WO2013004658A1

WO2013004658A1 - Formulations of 5-fluorocytosine and uses thereof.

Info

Publication number: WO2013004658A1
Application number: PCT/EP2012/062822
Authority: WO
Inventors: Jérôme HECQ
Original assignee: Transgene SA
Current assignee: Transgene SA
Priority date: 2011-07-01
Filing date: 2012-07-02
Publication date: 2013-01-10
Anticipated expiration: 2014-01-01

Abstract

The invention relates to granule formulations of 5-fluorocytosine (5-FC), and to their use for treating hyperproliferative diseases such as cancer and other disease, such as fungal diseases. The invention further relates to the manufacture of said granules.

Description

FORMULATIONS OF 5-FLUOROCYTOSINE AND USES THEREOF

TECHNICAL FIELD OF THE INVENTION

The invention relates to formulations of 5-fluorocytosine, and to use of them for treating hy erproliferative diseases such as cancer and other disease, such as fungal diseases. The invention further relates to the manufacture of said formulations.

BACKGROUND ART

Suicide gene transfer using thymidine kinase (TK) and ganciclovir (GCV) treatment or the cytosine deaminase (CDase)/5-fluorocytosine (5-FC) system represents the most widely used approach for gene therapy of cancer (Portsmouth et al., 2007, Molecular Aspects of Medicine, 28, 4-41).

CDase is involved in the pyrimidine metabolic pathway by which exogenous cytosine is transformed into uracil by means of a hydrolytic deamination. While CDase activities have been demonstrated in prokaryotes and lower eukaryotes, especially some fungal cells, they are not present in mammals. CDase is further able to deaminate an analogue of cytosine, the 5- fluorocytosine (5-FC), thereby forming 5-fluorouracil (5-FU), which is further converted to metabolites some of which (e.g. 5-fluoro-UMP (5-FUMP)) interfere with fungal RNA and DNA synthesis leading to strong cytotoxicity and cell death. Cells which lack CDase activity, either because of a mutation which inactivates the gene encoding the enzyme or because they are naturally deficient in this enzyme, as are mammalian cells, are resistant to 5-FC. By contrast, fungal cells which naturally express CDase activity or mammalian cells into which the sequences encoding CDase activity were transferred became sensitive to 5-FC.

The pyrimidine analog 5-fluorocytosine (5-FC) is a synthetic drug marketed by Valeant Pharmaceuticals in various countries under brand names such as Ancotil (Registered trademark) and Ancobon (Registered trademark). Both of these products are immediate release tablet and capsule formulation, respectively. 5-FC is well absorbed after oral administration, and penetrates into the body tissues well. This drug is used for treating severe systemic mycoses, such as cryptococcosis, candidosis, chromoblastomycosis and aspergillosis.

Similarly, 5-FC has been successfully used combined with vector based delivery and expression of exogenous CDase activity into cancer cells (see Erbs et al., 2008, Cancer Gene Therapy, 15, 18-28; Dias et al, Clin Cancer Res (2010),16,2540-9; Foloppe et al., 2008, Gene Ther., 15, 1361-1371). This approach to fight cancer consists in local chemotherapy directed by a suicide gene. More particularly, a viral-based vector carrying a suicide gene, e.g. the gene FCUl (see W099/54481), is injected directly into the tumor before 5-FC administration. This FCUl gene is encoding CDase activity combined with UPRTase activity. Phase I clinical trials are ongoing. Following the intra-tumoral injections of the viral-based vector, the recommended dose of 5-FC for human use (200 mg/kg/day) needs to be administered to patients for 14 days. Ancotil^® is currently available as 500 mg tablets or 1% intravenous solution bottles of 250 mL for systemic administration (or 250 mg and 500 mg capsules in the US under Ancobon^® trademark). To achieve the recommended dose, patients (average weight 60 kg) will have to take daily 12 g of 5-FC (4 intakes of 3.0 g each). This means that patients will need daily to swallow 24 Ancotil^® tablets (500 mg each) or to be injected with 1.2 L of the infusion solution, making the treatment not compliant with their welfare. Accordingly, there is a strong need to develop a new pharmaceutical formulation of 5-FC, much more patient friendly.

SUMMARY OF THE INVENTION A new 5FC pharmaceutical formulation has been developed. Main characteristics of pharmaceutical formulations are: (i) physical, such as size, hardness, friability, disintegration and dissolution; (ii) chemical, such as drug content; and (iii) stability and sensory, such as appearance, odor and taste. All of these three characteristics are equally important with respect to patient acceptance, preference and compliance. For example, drinkable formulation reduces patient acceptance, preference and compliance, especially for patients treated for cancer.

Accordingly, aspects of the present invention include pharmaceutical formulations that comprise at least one prodrug for oral administration useful in treating/preventing hyperproliferative diseases such as cancer, fungal and other disease. The formulation of the Invention is particularly useful for improving patient compliance with administration by reducing the drug intake difficulty.

In some aspects, the prodrug is 5-fluorocytosine (5-FC).

Several aspects of the invention described herein concern pharmaceutical formulations that comprise, consist essentially of, or that consist of granules comprising 5-fluorocytosine (5- FC).

In some embodiments, these granules comprise a core matrix and a coating.

In an embodiment, the core matrix comprises 5-fluorocytosine (5-FC) and at least one pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier included in the core matrix is a granulating agent.

In some embodiments, the carrier amount in the core matrix can range from about 5% to about 40%, advantageously from about 10% to about 15%, by weight of the core matrix.

According to one special embodiment said carrier comprises microcrystalline cellulose and polyvinyl pyrrolidone.

According to preferred embodiment, said carrier comprises microcrystalline cellulose, polyvinyl pyrrolidone and polyvinyl acetate.

According to specially preferred embodiment, said carrier comprises microcrystalline cellulose and copovidone.

In some embodiments, the weight ratio microcrystalline cellulose / copovidone is from about 2 to about 6, advantageously about 4.

In some embodiments, the core matrix of the granule formulation of the Invention comprises 5-fluorocytosine (5-FC), microcrystalline cellulose and polyvinyl pyrrolidone, and preferably further contains polyvinyl acetate.

