Pharmaceutical Compositions based on Crystalline Form I of 5-Azacytidine

ES 2 425 474 T3

DESCRIPTION

Methods for Isolating Crystalline Form I of 5-Azacytidine

Field of the invention

The invention relates to the isolation of crystalline polymorphic Form I of 5-azacytidine (also known as azacytidine and 4-amino-1-pD-ribofuranosyl-S-triazin-2 (1H) -one). 5-Azacytidine can be used in the treatment of diseases, including the treatment of myelodysplastic syndromes (MDS).

Background of the invention

Polymorphs exist as two or more crystalline phases that have different arrangements and / or different conformations of the molecule in a crystal lattice. When a solvent molecule (s) is contained within the crystal lattice, the resulting crystal is called a pseudopolymorph, or solvate. If the solvent molecule (s) found in the crystal structure is (are) a water molecule (s), then the pseudopolymorph / solvate is called a hydrate. Polymorphic and pseudopolymorphic solids exhibit different physical properties, including those due to packing, and various thermodynamic, spectroscopic, interfacial, and mechanical properties (see H. Brittain, Polymorphism in Pharmaceutical Solids, Marcel Dekker, New York, NY, 1999, pages 1- 2). The polymorphic and pseudopolymorphic forms of the drug substance (also known as the “active pharmaceutical ingredient” (IFA)) administered as such, or formulated as a pharmaceutical product (also known as the final or finished dosage form, or as the pharmaceutical composition ) are well known and can affect, for example, solubility, stability, flowability, fractability and compressibility of drug substances and to the safety and efficacy of pharmaceuticals (see, for example, Knapman, K Modem Drug Discoveries, March 2000: 53).

5-Azacytidine (also known as azacytidine and 4-amino-1-pD-ribofuranosyl-S-triazin-2 (1H) -one); Nation Service Center designation NSC-102816; CAS Registry Number 320-67-2) has undergone NCI-sponsored trials for the treatment of myelodysplastic syndromes (MDS). See Komblith et al., J. Clin. Oncol. 20 (10): 2441-2452 (2002) and Silverman et al., J. Clin. Oncol. 20 (10): 2429-2440 (2002). 5-Azacytidine can be defined as having a formula of C8H12N4O5, a molecular weight of 244.20 and a structure:

<img file="ES2425474T3_D0001.tif" />

In United States Patent Application Serial Number 10 / 390,578 (United States Patent Number

6,887,855) entitled "Forms of 5-azacytidine" filed March 17, 2003, describes eight different polymorphic and pseudopolymorphic forms of 5-azacytidine (Forms I-VIII), in addition to an amorphous shape. Each of Forms I-VIII have characteristic X-Ray Powder Diffraction (XRPD) patterns and are easily distinguished from one another using XRPD.

US 3,817,980 describes the synthesis of certain 5-azapyrimidine nucleosides by reacting a 1-O-acyl-1-O-alkyl derivative or a 1-halo derivative of a blocked sugar residue with a silylated 5azacytosine compound at presence of a Lewis acid. The residue containing the 5-azacytidine is crystallized from wet methanol.

MW Winkley and RK Robins, The Journal of Organic Chemistry (1970) 491-495, describe the synthesis of 5-azacytidine by direct glycosylation of 1,3,5-triazines. The material is crystallized from aqueous ethanol.

US 3,891,623 describes the synthesis of cytidine and cytidine derivatives by reacting a 4trialkylsilyloxyuridine derivative with ammonia or a primary or secondary amine, or with a salt of a primary or secondary amine in the presence of a tertiary amine.

US 6,723,728 describes polymorphic forms of (-) - and (±) -cis-FTC (4-amino-5-fluoro-1- (2- (hydroxymethyl) -1,3oxathiolan-5-yl) -2 (1H) -pyrimidinone). The hydrated crystalline form of (±) -cis-FTC is obtained by dissolving (±) -cis-FTC in

ES 2 425 474 T3 with water and recrystallizing the FTC.

GB 1 227 691 A describes the isolation of 1-glycosyl-S-azacytosine as a solid in certain reaction mixtures containing methanol.

A. Píscala et al., Nucleid Acid Chemistry (1978) 435-441 describe the isolation of 5-azacytidine as a solid in a reaction mixture. The reaction was carried out in methanol. The solid was then crystallized from water-acetone.

