- 1 -

SIB BI4214R

ASSOCIATION OF XANTHINE OXIDASE INHIBITORSANDMETFORMINAND USE THEREOF

DESCRIPTION

TECHNICAL FIELD OF THE INVENTION

Thepresent invention relates to the association of active principles, i.e. of a xanthine oxidase inhibitorwith a biguanide derivative, preferablymetformin, pharmaceutical compositions comprising saidactive principles, for use in a human or veterinary therapeutic treatment, and methods for the preparation thereof.

Such associations and such compositionsproved particularly effective in the treatmentof hyperglycemia,alone or in association withhyperuricemiaor to other disordersin the clinical context of the metabolic syndrome.

STATE OF THE PRIOR ART

Gout is an invalidating chronic diseasecharacterized by hyperuricemiaand deposition of monosodium urate crystalsin various tissues, mainly at the joint level and in the kidney. Hyperuricemiaandgoutare frequently associated to other cardiovascular risk factorssuch as hypertension, hypercholesterolemiaand other elements that are partof themetabolic syndrome, like obesity, fasting hyperglycemia, low HDL levels and high triglyceride levels.

Hence, the need to always havenovel means of treatmentin order to better managechronic therapy ofgoutand pathologies frequently correlated thereto.

A xanthine oxidase inhibitorwell-known in the literatureis allopurinol. More recently, other xanthine oxidase inhibitors have appeared on the market;among them, febuxostat is of particular relevance.

Febuxostat is a powerful non-purine selective inhibitor of xanthine oxidasewhich in clinical studies has been shown to reducehyperuricemiamore effectively than allopurinol.

Febuxostat is a thiazole derivative having formula (I), belonging to the class of xanthine oxidase inhibitors,and was originally described in EP513379.

( I )

In EP1020454 it is also described a polymorphic form of febuxostat and a process for obtaining it.

In addition to its use asanti-hyperuricemic agentand in the treatment of gout, references are also found to the potential use of febuxostat in other pathologies.

In WO2004060489 it is described the use ofxanthine oxidase inhibitors for increasing cardiac contractility in CHF (Chronic Heart Failure) patients.

In WO2007062028 febuxostat is used to reduce the QT intervalin patients in whichsuch interval is prolonged, and in the pathologies associated thereto.

In WO2008064015 the use ofxanthine oxidase,among which febuxostat,is indicated to preserve renal function.

In WO2007019153 it is claimed the use ofsomexanthine oxidase inhibitors, among which febuxostat,preferably for the treatment of prehypertensioncharacterized bysystolic pressure between 120 and 139 mmHg and diastolic pressure between 80 and 89 mmHg; here, xanthine oxidase inhibitors seem to be indicatedalso in the treatment of more marked hypertensions, though results obtaineddo not seem to be equal to those of already known anti-hypertensive agents.

Hyperglycemiais successfully treatedwithseveral drugs belonging to different therapeutic classes. Among them, Metformin, a biguanide derivative which has been used in type 2 diabetes therapy for nearly 50 years, must be considered of particular relevance.

Metforminreduces glycemia by decreasing hepatic production of glucose and enhancing insulin action at the level of the musclesand fat. Immediate-release formulationsare generally administered once or twice per day, whereas the prolonged-release formulations are administeredonce per day.

Butturini, U. et al. in Clinica Terapeutica (1976), 77 (6), 529-541, has shown that,in subjects with primary hyperuricemia, treatment with allopurinolandmetformincan gradually improve hydrocarbon utilization, as well as lipid metabolism.

SUMMARY OF THE INVENTION
The present invention is based on the surprising discoverymade by the Inventorsthat the association of a xanthine oxidase inhibitor, in particular febuxostat, or pharmaceutically acceptable salts thereofor polymorphic forms thereof, in combination with a biguanide derivative, preferablymetforminor pharmaceutically acceptable salts thereof,exhibits a synergistic therapeutic effectin the treatment ofhyperglycemia. In fact, experimental data reported in the present description demonstratethat the therapeutic effect resulting from the association of the twoactive principlesis greater than the sumof the therapeutic effectsresulting from the same dosagesof eachactive principleadministered alone.

A first object of the present inventionis an association of theactive principles:

a) xanthine oxidase inhibitor, febuxostat or pharmaceutically acceptable salts thereofor polymorphic forms thereof; and

b) metforminor pharmaceutically acceptable salts thereof

for use in a human or veterinary therapeutic treatment.

A second objectof the present inventionis a pharmaceutical compositioncomprising, as active principle,a mixture of:

a) xanthine oxidase inhibitor, febuxostat,or pharmaceutically acceptable salts thereofor polymorphic forms thereof;

b) metforminor pharmaceutically acceptable salts thereof, and

one or morepharmaceutically acceptable excipientsand/oradditivesand/ordiluents, for use in a human or veterinary therapeutic treatment.