In alternate embodiments, the core matrix of the granule formulation of the Invention comprises 5-fluorocytosine (5-FC), microcrystalline cellulose and polyvinyl acetate, and preferably further contains polyvinyl pyrrolidone.

Certain aspects of the pharmaceutical formulations of the Invention concern a core matrix that further comprises a sustained release agent.

In some embodiments, the sustained release agent is in an amount above 10 %, advantageously above 20%, by weight of the core matrix.

In preferred embodiments, said sustained release agent is a mixture of polyvinyl acetate and polyvinyl pyrrolidone.

According to alternate embodiments, said sustained release agent is at least one component selected in the group consisting of high molecular weight hydroxypropylmethylcellulose (HPMC 1000 to 100 000), high molecular weight hydroxypropyl cellulose, ethylcellulose, high molecular weight polyethylene glycol, compritol™, precinol, starch, carboxymethylcellulose, polyvinyl alcohol, methyl cellulose, alginate sodium or carraghenans. In some embodiments, the weight ratio polyvinyl acetate / polyvinyl pyrrolidone is 80%/20%.

In an embodiment, the sustained release agent is Kollidon SR (BASF-Laserson).

In several aspects of the invention described herein concern pharmaceutical formulations that comprise one first coating.

In some embodiments, the first coating comprises hydrophilic polymer.

In some embodiments, the first coating comprises hydroxypropylmethylcellulose

(HPMC).

In some embodiments, the first coating further comprises at least one lubricant.

In some embodiments, the first coating further comprises stearic acid.

In some embodiments, the first coating comprises, consists essentially of, or consists of hydroxypropylmethylcellulose (HPMC) and stearic acid.

In some embodiments, the first coating composition comprises at least 10%, advantageously at least 14%, or at least 30%, by weight of stearic acid.

In an embodiment, the first coating composition comprises 30% of stearic acid.

In an embodiment, the first coating composition is Sepifilm LP030.

In several aspects of the invention described herein concern pharmaceutical formulations that further comprise one second coating.

In some embodiments, the second coating composition comprises at least one water soluble polymer.

In some embodiments, the said water-soluble polymer is selected in the group consisting in polyvinylpyrrolidone, polyvinylpyrrolidone vinyl acetate, hydroxypropylmethyl cellulose and polyethylene glycol.

In some embodiments, the said water-soluble polymer comprises polyvinylpyrrolidone and polyethylene glycol.

In preferred embodiments, the po ly vi ny lpyrrol idonc is PVP K 1 7 or K90.

In preferred embodiments, the polyethylene glycol is PEG4000.

In some embodiments, the water-soluble polymer comprises polyvinylpyrrolidone and polyethylene glycol in weight ratio 10/3 (w/w), respectively. In some embodiments, the granule formulation comprises at least 1000 mg, preferably at least 1500 mg, of 5-FC.

In some embodiments, the granules show a 5-FC dissolution rate defined as follows:

• the dissolution is carried out by using USP type II apparatus with a paddle rotating speed of 75 rpm in 500 ml NaCl pH 6.8 and the time percent 5-FC release of the granule formulation of the Invention is :

5 min: at least 25%, in particular 25 %-45 % (preferably about 38.3%),

15 min: at least 70%, in particular 70%-87% (preferably about 80.20 %); and/or « the dissolution is carried out by using eq. 3g of 5-FC, USP type II apparatus with a paddle rotating speed of 75 rpm, 37°C, in 500 ml NaCl pH 4.5 and the time percent 5-FC release of the granule formulation of the Invention is :

5 min: at least 25%, in particular 15%-25% (preferably about 17.50

%),

15 min: at least 80%, in particular 80%-95% (preferably about 81.00 %).

In preferred embodiment, the granules exhibit a 5-fluorocytosine (5-FC) dissolution rate comparable to the commercially available 5-fluorocytosine (5-FC) tablet i.e. Ancotil^® tablets (500 mg).

Methods of making and using the 5FC pharmaceutical formulation described herein are also provided.

Preferred methods of making 5FC pharmaceutical formulation involve over- granulation step.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : Comparative analysis of batch 100255 to reference Ancotil® dissolution profiles

Figure 2 : Comparative analysis of batch 100257 to reference Ancotil® dissolution profiles

Figure 3 : Comparative analysis of batch 100283 to reference Ancotil®

Figure 4 : Comparative analysis of batch 100283 to batch 100257

Figure 5 : Comparative analysis of batch 100286 to reference Ancotil® Figure 6 : Comparative analysis of batches 100283, 100315 and 100317 and reference Ancotil^® tablets

Figure 7 : Redispersion analysis, batch 100333

Figure 8 : Comparative analysis of batch 100333 to reference Ancotil® pH 6.8 and 4.5 Figure 9 : Redispersion analysis, batch 100341

Figure 10 : Comparative analysis of batches 100340, 100341 and 100333 to reference

Ancotil®

Figure 11 : Redispersion analysis, batches 100356, 100357 and 100397

Figure 12 : Comparative analysis of batch 100356 to reference Ancotil® batch 80026952, pH 1.2 and 6.8 (mean values)

Figure 13 : Comparative analysis of batch 100356 to reference Ancotil® batch 80026952, pH 4.5 and 6.8 (mean values)

Figure 14 : Comparative analysis of batch 100397 to reference Ancotil® pH 4.5 and

6.8

Figure 15 : Comparative analysis of batches 100356 and 100397, pH 4.5 and 6.8

(mean values)

Figure 16: Particle size distribution analysis (sieving) of batch 100412

Figure 17: Particle size distribution analysis (sieving) of batch 100413

Figure 18: Redispersion analysis, batches 100412 and 100413

Figure 19: Comparative analysis of batch 100412 to reference Ancotil®, pH 4.5 and

6.8 (mean values)

Figure 20: Comparative analysis of batch 100413 to reference Ancotil®, pH 4.5 and 6.8 (mean values)

Figure 21 : Particle size distribution analysis (sieving) of batch 100397

Figure 22: Particle size distribution analysis (sieving) of batch 100426

Figure 23: Particle size distribution analysis (sieving) of batch 100428

Figure 24: Redispersion analysis, batches 100397, 100426 and 100428

Figure 25: Comparative analysis of batches 100426 and 100397, pH 4.5 and 6.8 (mean values)