A. Piscala et al., Collection of Czechoslovak Chemical Communications (1964) 2060-2069, describe the isolation of 5azacytidine as a solid in a reaction mixture. The reaction was carried out in methanol. The solid was then crystallized from water-methanol.

Beisler JA et al., Journal of Carbohydrates (1977) 281-299 describe the isolation of 5-azacytidine as a solid in certain reaction mixtures. Reactions were carried out in methanol.

Beisler JA et al., Journal of Medicinal Chemistry (1978) 204-208 describe the crystallization of 5-azacytidine in ethanowater.

In United States Patent Application Serial Number 10 / 390,578 (United States Patent Number

6,877,855) entitled "Forms of 5-azacytidine" filed March 17, it is demonstrated that this prior art procedure for the recrystallization of the crude synthesis product does not control the polymorphic forms of 5-azacytidine. Specifically, the prior art recrystallization process produces either Form I substantially free of other forms, or a mixed phase of Form I / II, that is, a solid material in which 5-azacytidine is present in a mixed phase of polymorphic Form I and polymorphic Form II. Thus, prior art methods do not allow Form I to be reliably targeted as the only polymorphic form in the drug substance. The present invention provides processes that allow 5azacytidine to be recrystallized as polymorphic Form I in a robust and reproducible manner.

Summary of the invention

The present invention provides methods for the robust and reproducible isolation of 5-azacytidine as a polymorphic Form I, substantially free of the other forms. The methods involve recrystallizing 5-azacytidine dissolved in a DMSO / co-solvent mixture and then collecting the resulting crystals. Pharmaceutical compositions comprising 5-azacytidine Form I together with a pharmaceutically acceptable excipient, diluent or carrier are also disclosed.

Detailed description of the preferred embodiments

Polymorphic 5-azacytidine Form I

In United States Patent Application Serial Number 10 / 390,578 (United States Patent Number

6,877,855) entitled "Forms of 5-azacytidine" filed March 17, describes Form I of 5-azacytidine. Table 1 provides the most prominent 2Θ angles, relative intensity separations for Form I observed using X-ray Powder Diffraction (XRPD) carried out according to the procedure of Example 4:

<td>Angle 2Θ (°)</td><td>Separation d (A)</td><td>Relative intensity</td>

<td> 12,182</td><td> 7,260</td><td> 39,1</td>

<td> 13,024</td><td> 6,792</td><td> 44,1</td>

<td> 14,399</td><td> 6,146</td><td> 31,5</td>

<td> 16,470</td><td> 5,378</td><td> 27,1</td>

<td> 18,627</td><td> 4,760</td><td> 16,0</td>

<td> 19,049</td><td> 4,655</td><td> 35,9</td>

<td> 20,182</td><td> 4,396</td><td> 37,0</td>

<td> 21,329</td><td> 4,162</td><td> 12,4</td>

<td> 23,033</td><td> 3,858</td><td> 100,0</td>

<td> 23,872</td><td> 3,724</td><td> 28,0</td>

<td> 26,863</td><td> 3,316</td><td> 10,8</td>

<td> 27,135</td><td> 3,284</td><td> 51,5</td>

<td> 29,277</td><td> 3,048</td><td> 25,6</td>

<td> 29,591</td><td> 3,016</td><td> 11,5</td>

<td> 30,369</td><td> 2,941</td><td> 10,8</td>

<td> 32,072</td><td> 2,788</td><td> 13,4</td>

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Table 1: 5-Azacitidine Form I - 2Θ angles, most prominent relative intensity separations (Ka radiation from Cu)

Isolation of Polymorphic 5-Azacytidine Form I by Recrystallization

Form I 5-azacitidine, substantially free of other forms, can be reproducibly isolated by recrystallizing dissolved 5-azacytidine and collecting the resulting crystals. Specifically, 5-azacytidine is fully dissolved in dimethyl sulfoxide (DMSO) first.

The 5-azacytidine used to form the solution can be synthesized by any method known in the art; An example synthesis scheme is provided in Example 1. Any polymorphic or pseudopolymorphic form (s) of 5-azacytidine can be used to form the solution, including mixed phases. Amorphous 5-azacytidine can also be used to form the solution. It is preferred, although not necessary, that the DMSO is preheated to an elevated temperature in order to ensure that the 5-azacytidine is fully dissolved. Most preferably, the dimethyl sulfoxide (DMSO) is preheated to a temperature in the range of about 40 ° C to about 90 ° C.