Another object of the present invention is a method for the preparation of the compositionaccording to the present description, wherein the active mixturecomprising

a) the xanthine oxidase inhibitor, febuxostat,or pharmaceutically acceptable salts thereofor polymorphic forms thereof;

b) metforminor pharmaceutically acceptable salts thereof,

is formulated in suitable dosage unitswith one or moreusual pharmaceutically acceptable excipients and/oradditives.

With respect to the state of the prior art, the invention entails the advantage of a greateranti-hyperglycemic activity compared to that observedusing the solemetforminor the solexanthine oxidase inhibitor. Moreover, a further advantage is givenby the possibility of obtainingsignificant effectsin the treatment of hyperglycemiawith a reduced amount ofmetforminwith respect to the monotherapy treatment.

DETAILED DESCRIPTION OF THE INVENTION

Thepresent invention relates toan association of theactive principles:

a) xanthine oxidase inhibitor, febuxostat,or pharmaceutically acceptable salts thereofor polymorphic forms thereof; and

b) metforminor pharmaceutically acceptable salts thereof

for use in a human or veterinary therapeutic treatment.

By "association" in the present description it is meantan association of theactive principles,both in the form of a physical mixtureconstituted by said active principles in a single dosage unitand in the form of dosage units physically separated for each active principle, but intended for a concomitant administration. In both cases, association must ensurea synergy of the therapeutic effectsobtained from the individualactive principleswith respect to the effect obtained in monotherapy.

According to the present invention,the non-purine xanthine oxidase inhibitorof said associationis preferably febuxostat, a thiazole derivative having formula (I), or pharmaceutically acceptable salts thereofor polymorphic forms thereof.

Pharmaceutically acceptable salts of xanthine oxidase inhibitors, and in particular of febuxostat, include but are not limited to cations of alkali metals and of alkaline earth metals, such as lithium, sodium, potassium, calcium, magnesium or aluminium salts, or non-toxic derivatives with quaternary ammoniumand cations of amines such as ammonium, tetramethylammonium, tetraethylammonium, methylammonium, dimethylammonium, trimethylammonium, or derive from the additionof organic aminessuch as ethylendiamine, ethanolamine, diethanolamine, piperazine, tromethamine, lysine, arginine and the like.

Polymorphic forms of febuxostat include, but are not limited to the forms described in European Patent EP1020454.

Febuxostat, its saltsor polymorphic forms thereofcould be obtained or preparedaccording to methods described in the known art, like e.g. in EP513379.

In case of some polymorphic forms,reference could be made to what is disclosed in European Patent EP1020454.

The secondactive principle of the associationis abiguanide derivative, i.e.metformin. Pharmaceutically acceptablesalts ofmetformin, having a basic function in the molecule, include but are not limited to those formally deriving from treatment with inorganic or organicacids selected from: hydrochloric, hydrobromic, sulfuric, phosphoric, carbonic acids, or organic acids, such as acetic, benzenesulfonic (besylate), methanesulfonic, maleic, malonic, succinic, aspartic, glutamic acid.

In a preferred embodimentthe pharmaceutically acceptable saltismetformin chlorhydrate.

In the association of the invention, the biguanide derivative, metforminor pharmaceutically acceptable salt thereofis associated to the xanthine oxidase inhibitor, febuxostat,or pharmaceutically acceptable salts thereofor polymorphic forms thereof, in a weight ratio of metformin/febuxostat comprised between0.4 and 150, or between 2 and 40.

E.g., the following amounts, expressed in grams per single dose, could be associated: febuxostat in an amount comprised between 10-200 mg, or better between 25-100 mg, in association with an amount ofmetformincomprised between 100-1500 mg, e.g. between 250-100 mg.

Where the association envisages a physical mixture of two compounds, as active principles,having the one an acid function and the other one a basic function, also the forming of an internal salt between the two is possible,in proportion to the respective amounts present in the mixture.

A further embodimentof the present inventionrelates to pharmaceutical compositions comprising, as active principle,a mixture of:

a) xanthine oxidase inhibitor, febuxostat,or pharmaceutically acceptable salts thereofor polymorphic forms thereof;

b) metforminor pharmaceutically acceptable salts thereof, and

one or morepharmaceutically acceptable excipientsand/oradditivesand/ordiluents, for use in a human or veterinary therapeutic treatment.