Figure 26: Comparative analysis of batches 100428 and 100397, pH 4.5 and 6.8 (mean values)

Figure 27: Comparative analysis of batch 100426 to reference Ancotil®, pH 4.5 and 6.8 (mean values)

Figure 28: Particle size distribution analysis (sieving) of batch 100333

Figure 29: Particle size distribution analysis (sieving) of batch 100432 Figure 30: Redispersion analysis, batches 100432 and 100433

Figure 31 : Comparative analysis of batches 100432 and 100333, pH 4.5 and 6.8 (mean values)

Figure 32: Comparative analysis of batch 100432 to reference Ancotil®, pH 4.5 and 6.8 (mean values)

Figure 33 Particle size distribution (sieving) of batch 100412

Figure 34: Particle size distribution (sieving) of batch 100412

Figure 35: Redispersion analysis, batches 100434 and 100412

Figure 36: Comparative analysis of batches 100434 and 100412, pH 4.5 and 6.8 (mean values)

Figure 37: Comparative analysis of batch 100434 to reference Ancotil®, pH 4.5 and 6.8 (mean values)

Figure 38: Dissolution results of selected formulation vs reference Ancotil tablets

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns in a first aspect a granule formulation comprising 5- fluorocytosine wherein said granule comprises :

(i) a core matrix, and

(ii) a coating.

Preferably, the core matrix comprises 5-fluorocytosine (5-FC) and at least one pharmaceutically acceptable carrier. Also preferably, the coating comprises a moisture barrier.

According to one special embodiment, the core matrix comprises 5-fluorocytosine (5- FC) and at least one pharmaceutically acceptable carrier, wherein said pharmaceutically acceptable carrier can be selected in the group consisting of solid diluents, disintegrants, binding agents, granulating agents, and optionally other formulating agents known in the art such as, for example, suspending agents, dispersing agents, solvents, preservatives or lubricants.

According to one preferred embodiment, said pharmaceutically acceptable carriers are granulating agents selected from the group consisting in cellulose derivatives, microcrystalline cellulose, gelatin, starches, sugar (e.g. glucose, sucrose, lactose, trehalose, maltose), polyols (e.g. sorbitol, mannitol), polyvinylpyrrolidone (polyvidone), copolymers of vinylpyrrolidone and vinylacetate (also referred to as copovidone), gums (e.g. tragacanth, xantham, acaciae), sodium alginate or combinations thereof. In preferred embodiment, the core matrix of the granule formulation of the Invention comprises 5-fluorocytosine (5-FC), microcrystalline cellulose and a cop o lymer o f vinylpyrrolidone and vinylacetate (also referred to as copovidone, e.g. Plasdone® S-630, a synthetic, 60:40, linear, random copolymer of N-vinyl-2-pyrrolidone and vinyl acetate).

The carrier amount in the core matrix can range from about 5% to about 50%, especially from about 10% to about 20%, more particularly from about 10% to about 15%, by weight of the core matrix.

According to particular embodiment, the weight ratio microcrystalline cellulose (e.g. Vivapur®) / copovidone is from about 2 to about 6, preferably about 4.

Preferably water is used in conjunction with the core matrix components as a wetting agent.

According to the Invention, the granule formulation further comprises one first coating which comprises a water soluble coat former. The first objective of this coating is to slow-down the 5-FC release from the granule for obtaining a dissolution rate profile comparable to the commercially available 5-fluorocytosine (5-FC) tablet i.e. Ancotil^® tablets.

According to special embodiment, said first coating composition comprises at least one binder chosen from starches, modified starches and the like, one or more compounds selected in the group consisting of natural wax either from vegetable or animal origin or synthetic wax, hydrophilic p o lymer such as hydroxypropylce llulo s e (HP C) , hydroxypropylmethylcellulose (HPMC - e.g. hypromellose 2910), hydroxypropylethylcellulose (HPEC), ethylcellulose, methylcellulose, cellulose acetate phthalate (CAP), microcrystalline cellulose or carrageenan, and mixtures thereof.

According to preferred embodiment, said first coating composition further comprises at least one hydrophobic compound chosen from the group consisting of magnesium stearate, aluminium stearate, calcium stearate, stearic acid, talc, hydrogenated castor oil, and mixtures thereof. Usually one hydrophobic compound is sufficient, namely stearic acid benefits the manufacture of the particular granule formulation of the Invention.

According to preferred embodiment, said first coating composition comprises hydroxypropylmethylcellulose (HPMC) and stearic acid. It may further contain microcrystalline cellulose. According to preferred embodiment, said first coating composition comprises at least 10% of stearic acid, preferably at least 14% of stearic acid, and more preferably at least 30%> by weight of stearic acid. In particular embodiment, it comprises 30% by weight of stearic acid.

According to preferred embodiment, said first coating composition shows pH- independent solubility.

According to preferred embodiment, said first coating composition is a mixture of hydroxypropylmethylcellulose (also referred to as hypromellose), microcrystalline cellulose, stearic acid, and optionally other components such as pigments and lakes. A specific example of a mixture of hydroxypropylmethylcellulose (also referred to as hypromellose), microcrystalline cellulose, stearic acid, pigments and lakes is Sepifilm™ LP030 (Seppic).

According to preferred embodiment, the granule formulation of the Invention is meant to be dispersed in a glass of water prior to administration to the patient.

According to more preferred embodiment, the 5-FC release from the granules during the reconstitution step (i.e. aqueous dispersion before administration period of 5 to 15 minutes) is avoided or as limited as possible.

According to advantageous embodiment, the core matrix may further comprise a sustained release agent, preferably a pH independent one. According to preferred embodiment this sustained release agent is in an amount above 10 % by weight of the core matrix, preferably above 20%, most preferably above 30 %, by weight of the core matrix.

