPG-110 SMDI Copolymer, and glycyrrhizin and/or its derivatives, in particular its active part without sugar, the glycyrrhetinic acid, as active principle, for the preparation of anti-inflammatory products in stable aqueous solution both for ophthalmic and nasal/oral use, in the technical form of spray or drops for disinfecting nasal and mouth cavities.

Description

PHARMACEUTICAL COMPOSITION BASED ON GLYCYRRHIZIN AND

EG56 POLYMER FOR THE PREPARATION OF ANTI-INFLAMMATORY PRODUCTS

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SUMMARY OF THE INVENTION

The present invention relates to a pharmaceutical composition based on glycyrrhizin and/or their salts or esters and . a thermo-sensitive polymer produced and marketed under the name of EG56 by PolymerExpert , whose thermoelastic features are such to ensure to said composition an extended contact onto body surfaces normally lubricated by biological fluids, such as the ocular, nasal and oral districts. Chemically the EG56 polymer is a Bis-Methoxy PEG-13 PEG-438/PPG-110 SMDI Copolymer .

Such composition is used to prepare an ophthalmic solution with anti-inflammatory action, adapted to adhere to the ocular surface sufficiently long the active principle, the glycyrrhizin or its active part without sugar, beta glycyrrhetinic acid, can exploit its action.

Further object of the present: invention is the use of the same composition based on EG56_. polymer and glycyrrhizin for the preparation of a nasal/oral solution in the form of spray or drops with antiinflammatory action.

DESCRIPTION

It is known the ability of the glycyrrhizin to specifically bind HMGBl protein which is an alarmin, that is an endogenous protein with a specific role as immune activator when, following a direct injury or infection, it accumulates in the intercellular spaces and in the body fluids (R. Sakamoto, M. Okano, H. Takena, K. Ohtsuki. Inhibitory effect of glycyrrizin on the phosphorylation and DNA-binding abilities of High Mobility Group proteins 1 and 2 in vitro. Biol. Pharm. Bull. 24(8) 906-911 (2001); L. Mollica et al. Glycyrrizin binds to high-mobility group box 1 protein and inhibits its cytokine activities. Chemistry & Biology 14,431-441). The released HMGB1 binds receptors of the plasmatic membrane of different cells (RAGE, TLR2, TLR4, TLR9) and such binding promotes immune response activation and pro-inflammatory cytokines releasing (Oppenheim, J. J.; Yang, D. Alarmins: chemotactic activators of immune responses. Curr. Opin. Immunol. 17, 359-365 (2005); Bianchi, M.E. DAMPs, PAMPs and alarmins: all we need to know about danger. J. Leukoc.. Biol. 81, 1-5 (2007)).

BACKGROUND OF THE INVENTION

The present invention has been established following a study carried out by prof. Alberto Chiarugi and his equip, which has for the first time highlighted the presence - of HMGBl protein in the tear fluid of patients affected by conjunctiva or eyelids inflammation.

Currently it has not been reported about the existence of ophthalmic solutions containing salts and/or esters of glycyrrhizin acid associated to a thermo-sensitive polymer according to the following claims .

^"AIM OF THE INVENTION Aim of the invention is treating inflammatory eye pathologies such as conjunctivitis, blepharitis and keratitis using a natural substance, the glicyrrhizin, by mechanically sequestering HMGB1 released in the extracellular space, without exerting any pharmacological, immune or metabolic action.

For the purpose of the present invention the terms "glycyrrhizin", "glycyrrhizic acid" and "glycirrhizinic acid" will be equally used as synonyms in the present description and are intended to denote the triterpene glycoside extracted from the dried rhizome and roots of various species of Glycyrrhiza (liquorice). While, the term "glycyrrhetinic acid" denotes trie hydrolysis product of the glycyrrhizin . In fact, glycyrrhizin is converted to glycyrrhetinic acid (aglycone) and two molecules of glucuronic acid (glycone) by hydrolysis. Such aglycone derivative of glycyrrhizin sometimes may be indicated also by the synonyms "glycyrrhetic acid", "18 beta-glycyrrhetic acid" and "18 beta-glycyrrhetinic acid".