After solvation of 5-azacytidine in DMSO, at least one cosolvent selected from the group consisting of ethanol, 2-propanol (isopropyl alcohol), acetonitrile, and methyl ethyl ketone is added to the 5-azacytidine solution. The use of mixtures of two or more of any of the aforementioned co-solvents is also within the scope of the invention.

It is preferred, although not necessary, that the co-solvents are preheated prior to mixing with the DMSO, preferably to a temperature below the temperature at which a substantial portion of the co-solvent would boil, most preferably, up to about 50 ° C. It is also preferred, although not necessary, that the co-solvent (s) be added gradually to the DMSO.

After mixing, the DMSO / co-solvent (s) mixture is then equilibrated at different temperatures in order to promote either slow recrystallization or rapid recrystallization of Form I 5-azacytidine, as described below.

Slow recrystallization means that the co-solvent / DMSO solution is allowed to equilibrate at a temperature in the range of about 0 ° C to about 40 ° C, preferably in the range of about 15 ° C to about 30 ° C and, more preferably, at about room temperature. Slow recrystallization of Form I from 5-azacytidine is accomplished using ethanol, isopropyl alcohol, methylethyl ketone, or acetonitrile.

Rapid recrystallization means that the co-solvent solution is allowed to equilibrate at a temperature below 0 ° C, preferably below about -10 ° C, and most preferably at about -20 ° C. Rapid recrystallization of Form I from 5-azacytidine is carried out with 2-propanol (isopropyl alcohol) or acetonitrile as co-solvent.

Examples of protocols for recrystallization of Form I according to the methods described herein are provided below in Examples 2 (slow recrystallization with DMSO as the main solvent and ethanol, isopropyl alcohol, acetonitrile, or methylethyl ketone as co-solvent. ) and 3 (rapid recrystallization with DMSO as main solvent and isopropyl alcohol or acetonitrile as co-solvent).

After recrystallization, Form I 5-azacytidine crystals can be isolated from the co-solvent mixture by any suitable method known in the art. Preferably, the Form I crystals are isolated using vacuum filtration through a suitable filter medium or by centrifugation.

Using the novel methods provided herein, for the first time it is possible to target Form I 5-azacytidine as a drug substance in a reproducible and robust manner. In particular, isopropyl alcohol and acetonitrile reliably produce Form I independent of cooling rate (either slow recrystallization or fast recrystallization) and are preferred as recrystallization co-solvents to recover Form I. Most preferably, Form I is isolated using isopropyl alcohol as a co-solvent since isopropyl alcohol has a Class 3 hazard rating (solvent of low toxic potential), while acetonitrile has a Class 2 hazard rating (solvent a limit) according to the International Conference of Harmonization's Guidelines for residual Solvents, July 1997. The use of the DMSO / isopropyl alcohol system allows 5-azacytidine Form I to be reliably recovered for the first time from low toxic potential solvents without the need to control the rate of recrystallization. In the most preferred embodiment, 5-azacytidine Form I can be recovered simply by dissolving 5-azacytidine in DMSO (preferably heated to a temperature in the range of about 40 ° C to about 90 ° C prior to the addition of 5-azacytidine ), adding isopropyl alcohol, and allowing the solvent mixture to equilibrate to about room temperature.

In some embodiments of the invention, Form I 5-azacytidine can be recovered from a mixture of DMSO / cosolvent (s) by initiating crystallization with a small amount of Form I 5-azacytidine either before or during the addition. of the co-solvent (s).

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By allowing isolation of a single polymorphic form, one skilled in the art will appreciate that the present invention enables for the first time the production of 5-azacitidine drug substance with uniform and consistent batch-to-batch properties, properties that include, but are not limited to, themselves, solubility and dissolution rate. In turn, this enables 5-azacytidine pharmaceuticals (see below) to be provided which also have uniform and consistent batch-to-batch properties.

Pharmaceutical formulations

For the most effective administration of the drug substance of the present invention, it is preferred to prepare a pharmaceutical formulation (also known as the "pharmaceutical product" or "pharmaceutical composition"), preferably in unit dosage form, comprising one or more of the polymorphs of 5-azacytidine and one or more pharmaceutically acceptable carriers, diluents or excipients. Most preferably, 5-azacytidine Form I prepared according to the methods provided herein is used to prepare the pharmaceutical formulation.