According to special embodiment, said sustained release agent is a mixture of polyvinyl acetate and polyvinyl pyrrolidone. According to alternate embodiments, said sustained release agent is at least one component selected in the group consisting of high molecular weight hydroxypropylmethylcellulose (HPMC 1000 to 100 000), high molecular weight hydroxypropyl cellulose, ethylcellulose, high molecular weight polyethylene glycol, compritol™ (a mixture of Glyceryl Dibehenate (CAS Number 94201-62-4 ), Tribehenin (CAS number 18641-57-1), and Glyceryl Behenate (CAS number 30233-64-8), precinol, starch, carboxymethylcellulose, polyvinyl alcohol, methyl cellulose, alginate sodium or carraghenans.

Preferably the weight ratio polyvinyl acetate / polyvinyl pyrrolidone is 80%/20%.

In special embodiment, said sustained release agent in the core matrix is a spray dried, non-hygroscopic powder consisting of polyvinyl acetate (8 parts w/w) and polyvinyl pyrrolidone (2 parts w/w). A specific example is Kollidon® SR. The preferred granule formulation of the Invention comprises approximately in the core matrix :

- 52.7% by weight of 5-fluorocytosine,

- 10.8% by weight of microcrystalline cellulose,

- 33.8 %> by weight of spray dried, non-hygroscopic powder consisting of polyvinyl acetate (8 parts w/w) and polyvinyl pyrrolidone (2 parts w/w)(e.g. kollidon® SR), and

-2.7%) by weight of copovidone.

According to advantageous preferred embodiment, the granule formulation of the invention further comprises a second coating. This second coating objective is to increase the wettability of the granules of the Invention, and thus to enhance the granule water-redispersion characteristics, because the developed granule formulation is meant to be dispersed in a glass of water prior to administration to the patient. Moreover, in order to maintain the granule 5-FC dissolution rate profile comparable to the commercially available 5-fluorocytosine (5-FC) tablet i.e. Ancotil^® tablets, it was requested for this granule formulation that 5-FC release from the granules during the reconstitution step (i.e. before administration - period of 5 to 15 minutes) should be avoided or as limited as possible.

According to special embodiment, said second coating composition comprises at least one water soluble polymer, i.e. polymeric composition, soluble in an aqueous solution.

According to special embodiment, said water-soluble polymer is selected in the group consisting in modified starch, gelatin, polyvinylpyrrolidone, copolymers of vinylpyrrolidone and vinylacetate (copovidone), cellulose derivatives (such as for example hydroxypropyl methylcellulose (HPMC) and hydroxypropyl cellulose (HPC)), polyethylene glycol, polyvinyl alcohol and mixtures thereof.

According to special embodiment, said water-soluble polymer is selected in the group consisting in polyvinylpyrrolidone and polyethylene glycol.

According to special embodiment, said water-s o lub l e p o lymer i s polyvinylpyrrolidone.

According to special embodiment, said water-soluble polymer is polyethylene glycol. According to advantageous and preferred embodiment, said water-soluble polymer comprises polyvinylpyrrolidone and polyethylene glycol. The term "polyvinylpyrrolidone" or PVP refers to any of the polymers of vinylpyrrolidone, or derivatives thereof. While PVP is typically made by a free radical polymerization process, any soluble grade polymer of vinylpyrrolidone can be used according to the invention. Typically, linear PVP polymers are water soluble and cross-linked PVP polymers are not water soluble.

According to the Invention, PVP is selected in the group consisting of grade K12, K15, K17, K25, K30, K60, and K90. According to preferred embodiment, it is PVP K17 or K90. "PVP Kn" refers to a PVP with a K value of n. The "K-value" of a polymer is a well- known constant, which is function of the average degree of polymerization and the intrinsic viscosity of the polymer, and may be determined easily by one skilled in the art by viscosity measurements.

The term polyethylene glycol (PEG) refers to any of the polymer of ethylene oxide or derivatives thereof.

According to the Invention, PEG is the range of the 300-20000, more particularly it is selected in the group consisting of PEGIOOO, PEG1500, PEG2000, PEG4000, PEG6000 and PEG20000. According to preferred embodiment, it is PEG4000. "PEGn" refers to a PEG having an average molecular weight of n.

According to advantageous and preferred embodiment, said water-soluble polymer comprises polyvinylpyrrolidone and polyethylene glycol in ratio 10/3 w/w, respectively.

In preferred embodiments, the granule formulation comprises at least 1000 mg, preferably at least 1500 mg, of 5-FC.

The preferred granule formulation of the Invention comprises approximately:

Composition Weight % Quantity mg/sachet

Granulation

5-fluorocytosine 38.22 1500.00

Macrocrystalline cellulose 7.84 307.69

spray dried, non-hygroscopic

powder consisting of polyvinyl

acetate (8 parts w/w) and

polyvinyl pyrrolidone (K30, 2

parts w/w)(e.g. Kollidon SR^®) 24.50 961.54

Copovidone 1.96 76.92

Total (phase granulation) 72.52 2846.15

Coating

mixture of

hydroxypropylmethylcellulose

(also referred to as hypromellose), 19.60 769.23 microcrystalline cellulose, stearic

acid, pigments and lakes (e.g.

Sepifilm^™ LP 030)

PVPK17/ 4.17 177.51

PEG 4000 1.36 53.25

Flavor

2 78.50

Total 100.00 3924.65

According to one embodiment, the granule formulation of the invention shows a 5-FC pH-independent solubility.

In preferred embodiments, the granule formulation according to the invention shows a 5-FC dissolution rate defined as follows:

a) the dissolution is carried out by using USP type II apparatus with a paddle rotating speed of 75 rpm in 500 ml NaCl pH 6.8 and the time percent 5-FC release of the granule formulation of the Invention is :

5 min: at least 25%, in particular 25 %-45 % (preferably about 38.3%),

15 min: at least 70%, in particular 70%-87% (preferably about 80.20 %);

and/or

b) the dissolution is carried out by using eq. 3g of 5-FC, USP type II apparatus with a paddle rotating speed of 75 rpm, 37°C, in 500 ml NaCl pH 4.5 and the time percent 5-FC release of the granule formulation of the Invention is :

5 min: at least 25%, in particular 15%-25% (preferably about 17.50 %),

15 min: at least 80%, in particular 80%-95% (preferably about 81.00 %).