A second task of the invention is employing the features of an innovative _^polymer allowing._ the natural substance to remain for a long time in the ocular district to maximally exploit the relative benefits, dramatically reducing its absorbance and avoiding hence any toxicity problem. It has to be considered that above 0,75% in weight, beta 18-glicyrrhetinic acid naturally aggregates into micelles: in presence of 5% in weight of beta 18-glicyrrhetinic and added EG56 polymer, to all intents and purposes the composition appears being as a medical device according to the European provisions.

DETAILED DESCRIPTION OF THE INVENTION

. The ocular district is prone to different types of inflammation, whom the most . common are among: conjunctivitis, blepharitis, dacryocystitis, keratitis, scleritis, episcleritis, uveitis. Treatment of such pathologies by administration of anti-inflammatory ophthalmic solutions to the lachrymalis sac presents limitation due to a feature of the ocular district itself, that is eye surface being covered with the tear film.

The tear fluid is made up of about 98% water and of 2% by different substances, mainly including sodium chloride. Furthermore, tear fluid contains lisozyme, a component with bactericide power providing disinfectant action, to protect the eyeball from microbial infections. Tear fluid production by lachrymal glands, takes place continuously, and thus fulfills three important functions:

1. washing the eyeball, always keeping it clear and clean from dust particles and tiny foreign particles, resulting in clarity of vision eye;

2. moistening permanently the eyeball, otherwise intended to reach out to dry in contact with air; 3. counteracting the engraftment of pathogenic microbes on the eyeball, thus reducing infection possibilities.

Two pivotal and ineradicable factors can decrease the efficacy of the ophthalmic solutions commonly used in order to moisten and lubricate eye surface, such as for instance the artificial tears: • tearing reflex (resulting in a further dilution of the solution itself) ;

• blinking reflex, caused by the application of eye drops in the conjunctival sac.

Previous studies have demonstrated that 90 seconds after the administration of various ophthalmic solutions, such as saline, or a solution containing polyvinyl alcohol, or a solution containing methylcellulose , in the lachrymalis sac have been found out only:

• 3% of saline,

• 5% of a solution containing polyvinyl

alcohol,

• 10% of a solution containing

methylcellulose.

According the study, following the instillation of artificial tears, the break up time (BUT) may increase and remain stable up to 120 seconds.

This shows that, in the formulation of a product in the form of ophthalmic solution to be installed in the eye, is absolutely essential for the activity of the product, to prolong the contact for a sufficient time for the active substance may exert its activity. Different Ophthalmic compositions based on viscoelastic substances able to increase the permanence of eye drops on eye surface are available on the market. In particular, polymeric materials which, increasing the viscosity of the preparations, prolong the retention time of eye promoting drugs absorbance, either with so- called mucoadhesive effect, i.e>. able to increase residence time of the drug without the preparation has a significant increase in viscosity.

For this purpose the most commonly used substances are: cellulose derivatives (MC, HEC, HPC, CMC, HPMC) , polyvinyl alcohol (PVA) , polyvinyl-pirrolidone (PVP) , hyaluronic acid, polyacrylics acids (Carbopol^®, Carbomer^®) , natural rubber (TSP^®) . Some examples of many preparations containing such substances already available on the market are: Vistagan^® (Levobunolo, PVA 1.4%, Allergan) , Pilocarpina 2% Plus^® (pilocarpine, methylcellulose. 0.5%, Allergan), Pilotonina^®

(pilocarpine, HPMC 0.33%, Farmila) , Piroftal^® (piroxicam, PVP, Bruschettini ) , Visumetazone^®

(dexamethazone, CMC 0.5%, Pharmec) , Tobradex^® (tobramycin and dexamethazone, HEC, Alcon) , Pilogel^® (pilocarpine, Carbopol 940 3.5%, Alcon).

The limit of use of this substances is often given by the concentration, in fact it is not possible increase the concentration above certain values as viscous gels are obtained, difficult to be sterilized by sterile filtration. Furthermore, too viscous solutions can cause vision problems.