Said pharmaceutical formulation can include a solid form of the present invention that is mixed with at least one pharmaceutically acceptable excipient, diluted by an excipient or surrounded by said vehicle that can be in the form of a capsule, a sachet, tablet, lozenge. , tablet, paper or other container. When the excipient serves as a diluent, it can be a solid, semi-solid or liquid material that acts as a vehicle, support or medium for the 5-azacytidine polymorph (s). Thus, the formulations can be in the form of tablets, lozenges, powders, elixirs, suspensions, emulsions, solutions, syrups, capsules (such as, for example, soft and hard gelatin capsules), suppositories, sterile injectable solutions and sterile packaged powders. .

Examples of suitable excipients include starches, acacia, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations may further include lubricating agents such as, for example, talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl and propyl hydroxybenzoates; sweeteners; or flavorings. Inert polyols, buffers and fillers can also be used. Examples of polyols include: mannitol, xylitol, sucrose, maltose, glucose, lactose, dextrose, and the like. Suitable buffers include phosphate, citrate, tartrate, succinate, and the like. Other inert fillers that can be used include those that are known in the art and are useful in the preparation of various dosage forms. If desired, the solid pharmaceutical compositions can include other components such as fillers and / or granulating agents, and the like. The compositions described herein can be formulated so as to provide rapid, sustained, controlled, or delayed release of the drug substance after administration to the patient using methods well known in the art.

For reference only, the 5-azacytidine polymorph (s) can be prepared in dosage unit form for oral administration. The 5-azacytidine polymorph (s) can be mixed with a solid, powdery carrier such as, for example, lactose, sucrose, sorbitol, mannitol, starch, amylopectin, cellulose derivatives or gelatin, as well as with an antifriction agent such as, for example, magnesium stearate, calcium stearate, and polyethylene glycol waxes. The mixture is then pressed into tablets or filled into capsules. If tablets, capsules or pulvules are desired, such tablets, capsules or pulvules can be coated with a concentrated solution of sugar, which may contain acacia, gelatin, talc, titanium dioxide or, with an enamel dissolved in the organic solvent or mixture. volatile organic solvents. Various colorants can be added to this coating in order to differentiate tablets with different active compounds or with different amounts of active compound present.

Soft gelatin capsules can be prepared in which the capsules contain a mixture of the 5-azacitidine polymorph (s) and vegetable oil or non-aqueous water-miscible materials such as, for example, polyethylene glycol and the like. Hard gelatin capsules may contain granules or powders of the 5-azacytidine polymorph with a solid powdery carrier, such as, for example, lactose, sucrose, sorbitol, mannitol, potato starch, cornstarch, amylopectin, cellulose derivatives, or gelatin. .

Tablets for oral use are typically prepared in the following manner, although other techniques may be employed. Solid substances are gently ground to a desired particle size and a binding agent is homogenized and suspended in a suitable solvent. The 5-azacytidine polymorph (s) and auxiliary agents are mixed with the binding agent solution. The resulting mixture is moistened to form a uniform suspension. Typically, wetting causes the particles to aggregate slightly, and the resulting mass is gently pressed through a stainless steel screen having a desired size. The layers of the mixture are then dried in controlled drying units for a predetermined period of time to achieve a desired particle size and consistency. The granules of the dried mixture are gently sieved to remove any dust present. To this mixture, disintegrating, anti-friction and anti-stick agents are added. Finally, the mixture is pressed into tablets using a machine with the appropriate punches and dies to obtain the desired tablet size.

In the event that the above formulations are for use for parenteral administration, said formulation typically comprises sterile aqueous and non-aqueous injection solutions comprising one or more

ES 2 425 474 T3 5-azacytidine polymorphs for which the preparations are preferably isotonic with the blood of the intended recipient. These preparations can contain antioxidants, buffers, bacteriostats, and solutes; which make the formulation isotonic with the blood of the desired recipient. Aqueous and non-aqueous suspensions can include suspending agents and thickening agents. The formulations may be present in single and multi-dose containers, for example, in hermetically sealed ampoules and vials. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the type described above.