In even more preferred embodiments, the granule formulation according to the invention shows a 5-FC dissolution rate defined as follows:

a) the dissolution is carried out by using USP type II apparatus with a paddle rotating speed of 75 rpm in 500 ml NaCl pH 6.8 and the time percent 5-FC release of the granule formulation of the Invention is : 2 min: 15%-25% (preferably about 17.50 %),

5 min: 25%-45% (preferably about 38.3%),

10 min: 45%-70% (preferably about 64.90 %),

15 min: 70%-87% (preferably about 80.20 %),

20 min: 87%-95% (preferably about 87.40%);

and/or

2 min: 15%-25% (preferably about 17.30 %),

5 min: 25%-58% (preferably about 36.6%),

10 min: 59%-79% (preferably about 65.00 %),

15 min: 80%-95% (preferably about 81.00 %).

In preferred embodiments, the granule formulation according to the invention exhibits a 5-fluorocytosine (5-FC) dissolution rate comparable to that of a matricial tablet comprising 500 mg 5-FC as an active ingredient, the matrix preferably comprising at least one binding agent (such as PVP and/or microcrystalline cellulose), a diluent (such as corn starch) and at least one lubricant (such as magnesium stearate and/or hydrated precipitated colloidal silica). More preferably, the granule formulation according to the invention exhibits a 5-fluorocytosine (5- FC) dissolution rate comparable to that of a matricial tablet comprising or consisting of:

- 500 mg of 5-FC,

15 mg of povidone (binding agent),

- 80 mg of corn starch (diluent),

- 75 mg of microcrystalline cellulose (binding agent),

5 mg of magnesium stearate (lubricant), and

25 mg hydrated precipitated colloidal silica (lubricant).

This composition is the composition of the commercially available 5-fluorocytosine (5-FC) tablet i.e. Ancotil^® tablet. The granule formulation according to the invention thus preferably exhibits a 5-fluorocytosine (5-FC) dissolution rate comparable to that of the commercially available 5-fluorocytosine (5-FC) tablet i.e. Ancotil^® tablet. According to preferred embodiment, said 5-fluorocytosine (5-FC) granule dosage form has a bioavailability equivalent to that of a matricial tablet as defined above, in particular the commercially available reference 5-fluorocytosine (5-FC) tablet i.e. Ancotil^® tablets.

According to one embodiment, the Invention provides a granule formulation showing similar 5-FC dissolution rate than a matricial tablet as defined above, in particular reference Ancotil^® tablets (see Experimental section).

Alternatively, the granule formulation according to the invention may show similar 5- FC dissolution rate than a capsule comprising 250 to 500 mg of 5-FC as an active ingredient, mixed with corn starch, lactose and talc, the capsule shells preferably comprising parabens (butyl, methyl, propyl) and sodium propionate and a coloring agent. This is the composition of commercially available reference Ancobon^® capsules. The granule formulation according to the invention thus preferably exhibits a 5-fluorocytosine (5-FC) dissolution rate comparable to that of the commercially available 5-fluorocytosine (5-FC) capsules i.e. Ancobon^® capsules (250 or 500 mg).

By "comparable" or "similar" dissolution rate is meant a dissolution rate from the granule formulation into liquid phase, measured under the same conditions, which is at least 80% of the dissolution rate of commercially available 5-fluorocytosine (5-FC) tablet i.e. Ancotil^® tablets (500mg), preferably at least 90% of the dissolution rate of commercially available 5-fluorocytosine (5-FC) tablet i.e. Ancotil^® tablets (500mg), even more preferably at least 100%) of the dissolution rate of commercially available 5-fluorocytosine (5-FC) tablet i.e. Ancotil^® tablets (500mg).

The Ancotil^® tablets and the Ancobon^® capsules being commercially available, it is within the reach of the one skilled in the art to compare dissolution rate of this reference compound with any other tested compound in comparable conditions.

In a second aspect, the invention also relates to a granule formulation comprising 5- fluorocytosine wherein said granule comprises:

(i) a core matrix, and

(ii) a coating,

wherein the granules exhibit a 5-FC dissolution rate as defined above.

Such a granule formulation may further have any feature or combination of features as defined above. The present invention further concerns composition comprising the said granule formulation.

The invention also provides methods for preparation of the granules and compositions comprising the granules. The granules of the Invention, in particular the preferred ones, can conveniently be manufactured by a wet granulation route, followed by dry blending and coating steps. "Granulation" is commonly defined as a size-enlargement process in which small particles are gathered into larger, permanent aggregates in which the original particles can still be identified. "Wet granulation" is a variation on this process, as refers to a granulation that adds solvents and binders to the enlargement process (see for example Lipps (1993) J. Pharm. Sci. 83: 937-947). A variety of blending, or mixing, or granulating, apparatus are commonly available. For example, the granulation can be done on a Fluid Bed Granulator, (e.g. Glatt Air Techniques Inc., N. J.). Preferably, the granulation is done on high shear granulator (e.g. GLATT TMG). The said granulator activity can further be completed by using granule drying apparatus, such as a fluidized bed drier (e.g. GLATT GPCG1) and a granule coating apparatus, (e.g. GLATT GPCG1). Method of the Invention are detailed in the example section.

The temperature during granulation can be set at any point as long as it does not exceed the melting point of any components in the formulation and the balance between spraying and drying is kept. Typically, the temperature is set in the range of 20° C to 50°C.

According to advantageous embodiment, the granule formulation of the invention are prepared by high shear wet granulation (HS WG) process, and more preferably said process comprises at least one step of over-granulation.

Over granulation of the formulation of the Invention is obtained by adding an excess of granulating solution after the point at which granulation of the blend is achieved. Over granulation allows obtaining granules with a greater particle size.

According to advantageous embodiment, the granule formulation of the invention present a particle size distribution such that at least 40% (in weight) of the particles present a particle size above 500 μηι, preferably above 700 μιη ; in some embodiments said particle size distribution is such that at least 50% (in weight) of the particles present a particle size above 500 μηι, preferably above 700 μιη; in some embodiments said particle size distribution is such that at least 60%> (in weight) of the particles present a particle size above 500 μιη, preferably above 700 μιη; in some embodiments said particle size distribution is such that at least 70% (in weight) of the particles present a particle size above 500 μηι, preferably above 700 μιη; in preferred embodiments said particle size distribution is such that at least 70% (in weight) of the particles present a particle size above 700 μιη.