According to the present invention, it has been tested the . thermo-sensitive polymer produced and marketed under the name EG56 by PolymerExpert , - that, dissolved in water at room temperature, produces a clear solution slightly viscous and easy filterable on industrial scale. This polymer is able to reversibly change its physical state following a change of temperature. In particular, the solution of the present composition, when brought to body temperature, as it is in contact with the ocular surface, increases its viscosity adhering to the surface for a much more prolonged time.

Chemically, such polymer is a Bis-Methoxy PEG-13 PEG-438/PPG-110 SMDI . Copolymer (EG56) .^■

It is therefore a first object of the present invention a pharmaceutical composition containing in association:

- the thermo-sensitive polymer EG56 having the above indicated chemical formula, soluble in aqueous solution, and able to increase its viscosity when brought to body temperature, able to adhere to a body surface for a time sufficient to have therapeutic efficacy;

- and glycyrrhizin and/or a their salt and/or a their ester, substances with known anti-inflammatory properties ,

for the preparation of ophthalmic solutions with anti-inflammatory action, able to adhere to the ocular surface for a . sufficient time in which the active principle can exerts its activity, preventing the absorption.

Further object of the present invention is using the same composition based on polymer EG56 and glycyrrhizin, for the preparation of a stable aqueous solution for nasal/oral application, in the form of spray or drops.

Due to the presence of EG56 which increases its viscosity in contact with the involved body surface, slows down the draining of the solution applied from the involved surface by the normal biological fluid (lachrymal liquid, saliva, nasal mucus) , and consequently increases the drug bio-availability.

Glycyrrhizin used in . the present invention is widely known in both the food and pharmaceutical technical field. In particular, the pharmaceutical use is documented only in some Asian countries, such as Japan.

Glycyrrhizin is obtained by liquorice root, which is known to be used for over 4000 years both as a sweetener in foods and drinks and as a flavouring agent in tires and cigarettes; sometimes it has been used to mask the bad taste of medicinal preparations. Liquorice extracts have been further utilized for treating dyspepsia and ulcer dyspeptic. The most common use relates the exploitation of the anti-inflammatory activity.

Glycyrrhizin is a triterpene glucoside and along with its aglycone, the 18- -glycyrrhetinic acid, a pentacyclic triterpene belonging to the β-amirin serie, is the most important extract obtained from the liquorice root. Both glycyrrhizin and its aglycone, the 18- -glycyrrhetinic, have _a wide range of activities including the most well known are the anti-inflammatory activity, anti-ulcer, anti-allergic, anti-oxidant, anti-viral. In Japan, for instance, glycyrrhizin, is utilized intravenously for treating chronic hepatsis B and C.

Glycyrrhetinic acid comes in the form of two stereoisomers, i.e. stereoisomer a and stereoisomer β. Alpha-glycyrrhetinic acid is more soluble in water and tends to form gels, while, beta-glycyrrhetinic acid is sweet and has a wider spectrum with regard to the physiological activities. Different glycyrrhizic acid derivatives are known, in particular esters, amines and salts, for which there was an increase of the anti- inflammatory activities.

Beta-glycyrrhetinic acid structure is similar to that of cortison: both molecules are flat and similar in positions 3 and 11. Initially, the anti-inflammatory activity has been related to such similarity, however subsequent studies have demonstrated a different . action mechanism, likely connected to the glycyrrhizin ability to interact with HMGBl protein.

Recent studies show the HMGBl protein role in the pathogenesis of ocular disorders. Increased levels of extracellular HMGBl have been found in the vitreous and in the epiretinal membrane of patients with diabetic retinopathy and vitreoretinopathy . (Pachydaki et al. Up-regulation of RAGE and its ligands in proliferative retinal disease. Exp. Eye Res 82, 807-815 (2006)). Furthermore, there is about a 70 times increase in the level of HGMB1 in the vitreous of patients affected by endophthalmitis . Under physiological ...conditions , jLn a _ rat experimental model the protein level is not measurable in the aqueous humor, while increases considerably in case of uveoretinitis . (Arimura et al. High mobility group box 1 protein in endophtalmitis . Graef's Arch. Clin. Exp. Cphtalmol. .246, 1053 - 1058, 2008). HMGBl content in the vitreous also increases in subjects with retinal, detachment (Arimura et al . Intraocular expression and release of high mobility group box 1 protein in retinal detachment. Laboratory Investigation 89, 278-287, 2009) . All this findings indicate . an active role of HMGB1 protein in inflammatory and degenerative pathologies of the eye.

