Peptides and peptide mimetics to treat pathologies characterized by an inflammatory response

NEW POLYMORPHIC FORMS OF RIFAXIMINE, THEIR METHODS OF PREPARATION AND THEIR USE IN MEDICINE

Context of the invention

Rifaximin (INN; see The Merck Index, XIII Ed., 8304) is an antibiotic belonging to the class of rifamycin, it is exactly a pyrido-imidazo rifamycin described and claimed in Italian Patent IT 1154655, while European Patent EP 0161534 describes and claims a process for its production starting from rifamycin O (The Merck

Index, XIII Ed., 8301).

All of these patents describe the purification of Halifaximine in a generic manner indicating that crystallization can be carried out in suitable solvents or solvent systems and showing roughly in some examples that the product from the reaction can be crystallized from 'a 7: 3 mixture of ethyl alcohol / water and can be dried both under atmospheric pressure and under vacuum without indicating anyway nor the experimental conditions of crystallization and drying, nor any distinctive crystallographic characteristic of the product obtained. The presence of different polymorphs had not been correctly noted and consequently the experimental conditions described in all the patents had been developed with the aim of obtaining a homogeneous product having a suitable purity from the chemical point of view, independently of the crystallographic aspects of the product himself. It was discovered, out of the blue, that certain polymorphic forms exist, the formation of which, besides the solvent, depends on the conditions of time and temperature at which crystallization and drying are carried out.

These polymorphic forms in order will later be designated conventionally by rifaximin δ (figui'e 1) and rifaximin ε (Figure 2) on the basis of their respective specific diffractograms reported in the present application.

The polymorphic forms of rifaximin have been characterized by the X-ray powder diffiaction technique. The identification and characterization of these polymorphic forms and, at the same time, the definition of the experimental conditions to obtain them is very important for a compound endowed with pharmacological activity which, like rifaximin, is marketed as a medical preparation, at both for human and veterinary use. In fact, it is known that the polymorphism of a compound which can be used as an active principle contained in the medicinal preparation can influence the pharmacotoxicological properties of the drug. The different polymorphic forms of an active principle administered as a medicament in oral or topical form can modify several of their properties such as bioavailability, solubility, stability, color, compressibility, fluidity and suitability for use. with the consequent modification of toxicological safety profiles,] clinical efficacy and productive efficacy.

What is mentioned above is confirmed by the authority by the fact that the authorities which regulate the issuance of the authorization for the admission of medicinal products to the market require that the manufacturing processes of the active ingredients are standardized and controlled. in such a way that they give homogeneous and fair results in terms of polymorphism of the production batches (CPMP / QWP / 96, 2003 - Advice note on the chemistry of the new Active Substance: CPMP / ICH / 367/96 - Guidance Note on Specifications: Testing Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Chemicals; Date of entry into force: May 2000).

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The need for the above stated standardization has been further strengthened in the area of ​​Kenwood s rifamycin antibiotics. Q., de Villiers Μ.Μ., Liebenberg w. and Ldtter Α.Ρ., Drug Development and Industrial Pharmacy, 26 (4), 403-408, (2000), who established that different production batches of rifampicin (INN) made from different manufacturers which differ between them because they show different polymorphic characteristics, and therefore, they show different dissolution profiles as well as the consequent change in the respective pharmacological properties.

By applying the crystallization and drying processes disclosed generically in previous patents IT 1154655 and EP 0161534, it has been discovered that under some experimental conditions the poorly crystalline form of rifaximin is obtained while under other experimental conditions the others. Polymorphic crystalline forms of rifaximin are obtained. Moreover, it has been discovered that some parameters, not absolutely revealed in the above patents, such as for example the storage conditions and the relative humidity of the environment, have a surprising effect in determining the polymorph form.

The polymorphic forms of rifaximin, the subject of the present patent application, have never been seen or presumed, whereas it is believed that a single homogeneous product would always have been obtained by any process which would have been chosen in the range of conditions described, independent of the conditions used for crystallization, drying and storage.

is now discovered that the formation of the δ and ε forms depends on the presence of water in the crystallization solvent, on the temperature at which the product is crystallized and the amount of water present in the product at the end of the drying phase. The δ form and the ε form of rifaximin were then synthesized and are the subject of the invention.

In particular the δ form is characterized by the residual water content in the dried solid matter ranging from 2.5% to 6% (w / w), more ideally from 3% to 4.5٥ / ο, while the form ε is the result of a polymorphic transition under a controlled temperature moving from the form δ.