The pharmaceutical compositions according to the present inventionmay be formulated in a variety of waysdepending on the selected administration route. According to a specific embodiment of the invention, the pharmaceutical compositionis suitable for oral administration of solid formsand may include formssuch as capsules, tablets, pills, powders and granules. In these solid forms the two active principles, xanthine oxidase inhibitorand metformin, can be mixed with one or more pharmaceutically acceptable inert excipients. Such excipients may be selected among those commonly known in the state of the art and include, but are not limited to: a) carriers, such as sodium citrate and calcium phosphate, b) fillers, such as starch, lactose, microcrystalline cellulose, sucrose, glucose, mannitol and colloidal silica, c) moistening agents, such as glycerol, d) disintegrating agents, such as alginates,calcium carbonate, starches,derivatives of starch, of celluloseand polyvinylpyrrolidone, silicates and sodium carbonate, e)binders, such as carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, polymeric derivatives of cellulose, starch derivatives, f)retarding agents, such as paraffin,cellulose polymers, fatty acid esters, g) absorption accelerators, such as quaternary ammonium compounds, h) wetting agents and surfactants, such ascetyl alcohol and glycerol monostearate, i) adsorbents, such as bentonite clays and kaolin, k) lubricants, such astalc, calcium stearate, magnesium stearate,polyethylene glycol, sodium lauryl sulfate, sodium stearyl fumarate, j)glidants, such as talc, colloidal silica.

In case the selected compositions constitute the filling of gelatin capsules, the excipients include but are not limited to compoundsof the type: lactose, high molecular weight polyethylene glycol, and the like.

Solid-dosage forms, such as tablets, capsules, pills and granules, may be coated with enteric, gastric coatings, or coatings of other typewell-known in the state of the art. They may contain matting agentsand may beof the typesuch as to allow the release of active ingredientsonly or preferably in a certain section of the intestine, optionally in a delayed manner. Substances capable of allowing such a delayed use include, but are not limited to polymers and waxes.

Liquid forms suitable for oral administration are emulsions, solutions,prepared or extemporary suspensions, syrups andelixirs. Excipients suitable for the formulations according to the present inventionin liquid forms for oral useinclude but are not limited to diluentscommonly used in the art, such as water or other solvents, solubilizing and emulsifying agentsselected fromethyl alcohol, polyalcohols, propylene glycol, glycerol,polyethylene glycol and sorbitan esters. These formulations can also containsweeteners and aromasselected fromthose well-known in the state of the art.

Compositions suitable for pharmaceutically acceptable parenteral injectionsmay comprise sterile aqueous solutions, sterile dispersions, suspensions or emulsions or powdersfor a reconstitution ininjectable solutions or dispersions; examples of excipients suitable thereforinclude, but are not limited to aqueous or non-aqueous carriers, diluents, solvents or vehiclesselected from: water, ethanol, polyoils (propylene or polyethylene glycol, glycerol, and the like), polyalcohols, isopropyl alcohol, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1.3-butylene glycol, dimethylformamide, vegetable oils (in particularof olive, cotton, peanut, corn, wheat germ, olive, castor, sesame), organic esters such as ethyl oleate or the like.

These compositions may also contain preservativesof antibacterial or antifungal type, selected, yet not exclusively, from: paraben, chlorbutanol, phenol, sorbic acid and the like. It may also be useful to include an isotonic agent, e.g., a sugar, sodium chloride or the like. Moreover, pharmaceutical forms with a delayed absorption may be obtainedwith agents such as, for instance, yet not exclusively, aluminium monostearate and gelatin.

The suspensions, beside the active principles (xanthine oxidase inhibitorsandmetformin), may contain suspending agents such as, for instance, yet not exclusively,ethoxylated isostearic alcohols, polyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminium hydroxide, bentonite, alginates and cellulose derivativesin general or the like.

The right fluidity can be maintained with a coating materialsuch as lecithin, with the maintaining of the right particle sizes in the dispersions or with the use of surfactants.

Also slow-release formulations can be prepared, by the techniques and products well-known in the state of the art.

The pharmaceuticalassociations and compositionsof the inventionare extremely effectivein the treatment, prophylactic as well as therapeutic,of hyperglycemia, in humans and in animals.

Hyperglycemiacan be associated or not associatedto other pathologies or syndromes and symptoms. In particular, the associations and compositions described hereinare useful also in thetherapeutic treatment of hyperglycemiaassociated to hyperuricemia.

Manifestationssuch ashyperglycemiaandhyperuricemia, individually or in combination, can also be associated to specific syndromeslike themetabolic syndrome.

By"metabolic syndrome"it is meant a clinical condition accompanied by various manifestationssuch as obesity.

The association described hereincan therefore be usedin the therapeutic treatment of hyperglycemiaassociated to hyperuricemiaor other pathologiesin the context of the metabolic syndrome.

Dosage may vary depending on the patient's age and general conditions, the nature and seriousness of the pathology or disorderand of the administration route and type. Dosage should therefore take into accountthe specific condition to be treated (e.g., hyperglycemia alone or in association withhyperuricemia), the severity of the condition to be treated, the age, weight and general physical conditions of the specific patient, as well as other drugs that the patient is taking, as is well-known to those skilled in the art. Moreover, it is evident that said effective amountmay, when required, be lowered or raisedaccording to the responses of the treated patientand/oraccording to the assessment of the physician prescribing the compoundsof the present invention.