There are several options to block the HMGB1 protein pro-inflammatory signal, either through a release block of the protein itself, or through a receptor competition, but both processes could lead to significant side effects (in the first case) or difficulties of receptor selectivity (in the second case) .

The strategy followed by the inventors has been trying to seize HMGBl released in the tissues so as to prevent binding to the receptors. In particular, it has been thorough the study of the action mechanism of glycyrrhizin in the binding to HMGBl, and so, its capability to block the pro-inflammatory signals of the protein itself.

In order to better describe such mechanism the inventor has introduced and uses a new term, "flogolitic" to indicate an anti-inflammatory action carried our by glycyrrhizin through a different mechanism from the pharmacological, immune_:_ and metabolic ones.

As already mentioned, recent studies have demonstrated the ability of the glycyrrhizin to recognize and to bind HMGBl, thus blocking the proinflammatory effects of the protein itself. (Mollica et al. Glycyrrhizin binds to high-mobility group box 1 protein and inhibits its cytokine activities. Chem. Biol. 14, 431-441, 2007). The same studies have demonstrated that the triterpenic portion of the glycyrrhizin, the 18-β glycyrrhetinic acid, is responsible of the specific recognition at HMGBl surface .

During the research carried out in support of the present application, the affinity of 18 beta-_, glycyrrhetinic acid for HMGBl has been specifically determined through an ELISA assay. In this assay, addition of 10 beta-glycyrrhetinic acid and HMGBl to the reagents has inhibited the binding HMGBl-antibody anti-HMGBl in a concentration dependent manner, IC₅₀ = 29±4 mM. Once it has been so established the capability of 18 beta-glycyrrhetinic to bind the HMGBl protein, it has been evaluated the effect of the same in preventing the inflammatory response caused by the protein itself.

In particular, it has been demonstrated the inhibition of HMGBl-dependent COX-2 induction in human monocytes obtained from peripheral blood mononuclear cells pre-treated with HMGBl.

The attached figures show the results of the experiments carried out, .and in particular

- Figures 1A and IB show HMGBl levels in tear fluids., of _healthy. subjects . and—patients with bacte.ri.al- or allergic conjunctivis or blepharitis;

Figure 2A shows glycyrrhizin structure, (glucuronic acid residues and the., structure of 18-beta- glycyrrhetinic acid (aglycon) are indicated;

- Figure 2B shows the effect of the aglycon on HMGBl amount through an ELISA assay wherein the aglycon has been added during the 12 hours incubation of HMGBl with its primary antibody, or following this incubation along the secondary antibody. Figure 3A _. shows the effect of . 18-beta- glycyrrhetinic acid on COX-2 expression following HMGBl 1 μΜ stimulation carried out for 16 hours, using β- actin as positive control;

t -_. Figure 3B shows the effect of the aglycon part on HMGBl induced human monocytes aggregation.

Thanks to the special activity features, glycyrrhizin and, in particular, 18-beta-glycyrrhetinic acid have thus an important therapeutic potential because they are able to block the inflammatory process through a mechanism of seizure of the agent propagator and not through an inhibition at receptor level.

It is therefore essential to prolong at maximum level the possible contact time between the . active principle and the biological liquid under examination.

Object of the present invention is thus the combination of glycyrrhizin and/or its salts and/or its esters with EG56 polymer, able to ensure a long enough permanence at the surface of interest, allowing the use of the active principle for therapeutic purposes.

The possible application areas have been identified, they are:

• district of the eye;

• district of the mouth;

· district of the nasal cavity.

According to the present invention products with anti-inflammatory effects have been developed in the form of collyrium, spray or nasal and oral drops, whose mechanism of action is based on the ability of glycyrrhizin, maintained in contact with the involved surface for a long enough time, to remove the HMGBl protein from the biological fluids, reducing or even eliminating the inflammation induced by it.