According to preferred embodiment, the final pharmaceutical form of the formulation of the Invention is intended to be a sachet with granules to be reconstituted in liquid, preferably water, before administration, and flavouring can be added in order to taste-mask the bitterness of 5-fluorocytosine contained in the granules and to reach good patient compliance. A non- limiting representative list of examples includes toffee, orange, lime, lemon, grapefruit, pinenapple, or combination thereof. According to special embodiment, said flavoring compound can represent about 1% to 2% of the final granule formulation.

In a further aspect, the invention concerns the use of a granule formulation comprising

5-fluorocytosine (5-FC) according to the invention for the preparation of a medicament for use in the treatment of fungal diseases, proliferative diseases, especially cancer or diseases associated to an increased osteoclast activity (e.g. rheumatoid arthritis, osteoporosis), infectious disease and other disease therapies, preferably for use in the treatment of cancer.

The present invention also relates to a granule formulation comprising 5- fluorocytosine (5-FC) according to the invention, for use in the treatment of fungal diseases, proliferative diseases, especially cancer or diseases associated to an increased osteoclast activity (e.g. rheumatoid arthritis, osteoporosis), infectious disease and other disease therapies, preferably for use in the treatment of cancer.

The present invention also concerns a kit of parts, comprising:

a) a granule formulation comprising 5-fluorocytosine (5-FC) according to the invention, and

b) a polypeptide comprising cytosine deaminase activity, a polynucleotide encoding a polypeptide comprising cytosine deaminase activity, or an expression vector to induce expression of a polypeptide comprising cytosine deaminase activity,

wherein the granule formulation and the polypeptide comprising cytosine deaminase activity or expression vector thereof are intended to be administered simultaneously (in a single composition or in distinct compositions by various administration routes) or sequentially (one after the other) in any appropriate order. Said kit of part is also intended for use in the treatment of fungal diseases, proliferative diseases, especially cancer or diseases associated to an increased osteoclast activity (e.g. rheumatoid arthritis, osteoporosis), infectious disease and other disease therapies, preferably for use in the treatment of cancer. The present invention further provides methods of treating a fungal infection by administering a granule formulation of the invention. The present invention also provides a method of treating proliferative diseases, cancer, diseases associated to an increased osteoclast activity, infectious disease or other disease in a mammal by administering a granule formulation of the invention in conjunction with a polypeptide comprising cytosine deaminase activity or in combination with a polynucleotide encoding such a polypeptide.

According to one embodiment, the present invention concerns a method of treating a fungal disease. The method comprises administering to a subj ect in need thereof a therapeutically effective amount of a granule formulation of the invention comprising 5- fluorocytosine. The method can be used to treat any applicable fungal disease, including a fungal disease which is a brain fungal disease, an internal organ fungal disease, vaginal fungal disease, mouth fungal disease foot fungal disease and/or which is an infection by a fungus. In treating a fungal disease with a granule formulation of the invention comprising 5- fluorocytosine, it can be beneficial to coadminister another anti-fungal agent. Any appropriate antifungal agent may be coadministered with the granule formulation of the invention comprising 5- fluorocytosine, including amphotericin B and azole antifungals such as fluconazole and itraconazole. Such additional agents may be formulated with the granule of the invention or administered separately.

According to another embodiment, the present invention concerns a method for treating a hyperproliferative disease. The method comprises administering to a subject in need thereof a sufficient amount of (i) a polypeptide comprising cytosine deaminase activity or (ii) an expression vector to induce expression of a polypeptide comprising cytosine deaminase activity in hyperproliferating cells, and a therapeutically effective amount of a granule formulation of the invention comprising 5-fluorocytosine. The method can be used to treat any applicable hyperproliferative disease such as cancer, restenosis, diseases associated to an increased osteoclast activity, rheumatoid arthritis, Crohn's disease, chronic obstructive pulmonary disease, benign prostate hyperplasia, and others. In treating a hyperproliferative disease with a granule formulation of the invention comprising 5-fluorocytosine, it can beneficial to coadminister another antiproliferative disease agent. Any appropriate anti-proliferative disease agent may be coadministered with the 5- fluorocytosine containing formulation of the invention, including, but not limited to methotrexate, cyclophosphamide, leucovorin, steroids and cancer drugs. Such additional agents may be formulated with the granule of the invention or administered separately. According to special embodiment, the present invention concerns a method for treating cancer. The method comprises administering to a subject in need thereof a sufficient amount of (i) a polypeptide comprising cytosine deaminase activity or (ii) an expression vector to induce expression of a polypeptide comprising cytosine deaminase activity in cells of the cancer and a therapeutically effective amount of a granule formulation of the invention comprising 5-fluorocytosine. The method can be used to treat any applicable cancer, including brain cancer (e.g. glioblastoma multiforme), lung cancer, colon-rectum cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer, lymphoma, oral cancer, pancreatic cancer, leukemia, melanoma, stomach cancer, ovarian cancer, cancers of the adrenal gland, bladder cancer, bone cancer, bone marrow cancer, cervix cancer, ganglia cancer, kidney cancer, liver cancer, salivary glands cancer, skin cancer (e.g., melanoma), spleen cancer, testis cancer, thymus cancer, and thyroid cancer. Cancers to be treated include solid tumors including metastases to a brain cancer, lung cancer, colon-rectum cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer lymphoma, oral cancer, pancreatic cancer, leukemia, melanoma, stomach cancer and ovarian cancer. In treating a cancer with a formulation comprising 5- fluorocytosine of the invention, it can be beneficial to coadminister another anticancer agent. Any appropriate anti-cancer agent may be coadministered with the 5- fluorocytosine -containing formulation of the invention, including, but not limited to leukovorin, busulfan, cisplatin, mitomycin C carboplatin; antimitotic agents, such as colchicine, vinblastine, paclitaxel, and docetaxel; topo I inhibitors, such as camptothecin and topotecan; topo II inhibitors, such as doxorubicin and etoposide; RNA/DNA antimetabolites, such as 5- azacytidine, 5-fluorouracil and methotrexate; DNA antimetabolites, such as 5-fluoro-2 ' deoxy- uridine, hydroxyurea and thioguanine; EGFR inhibitors, such as Iressa™ (gefitinib) and Tarceva™ (erlotinib) ; proteosome inhibitors; antibodies, such as campath, Herceptin™ (trastuzumab), Avastin™ (bevacizumab), or Rituxan™ (rituximab) ; steroids and alkalating agents such as temodar, melphalan, chlorambucil, cyclophosamide, ifosfamide, vincristine, mitoguazone, epirubicin, aclarubicin, bleomycin, mitoxantrone, elliptinium, fludarabine, octreotide, retinoic acid, tamoxifen, Gleevec™ (imatinib mesylate) and alanosine. Such additional agents may be formulated with the granule of the invention or administered separately.