These results are of remarkable importance since they determine the conditions for the industrial manufacture of certain work steps which could not be critical for the determination of the polymorphism of a product, such as, for example, maintaining a quantity of water for a crystallized product. in a rigorous range of values, or the process of drying the final product, in which a shape, denoted by the form δ, must be obtained before continuing the drying to obtain the form ε, or the storage conditions of the final product, or the characteristics of the container in which the product is stored.

Rifaximin exerts its broad antibacterial activity in the gastrointestinal tract against localized gastrointestinal bacteria that cause infectious diarrhea including anaerobic strains. Reports have indicated that rifaximin is characterized by negligible systemic absorption, due to its chemical and physical characteristics (Descombe JJ. Et al. Pharmacokinetic study of rifaximin after oral administration in healthy volunteers. Int ل Clin. Pharmacol. Res., 14 (2), 51-56, (1994))

We have now found that it is possible on the basis of two identified polymorphic forms of rifaximin to modulate its level of systemic absorption, and this is a part of the present invention, by administration of distinct polymorphic forms of rifaximin, designated as rifaximin δ and rifaximin ε. It is possible to have a difference in adsorption of almost 100 times in the range 0.001 to 0.3 pg / ml in blood. The demonstrated difference in bioavailability is important because it can differentiate the pharmacological and toxicological behavior of two polymorphs of rifaximin δ and ε.

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In fact, rifaximin ε is carelessly absorbed by the oral route while rifaximin δ shows mild absorption.

Rifaximin ε is not practically absorbed, and could react only by topical action, including the case of the gastrointestinal tract, with the benefit of lower toxicity. On the other route, rifaximin δ, which is moderately absorbed, we can find an advantageous use against systemic microorganisms, able to hide and partially elude the action of topical antibiotics.

Regarding the possible adverse effects coupled with the therapeutic use of rifaximin of particular importance is the induction of bacterial resistance to antibiotics. Generally speaking, it is still possible in antibiotic therapeutic practice to induce bacterial resistance to the meme or another antibiotic by selection of resistant strains.

In the case of rifaximin, this aspect is particularly relevant, since rifaximin belongs to the rifamycin family, a member of which, rifampicin, is widely used in the therapy of tuberculosis. The current short-term treatment of tuberculosis is combination therapy involving four active pharmaceutical ingredients: rifampicin, isoniazid, ethambutole and pyrazinamide and among them rifampicin plays an essential role. Therefore, any drug that has endangered selection therapy for rifampicin resistance would be harmful. (Kremer L, et al. Reemergence of tuberculosis: strategies and treatment. Expert Opin nvestig.Drugs, 11 (2), 153157, (2002)).

In principle, by looking at the structural similarity between rifaximin and rifampicin, it might be possible using rifaximin to select resistant strains of M. tuberculosis and introduce cross resistance to rifampicin. In order to avoid this negative event, it is crucial to have a check on the amount of rifaximin systemically absorbed.

From this point of view, the difference found in the systemic absorption of the δ and ε forms of rifaximin is considerable, since also at a concentration under inhibitory rifaximin, such as in the range of 0.1 to 1 gg / ml , the selection of resistant mutants has been shown to be possible (Marchese A. et al. In vitro activity of rifaximin, metronidazole and vancomycin against Clostridium difficile and the rate of selection of spontaneously resistant mutants against representative anaerobic and aerobic bacteria, including ammonia- producing species. Chemotherapy, 46 (4), 253-266, (2000)).

According to the above, the importance of the present invention, which has led to the knowledge of the existence of the polymorphic forms of rifaximin mentioned above and to various industrial routes for the manufacture of pure single forms having different pharmacological properties. , is clearly reinforced.

The δ and ε forms mentioned above can be used advantageously as pure and homogeneous products in the manufacture of pharmaceutical preparations containing rifaximin.

As already stated, the process for manufacturing rifaximin from rifamycin O disclosed and claimed in EP 0161534 is insufficient from the point of view of purification and identification of the detailed product; it shows certain limits also from the synthetic point of view with regard to, for example, very long reaction times, from 16 to 72 hours, very unsuitable for industrial use and moreover because it is not intended for in situ reduction of oxidized rifaximin which may be formed in the reaction mixture.

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Therefore, another object of the present invention is an improved process for the industrial manufacture of the δ and ε forms of rifaximin, claimed herein as products and usable as defined and homogeneous active ingredients in the manufacture of medicinal preparations containing such an active ingredient.

Description of the invention

As already said, the δ form and the ε form of the antibiotic known as rifaximin (INN), the processes for their production and their use in the manufacture of medicinal preparations for oral or topical use, are the subject of the present invention.