EXAMPLES

Examples of the formulation of the composition object of the invention are shown below, provided for illustrative purposes only, without in any way limiting the invention.

Type formula for lOOg of ophthalmic solution:

• glycyrrhizic acid and/or derivatives: 0.5-7g

• EG-56 polymer: 0.5-5%

• buffer solution pH 7-7.5: up to lOOg

• preserving agent and/or EDTA: 0.01-0.5g

• deionized water: up to lOOg

Type formula for lOOg of nasal/oral solution in the form of spray or drops:

• glycyrrhizic acid and/or derivatives: 0.5-7 g

• EG56 polymer: 0.5-5%

• buffer solution pH 7-7.5: up to 100 g

• preserving agent and/or EDTA: 0.01-0.5 g

• flavouring agent: ad libitum

• deionized water: up to 100 g

In_ particular,., the formulation of the ophthalmic solution is the following:

• glycyrrhizic acid potassium salt: 5 g

• EG56 polymer: 3.5 g

• buffer solution pH 7-7.5: up to 100 g

• preserving agent: 0.004 g

• EDTA: 0.1 g

• deionized water: up to lOOg

In particular the formulation of the nasal/oral solution in the form of spray or drop is the following: • glycyrrhizic acid potassium salt: 5 g · EG56 polymer: 3.5 g

• buffer solution pH 1 - 7.5: up to 100 g

• preserving agent: 0.1 g

· EDTA: 0.1 g

• Flavouring agent: 0.1 g

• deionized water: up to 100 g

Claims

1. A pharmaceutical composition, characterized in that it contains . the thermosensitive polymer produced and marketed under the tradename EG56, corresponding to the chemical formula bis-methoxy PEG- 13 PEG-438/PPG-110 SMDI copolymer, and glycyrrhizin and/or its derivatives as active principle, for the preparation of products with anti-inflammatory action in stable aqueous solution.

2. The composition according to Claim 1, characterized in that the active principle is a salt and/or dster of glycyrrhizic acid.

3. The composition according to Claim 1, characterized in that the active principle is 18-beta- glycyrrhetinic acid and/or its salts and/or esters.

4. The composition according to Claims 1 to 3, characterized in that it is in the technical form of ophthalmic solution for the treatment of forms of keratitis, blepharitis, dacryocystitis, scleritis, episcleritis, uveitis.

5. The composition according to Claims 1 to 3, characterized in that it is in the technical form of drops for the disinfection of the nasal cavities and of the mouth.

6. A type formulation for 100 g of ophthalmic solution, comprising: glycyrrhizic acid and/or derivatives in a range of between 0.5 - 7 g; EG56 polymer in a range of between 0.5 - 5%; buffer solution^" with pH of 7 - 7.5 as required to reach 100 g; preserving agent and/or EDTA in an amount of 0.01 - 0.5 g; deionized water as required to reach 100 g.

7. A type formulation for 100 g of nasal/oral solution in the form of spray or drops comprising: glycyrrhizic acid and/or derivatives in a range of' between 0.5 - 7 g; EG56 polymer in a range of between 0.5 - 5%; buffer solution with pH of 7 - 7.5 as required to^' reach 100 g; preserving agent and/or EDTA in an amount of 0.01 - 0.5 g; flavouring agents required; deionized water as required to reach 100 g.

8. A formulation for ophthalmic solution comprising: glycyrrhizic acid potassium salt: 5 g; EG56 polymer: 3.5 g; buffer solution with pH of 7-7.5 as required to reach 100 g; preserving agent: 0.004 g; EDTA: 0.1 g; deionized water as required to reach 100 g- 9. A formulation for nasal/oral solution in the form of spray or drops comprising: glycyrrhizic acid potassium salt: 5 g; EG56 polymer: 3.5 g; buffer solution with pH of 7-7.5 as required to reach 100 g; preserving agent: 0.1 g; EDTA: 0.1 g; aroma: 0.1 g; deionized water as required to reach 100 g.