According to another special embodiment, the present invention concerns a method is provided for treating diseases associated to an increased osteoclast activity. The method comprises administering to a subject in need thereof a sufficient amount of (i) a polypeptide comprising cytosine deaminase activity or (ii) an expression vector to induce expression of a polypeptide comprising cytosine deaminase activity in targeted cells, and a therapeutically effective amount of a granule formulation of the invention comprising 5- fluorocytosine. The method can be used to treat any applicable diseases associated to an increased osteoclast activity, including osteoporosis, hypercalcemia of malignancy, rheumatoid arthritis, tumor metastases and Paget's disease, periodontal disease, fibrous dysplasia, bone resorption by osteoclasts exceeds, bone formation by osteoblasts leading to decreased bone mass, skeletal fragility and bone fracture. In treating a disease associated to an increased osteoclast activity with an extended release formulation comprising 5- fluorocytosine, it can be beneficial to coadminister another agent of interest for treating this type of diseases. Any appropriate of these agent may be coadministered with the granule formulation of the invention comprising 5- fluorocytosine, including, but not limited to biphosphonate, selective oestrogen receptor modulators (SERMs), parathyroid hormone (PTH) (e.g. teriparatide (Forteo)), strontium ranelate, Denosumab™ or calcitonin, and others or a combination thereof. Such additional agents may be formulated with the granule of the invention or administered separately.

According to another embodiment, the present invention concerns a method for treating infectious diseases. The method comprises administering to a subject in need thereof a sufficient amount of (i) a polypeptide comprising cytosine deaminase activity or (ii) an expression vector to induce expression of a polypeptide comprising cytosine deaminase activity in infected cells, and a therapeutically effective amount' of a granule formulation of the invention comprising 5- fluorocytosine.The method can be used to treat any applicable non-fungal infectious disease, including viral, bacterial and mycoplasma based diseases. Examples of such diseases include HIV infection, HBV infection, HCV infection, HPV infection, herpes viral infection, Tuberculosis. In treating an infectious disease with an extended release formulation comprising 5- fluorocytosine, it can be beneficial to coadminister another anti-infectious disease agent. Any appropriate anti-infectuous disease agent may be coadministered with the granule formulation of the invention comprising 5-fluorocytosine, including, but not limited to antibiotics, and antivirals such as valcyclovir, neverapin, anti-HIV drug combos, ribovirin and others. Such additional agents may be formulated with the granule of the invention or administered separately.

As used herein the term 'therapeutically effective amount' means the amount of a prodrug and/or other biological substance necessary to induce a desired pharmacological effect. The amount can vary greatly according to the effectiveness of a particular active substance, the age, weight, and response of the individual host as well as the nature and severity of the host's symptoms. Other factors that can be taken into account include the expression rate of a cytosine deaminase polynucleotide from a vector and the activity of a cytosine deaminase for 5-FC. Accordingly, there is no upper or lower critical limitation upon the amount of the prodrug or active substance. The therapeutically effective amount to be employed in the methods of the disclosure can readily be determined by those skilled in the art.

According to special embodiment, said polynucleotide or expression vector encoding a polypeptide comprising cytosine deaminase activity can comprise natural or synthetic nucleotides, be an oligonucleotide, be RNA or DNA, and be single or double stranded.

In one preferred aspect, the polynucleotide is delivered using an expression vector, and more particularly, a viral or viral derived expression vector. The viral or viral derived expression vector can be replicating or non-replicating, especially in human cells, can be delivered as a viral particle or as polynucleotides encoding the viral vector, and can be an adenoviral vector, a measles vector, a herpesvector, a retroviral vector, a poxvirus vector (including animal pox or vaccinia derived vectors), a parvovirus vector or any other viral vector known to one skilled in the art.

In one preferred embodiment, the viral vector is a replication competent vector capable of infecting mammalian cells, particularly dividing cells (i.e. oncogenic vectors), and more specifically is replication competent poxviral vector.

In one preferred embodiment, the viral vector is oncolytic viral vector. In an embodiment of this invention the oncolytic virus is selected from the group consisting of a Newcastle Disease Virus (NDV), a Mumps Virus, a Measles Virus, a Vesicular Stomatitis Virus, a Para-influenza Virus, an Influenza Virus, an Adenovirus, a Herpes I Virus, a Vaccinia Virus, and a Reovirus. In one preferred embodiment it is a vaccinia virus, for example, Vaccinia virus strains Dairen I, IHD-J, L-IPV, LC16M8, LC16MO, Lister, LIVP, Tashkent, WR 65-16, Wyeth, Ankara, Copenhagen, Tian Tan and WR can be used. According to a particularly preferred embodiment, the poxvirus according to the invention is a Vaccinia virus strains Copenhagen or WR (see for example WO2009/065547, WO 2009/065546 or W09531105).