The object of the process of the present invention involves the reaction of one molar equivalent of rifamycin O with an excess of 2-amino-4-methylpyridine, preferably 2.0 to 3.5 molar equivalents, in a mixture of solvent. makes water and ethyl alcohol in volumetric ratios between 1: 1 and 2: 1, for a period of between 2 and 8 hours at a temperature between 40 C and 6O٥C.

At the end of the reaction the reaction mass is cooled to room temperature and it is added with a solution of ascorbic acid in a mixture of water, ethyl alcohol and aqueous concentrated hydrochloric acid, under rigorous agitation, in order to reduce the small amount of oxidized rifaximin which forms during the reaction and finally the pH is brought to about 2.0 by means of further addition of a concentrated aqueous solution of the hydrochloric acid, in order to remove most preferably the excess of 2-amino-4methylpyridine used in the reaction. The suspension is filtered and the solid obtained is washed with the same mixture of water / ethyl alcohol solvent used in the reaction. Such semi-finished product is called crude rifaximin.

The crude rifaximin can be subjected directly to the next purification step. Alternatively, if long periods of preservation of the semi-finished product are expected, the crude rifaximin can be vacuum dried at a temperature below 65٥c for a period between 6 and 24 hours, such semi-finished product is called rifaximin. raw dried.

The bmte rifaximin thus obtained and / or the crude dried rifaximin are purified by dissolving them in ethyl alcohol at a temperature between 45٥c and 65٠c and by crystallizing them by the addition of water, preferably in quantities by weight between 15%. and 70% relative to the amount by weight of ethyl alcohol used for the dissolution, and keeping the suspension obtained at a temperature between 5O٥C and o٥c with stirring for a period between 4 and 36 hours.

The suspension is filtered and the solid obtained is washed with water and dried under vacuum or at normal pressure, with or without drying agent, at a temperature between room temperature and 10C for a period of time of 2 and 72 hours. .

The fulfillment of the δ and ε forms depends on the conditions chosen for crystallization. In particular, the composition of the solvent mixture from which crystallization is carried out, the temperature at which the reaction mixture is maintained after crystallization and the period of time at which this temperature is maintained, have been proven to be critical.

More precisely, the rifaximin δ and ε are obtained when the temperature is first brought to a value between 28٠c and 32٥c in order to cause the beginning of crystallization, then the suspension is brought to a temperature between 4O٥C and 5O٥C and kept at this value. for a period between 6 and 24 hours, then the suspension is cooled rapidly to o dansc, in a period of time between 15 minutes and one hour, is filtered, the solid is washed with water and it is then dried.

Λ

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The drying step has an important part in obtaining the polymorphic forms δ and ε of rifaximin and must be verified by means of a suitable method suitable for the determination by water, such as for example the Karl method. Fisher, to verify the amount of water present in the product under drying.

Obtaining rifaximin δ during drying actually depends on the amount of water remaining at the end which should be between 2.5% (w / w) and 6% (w / w), preferably between 3% and 4.5%, and not under experimental conditions of pressure and temperature at which this critical limit of percent water is achieved.

To obtain the poorly absorbed ε form, it is necessary to start with the δ form and it must continue drying under vacuum or at atmospheric pressure, at room temperature or at high temperatures, in the presence or absence of drying agent, at provided that the drying is prolonged for the time necessary so that the transformation into ε form is completed.

Both the δ and ε forms of rifaximin are hygroscopic, they absorb water in a reversible manner over time under the presence of suitable conditions of pressure and humidity at room temperature and are susceptible to transformation at other forms. Transitions from one form to another are found to be very important within the scope of the invention, as they can be an alternative manufacturing process for obtaining the desired form for the production of medicinal preparations. Accordingly, the process which allows rifaximin δ to be returned to rifaximin ε in a valid industrial manner is an important part of the invention. The process of converting rifaximin δ to rifaximin ε involves drying the rifaximin δ under vacuum or at atmospheric pressure, at room temperature or at elevated temperatures, in the presence or absence of drying agents, and in holding it for a period of time until transformation is achieved, usually between 6 and 36 hours. From the above, it follows that during the preservation phase of the product special care must be taken so that the ambient conditions do not change the water content of the product, by preserving the product in an ambient temperature having a controlled humidity or in closed containers which do not allow significant exchange of water with the external environment. The polymorph called rifaximin δ is characterized by a water content in the range between 2.5% and 6%, preferably between 3.0٥ / ο and 4.5% and an x-ray difftactogram of the powder (reported in figure 1) which shows peaks at 2Θ diffraction angle values ​​of