Liquid preparations for oral administration are prepared in the form of solutions, syrups or suspensions, the latter two containing, for example, 5-azacytidine polymorph (s), sugar and a mixture of ethanol, water, glycerol and propylene glycol. If desired, such liquid preparations contain colorants, flavors and saccharin. Thickening agents such as carboxymethylcellulose can also be used.

As such, the pharmaceutical formulations described herein are preferably prepared in unit dose form, each unit containing from about 5 mg to about 200 mg, more usually about 100 mg of the 5-azacytidine polymorph (s). . In liquid form, the unit dose contains from about 5 to about 200 mg, more usually about 100 mg of the 5-azacytidine polymorph (s). The term "unit dosage form" refers to physically discrete units suitable as unit doses for human or other mammalian subjects / patients, each unit containing a predetermined quantity of 5-azacitidine polymorph calculated to produce the desired therapeutic effect, in association with, preferably, at least one pharmaceutically acceptable carrier, diluent or excipient.

Examples

Example 1 (comparative)

Prior Art Procedure for Synthesis and Recrystallization of Drug Substance 5-Azacytidine

5-Azacytidine can be synthesized using commercially available 5-azacytosine and 1,2,3,5-tetra-O-acetyl-pD-ribofuranose (RTA) according to the following route:

<img file="ES2425474T3_D0002.tif" />

The crude synthesis product is dissolved in DMSO (preheated to about 90 ° C), and then methanol is added to the DMSO solution. The co-solvent mixture is equilibrated to about -20 ° C to allow the formation of 5-azacytidine crystals. The product is collected by vacuum filtration and allowed to air dry.

Example 2

5-Azacytidine Form I: Slow recrystallization of 5-Azacytidine in co-solvent systems

Approximately 250 mg of 5-azacytidine was dissolved with approximately 5 ml of dimethyl sulfoxide (DMSO), preheated to approximately 90 ° C, in separate 100 ml beakers. The solids were allowed to dissolve to a clear solution. Approximately 45 ml of ethanol, isopropyl alcohol, acetonitrile or methylethyl ketone was added to the solution as a co-solvent, preheated to approximately 50 ° C, and the resulting solution was mixed. The solution was covered and allowed to equilibrate under ambient conditions. The product was collected by vacuum filtration using a Buchner funnel.

Example 3

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5-Azacytidine Form I: Rapid recrystallization of 5-Azacytidine in co-solvent systems

Approximately 250 mg of 5-azacytidine was dissolved with approximately 5 ml of DMSO, preheated to approximately 90 ° C, in separate 100 ml beakers. The solids were allowed to dissolve to a clear solution. About 45 ml of isopropyl alcohol or acetonitrile was added to the solution as a co-solvent, preheated to about 50 ° C, and the resulting solution was mixed. The solution was covered and placed in a freezer to equilibrate at about -20 ° C to allow the crystals to form. The solutions were removed from the freezer after crystal formation. The product was collected by vacuum filtration using a Buchner funnel.

Example 4

X-ray powder diffraction of recrystallized 5-azacytidine

X-ray powder diffraction (XRPD) patterns were obtained for each sample on a Scintag XDS 2000 or Scintag X2 Θ / Θ diffractometer operating with copper radiation at 45 kV and 40 mA using a Kevex Psi Peltier cooled silicon detector or a Thermo ARL Peltier cooled solid state detector. Source slits of 2 or 4 mm and detector slits of 0.5 or 0.3 mm were used to obtain data. The recrystallized material was gently ground for approximately 1 minute using a mincer and agate hand. The samples were placed in stainless steel or silicon sample holders and leveled using a glass microscope slide. The powder diffraction patterns of the samples were obtained from 2 to 42 ° 2Θ at 1 ° / minute. The calibration of the X2 diffractometer was verified using a silicon powder standard.

XRPD carried out according to this method revealed that Form I 5-azacytidine was isolated in Example 2 by slow recrystallization using ethanol, isopropyl alcohol, acetonitrile or methylethyl ketone as co-solvent and, in Example 3 by recrystallization using isopropyl alcohol or acetonitrile as a co-solvent. The results indicate that Form I 5-azacytidine can be reliably recovered in DMSO / isopropyl alcohol and DMSO / acetonitrile solvent systems without controlling the rate of recrystallization.