Typically,compositions for oral use in solid formcan contain an amount ofxanthine oxidase inhibitor, specifically febuxostat, of between 10 and 200 mg per single dose, and preferably of between 25 and 120 mg, and an amount ofmetformin, preferablymetforminhydrochloride, of between 100 and 1500 mg per single dose, preferablyof between 250 and 1000 mg.

By the term "dosage unit" in the present description it is meant the unitary formulation for a single administration, e.g. a tablet, capsule, etc.

By "unit dosage" it is meantthe amount of active principle for a single administration.

The pharmaceutical mixtures and compositions of the inventioncould be prepared according to techniques known in the field,both using the previously prepared association of active principles, and mixing the individual compounds directly during the preparation of the composition.

In particular, the association of active principles may be obtained by a step of mixingmetforminor pharmaceutically acceptable salts thereofwiththe xanthine oxidase inhibitor, febuxostat,or pharmaceutically acceptable salts thereofor polymorphic forms thereof,in a weight ratio comprised between0.4 and 150, or between 2 and 40.

For the preparation of the pharmaceutical compositions described hereinthe mixture of active principlesis formulated in suitable dosage unitswith one or morepharmaceutically acceptable excipients and additives.

Testing

Testing demonstrating activity of the associations according to the invention is reported hereinafter.

1. Biological activity measurement

Hypoglycemizing activity ofmetformin, alone or in association with febuxostat, was assessedin Wistar rats having a weight of 240-260 g (Harlan Laboratories, Udine, Italy). To induce diabetes, animals were treatedwith a single intraperitoneal injectionof streptozotocin (STZ, 65 mg/kg) dissolved in citrate buffer at pH 4.5. The control groupreceived an equivalent volume of citrate buffer. Fasting plasma glucose levelswere determined with a commercial device (Glucocard G-meter, Menarini Diagnostics). Blood was collected from a tail vein. At+4 weeksfrom the treatmentwith STZ a significant increase of fasting glycemiawas measured, from 4.17±0.45 mmol/l (75 mg/dl) in the controlsto 11.57±0.70 mmol/l (208 mg/dl) in diabetic rats, with a 180% increase (n=5). These values were maintained in the two successive weeks.

Two administration protocols were performed, an acute one and a chronic one, for two weeks.

Acute model. Metforminat the dose of 100 mg/kg per osdid not significantly modifythe hyperglycemic curvein the 5 hours of observation, whereas at the doseof 300 mg/kg per osit reduced by 70% the high bloodglucose levels in the period between2 and 5 hoursafter administration. Febuxostat, with a single dose of 5 mg/kg per os, did not show positive effects onhyperglycemia, whereas coadministrationwithmetformin (100 mg/kg per os) yielded a hypoglycemizing effect comparable to that ofmetformin at 300 mg/kg per os.

Chronic model. Febuxostat and/ormetformin, alone or in association, were administered orally once per dayfor 2 weeks, at the dose of 5 mg/kg for febuxostat and of 100-300 mg/kg formetformin. Plasma levels of glycemiawere measured at the end of thefirst and of the second week. Febuxostat did not significantly modifyhyperglycemiavalues in the 2 weeks of observation, whereasmetforminshowed a consistent reduction of blood glucoseat the dose of 300 mg/kg per osalready at the end of the first week of treatment, reduction that was maintained also in the second week. The combined administrationof febuxostat andmetforminyielded maximal resultsin reducing the high levels of glycemia at the daily doses of 5 mg/kg per osof febuxostat and of 100 mg/kg per osofmetformin, comparable to the result obtainedwith 300 mg/kg per os.

The results obtainedwith both experimental protocolsdemonstrated a surprising synergistic effect, suggesting that the two compounds have a favourable and unexpectedinteractionthat can yield positive effectson hyperglycemia, reducing the effective doses ofmetformin.

This observation may have interesting repercussions at the therapeutic level on diabetes control.

Example 1 (with immediate-releasemetformin)

tablet for oral administration, containing:

febuxostat 120 mg

metformin HCl 1000 mg

macrogol 4000 24.7 mg

lactose 40 mg

microcrystalline cellulose 4.8 mg

colloidal silica 0.5 mg

croscarmellose sodium (disintegrant) 10 mg

magnesium stearate (lubricant) 0.8 mg

yellow iron oxide 1.0 mg

red iron oxide 1.0 mg

Example 2 (with immediate-releasemetformin)

tablet for oral administration, containing:

febuxostat 80 mg

metformin HCl 850 mg

macrogol 4000 24.7 mg

lactose 60 mg

microcrystalline cellulose 7.2 mg