,12.1٥±0.2 ,11.3٥±0.2 ,10.8°±0.2 ,10.4٥±0.2 ,8.7٥±0.2 ,8.0٥±0.2 ,7.1٥±0.2 ,6,7٠±0,2 ,5,7٠±0,2

17.0٥ ± 0.2, 17.3٥ ± 0.2, 17.5٥ ± 0.2, 18.5٥ ± 0.2, 18.8٥ ± 0.2, 19.1٥ ± 0.2, 21.0٥ ± 0.2, 21.5٠ ± 0.2. The polymorph called rifaximin ε is characterized by the X-ray difftactogram of the powder (reported in Figure 2) which shows peaks at 2Θ diffraction angle values ​​of

ة 0.2 لجه 14.5, 0.2 ± 12.4٥, 0.2 ± 11.7٥, 0.2 ± 1٥ .0.2,11 ± 10.3٥, 0.2 ± 8.7٥, 0.2 ± 8.2٥, 0.2 ± 7.3٥, 700.2

163Ο.2, 17.2٥ ± 0.2, 18.0٥ ± 0.2, 19.4٥ ± 0.2.

The diffractograms were performed using the Philips X'Pert instrument with Bragg-Brentano geometry and under the following operating conditions:

X-ray tube tube: Copper

Radiation used: K (al), K (a2)

Generator voltage and current: KV 40, mA 40

Monocromateur: Graphite step size: 0.02

Time per step: 1.25 seconds

Start and end angle 2 © value: 3.0٥ 0.0 °

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The evaluation of the water content present in the samples analyzed has always been carried out by means of the Karl Fisher method. Rifaximin δ and rifaximin ε also differ from each other because they show significant differences in bioavailability.

The bioavailability study of two polymorphs was carried out on female Beagle dogs, they are treated orally with a dose of 100 mg / kg in a capsule from one of the polymorphs, by collecting blood samples from the jugular vein of each animal before each assay and 1,2,4,6,8 and 24 hours after each assay, the samples are transferred to tubes containing heparin and the plasma is separated by centrifugation.

Plasma was tested for rifaximin on valid LC-MS / MS method and observed maximum plasma concentration (Cmax), time to reach Cmax (tmax), and area below concentration curve - time (AUC) were calculated.

The experimental data reported in the following Table 1 clearly show that rifaximin ε is absorbed negligibly, while rifaximin δ is absorbed at a value (Cmax = 0.308 pg / ml) in the range of 0.1 to 1.0 pg / ml .

Table 1

Pharmaceutical Parameters for Polimorphs of Rifaximin Polimorphs Following Single Oral Administration of 100 mgkg per Capsule to Female Dogs

<td></td><td>w</td><td>Tmax h</td><td>AUCO-24 Ng.hAnl</td>

<td></td><td>Average</td><td>Average</td><td>Average</td>

<td>Polymorphic δ</td><td> 308.31</td><td> 2</td><td> 801</td>

<td>'Polymorphic ε</td><td> 6.86</td><td> 4</td><td> 42</td>

The above experimental results further show the differences existing between two polymorphs of rifaximin.

The δ and ε forms can be advantageously used in the production of medicinal preparations having antibiotic activity, containing rifaximin, for both oral and topical use. Medicinal preparations for oral use contain rifaximin δ and ε together with the usual excipients as a diluting agent such as mannitol, lactose and sorbitol; fixing agents such as starches, gelatins, sugars, cellulose derivatives, natural gums and polyvinylpyrrolidone; lubricating agents such as talc, stearates, hydrogenated vegetable oils, polyethylenglycole and colloidal silicone dioxide; disintegrating agents such as starches, celluloses, alginates, gums and crosslinked polymers; coloring, fragrance and sweetening agents.

All solid orally administrable preparations can be used within the scope of the present invention, for example coated and uncoated tablets, capsules made of hard and soft gelatin, sugar coated pills, lozenges, wafer sheets. , lozenges and powders in closed packages.

Medicinal preparations for topical use contain rifaximin δ and ε together with the usual excipients such as white petrolatum, white wax, lanolin and their derivatives, stearyl alcohol, propylenglycol, sodium lauryl sulfate, fatty polyoxyethylene alcohol ethers, fatty polyoxyethylene acid esters, sorbitan monostearate, glyceryl monostearate, propylene glycol monostearate, polyethylene glycols, methylcellulose, hydroxymethylpropylcellulose,

٠ΜΑ 29488Β1 sodium carboxymethylcellulose, colloidal aluminum magnesium silicate, sodium alginate.

All topical preparations can be used within the scope of the present invention, for example ointments, ointments, creams, gels and lotions.

The invention of the present is illustrated below by some examples which should not be taken as a limitation of the invention: from what is described it results in evidence that the forms δ and ε can be obtained by suitably combining the above mentioned conditions of crystallization and drying.

Example 1

Preparation of crude fifaximine and crude rifaximin

In a three-necked flask equipped with a mechanical stirrer, a thermometer and a reflux condenser, 120 ml of demineralized water, 96 ml of ethyl alcohol, 63.5 g of rifamycin o and 27.2 g of 2-amino-4- methylpyridine are charged in succession at room temperature. After loading, the mass is heated to 473 ؛ OC, is kept under stirring at this temperature for 5 hours, then it is cooled to 2O + 3OC and, for 30 minutes, it is added to a mixture, prepared separately, made of 9 ml of demineralized water, 12.6 ml of ethyl alcohol, 1.68 g of ascorbic acid and 9.28 g of concentrated aqueous hydrochloric acid. At the end of the addition, the mass is kept under stirring for 30 minutes at an internal temperature of 2O ± 3٥C and then, at the same temperature, 7.72 g of concentrated hydrochloric acid are drained to a pH equal to 2.0 .

At the end of the addition, the mass is kept under stirring, still at an internal temperature equal to 2O٥C, for 30 minutes, then the precipitate is filtered and washed by means of a mixture made of 32 ml of demineralized water and of 25 ml of ethyl alcohol, the crude rifaximin thus obtained (89.2 g) is dried under vacuum at room temperature for 12 hours, obtaining 64.4 g of dry crude rifaximin which shows a water content equal to 5.6%. The product by further drying under vacuum up to the weight of 62.2 g of dry crude rifaximin having a water content equal to 3.3%, the diffractogram of which corresponds to the polymorphic form δ characterized by a diffractogram of powder subjected to rays X showing peaks at 2Θ angle values ​​of 5.7٥ ± 0.2, 6.7٠ ± 0.2.7. 7.3٥ ± 0.2, 8.0٥ ± 0.2,

,17.5٥±0.2 ,17.3٥±0.2 ,17.0٥±0.2 ,12.1٥±0.2 ,11.3٥±0.2 ,10.8٥±0.2 ,10.4٠±0.2 ,8.7٠±0.2

18.5 ° ± 0.2, 18.8٥ ± 0.2, 19.1٠ ± 0.2, 21.0٠ ± 0.2,21.5٥ ± 0.2. The product is hygroscopic.

Example 2

Preparation of rifaximin ε

Example 1 is repeated and after obtaining the forme form, the solid powder is further dried under vacuum for 24 hours at a temperature of 65٥c. The product obtained is rifaximin ε characterized by the diffractogram of powder subjected to X-rays showing peaks at 2Θ angle values ​​of 7.0٥ ± 0.2, 7.3٥ ± 0.2, 8.2٥ ± 0.2, 8.7٥ ± 0.2, 10.3٥ ± 0.2, 11.1٥ ± 0.2, 11.7٥ ± 0.2, 12.4٥ ± 0.2, 14.5٥ ± 0.2, 16.3٥ ± 0.2, 17.2٥ ± 0.2, 1800.2, 19.4٥Ο.2.

Example 3

Oral bioavailability in dogs

Eight purebred female Beagle dogs aged 20 weeks and weighing between 5.0 and 7.5 kilograms were divided into two groups of four. The first of these latter groups were treated with rifaximin δ, the second with rifaximin ε according to the following procedure.

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Each dog was orally administered 100mg / kg of one of the rifaximin polymorphs in capsules and blood samples of 2ml each were collected from the jugular vein of each animal prior to dispensing and 1.2 times. 4.6.8 and 24 hours after administration.

Each sample was transferred to a tube containing heparin as an anticoagulant and centrifuged; the plasma was divided into two aliquots, each 500 μ.1, and was frozen at -2O٥C. Plasma rifaximin was analyzed using the validated LC-MS / MS method and the following parameters were calculated according to the standard non-compartmental analysis:

Cmax = maximum observed plasma concentration of rifaximin in plasma; Tmax = time at which Cmax is reached;

AUC = area below the concentration-time curve calculated by the linear trapezoidal rule.

The results reported in Table 1 clearly show how rifaximin δ is also more absorbed, more than 40 times, compared to rifaximin ε, which is virtually unabsorbed.