The prodrug 5-FC is converted to a cytotoxic drug by the action of enzymes inherent in a microorganism or which have been otherwise recombinantly introduced into an organism. For example, the yeast, or bacterial, cytosine deaminase converts the innocuous antibiotic prodrug 5-FC into the cytotoxic chemotherapeutic agent 5-fluorouracil (5-FU). Humans (and mammals in general) are not known to have a naturally occurring gene encoding an enzyme with significant cytosine deaminase activity. Yeast and bacterial cytosine deaminase have gained recognition in the treatment of cancers using gene delivery and viral vectors for the delivery of the enzyme followed by treatment with 5-FC, which is then converted by the enzyme to a cytotoxic drug.

The cytosine deaminase polypeptide which will catalyze the conversion of 5- fluorocytosine to 5-fluorouracil includes those from bacteria and fungi, such as cytosine deaminase from E.coli or Saccharomyces cerevisiae as well as improved recombinant cytosine deaminases (e.g. WO 99/54481 and WO05/07857) can be used. According to special embodiment the cytosine deaminase activity is combined to UPRTase activity.

The methods described according to the present invention can be used in any mammalian species, including human, monkey, cow, sheep, pig, goat, horse, mouse, rat, dog, cat, rabbit, guinea pig, hamster and horse. Humans are preferred.

According to the methods described herein, an granule formulation comprising 5- fluorocytosine of the invention can be administered to the host by an appropriate route, either alone or in combination with another drug. A 'therapeutically effective amount' of granule formulation comprising 5- fluorocytosine of the invention is administered. A 'therapeutically effective amount' sufficient to achieve the desired therapeutic effect under the conditions of administration, such as an amount sufficient to reduce or eliminate a fungal infection, reduce or eliminate a nonfungal infectious disease, or to slow the growth, reduce or eliminate cancer cells or cells of a hyperproliferative disease.

In one embodiment, the 5-FC formulation of the invention is used to treat a subject for 7 days out of every month for months or years. In another embodiment, the dose can be from about 1500 to 6000 mg administered 1-4 doses per day. In yet another embodiment, the 5-FC formulation of the invention is administered from about 1500 to 3000 mg one or more times per day. In one aspect, the dose of 5-FC is adjusted based upon the activity of cytosine deaminase activity within a subject, tissue or cell. EXAMPLES

Example 1 - 5-FC dissolution from commercially available reference Ancotif 500 mg tablets and Ancobon^® 500 mg capsules

• Ancotif Batch 80026952

USP type II apparatus

Dissolution medium: NaCl pH 1.2

5-FC strength tested: 3000 mg

Paddle rotation speed 75 rpm Volume 500 ml

Mean value has been determined based on 6 independent experiments.

Dissolution Ancotil batch 80026952. buffer pH 1.2. 500 ml. 75 rpm

Ancotil^® Batch 80026952

USP type II apparatus

Dissolution medium: buffer pH 6.8

5-FC strength tested: 3000 mg

Paddle rotation speed 75 rpm

Volume 500 ml

Mean value has been determined based on 6 independent experiments.

Dissolution Ancotil batch 80026952. buffer pH 6.8. 500 ml. 75 rpm

Ancotif Batch 80026952

USP type II apparatus

Dissolution medium: buffer pH 4.5

5-FC strength tested: 3000 mg

Paddle rotation speed 75 rpm Volume 500 ml

Mean value has been determined based on 6 independent experiments.

Dissolution Ancotil batch 80026952. buffer pH4.5. 500 ml. 75 rpm

Ancobon^® Batch CI 261

Dissolution medium: NaCl pH 1.2

5-FC strength tested: 500 mg

Paddle rotation speed 75 rpm

Volume 900 ml

Mean value has been determined based on 6 independent experiments.

Dissolution Ancobon^® batch CI 261. NaCl pH 1.2. 900 ml. 75 rpm:

Time (min) Mean (% released) RSD (%)

0 0.0 —

5 22.9 17.8

10 51.5 18.8

15 72.1 16.1

20 85.3 12.3

25 92.3 7.8

30 95.8 4.0

35 98.0 1.8

40 99.5 1.9

45 100.2 2.6

50 100.5 3.1

55 100.5 3.2

60 100.6 3.2 Example 2 - Influence of the Sepifllm LP grade on 5-FC dissolution from coated granules

Two different grades of Sepifllm LP (SEPPIC) were used to investigate the influence of the stearic acid content on 5-FC dissolution from the coated granules. Stearic acid is an hydrophobic agent present in the Sepifilm^™ LP excipients that allows to protect humidity- sensitive active pharmaceutical ingredients against moisture and also to slightly delay drug dissolution from coated cores (granules, tablets...).

Sepifilm LP 014 and Sepifllm LP 030 have been tested.

Qualitative and quantitative composition of batches 100255 and 100257 :

Process

Preparation of granulating solution:

- Solubilize copovidone in 100 grams of deionised water under magnetic stirring (30 minutes at 300 rpm).

Blending step: Add 5-fluorocytosine (sieved on 1 mm screen) and microcrystalline cellulose (sieved on 1mm screen) and mix 4 minutes in a high shear granulator GLATT TMG (1 liter tank, impeller speed 200 rpm and chopper speed 300 rpm).

Granulation step (GLATT TMG 1 liter tank):

Batch 100255

Impeller speed Chopper

Quantity (g) Time

(rpm) speed (rpm)

Wetting (granulating

125.00 200 300 solution) 2min OOsec Granulation 3min OOsec 200 300

Wetting (additional

80.48 200 300 deionised water) Omin 50sec

Granulation 4min lOsec 200 300

Granulation 2min OOsec 300 300

Batch 100257

Drying step (GLATT GPCG1 2.4 liter Wurster tank):

An airflow rate of 70 m /h was found sufficient for adequate fluidization of the granules.

Calibration step: Granules of batch 100255 were calibrated manually (forced sieving) on a 2 mm screen. Batch 100257 was calibrated (forced sieving) manually on a 1.6 mm screen before the drying operation.

Coating step (GLATT GPCG1 2.4 liter Wurster tank - 0.8 mm nozzle):

- Preparation of Sepifilm^™ LP coating solution:

Batch 100255 Batch 100257

Sepifilm^™ LP014 12%

Sepifilm^™ LP030 10%

Deionised water 88% 90%

Total 100% 100% Progressively add the prescribed amount of Sepifilm LP in the deionised water under stirring using a de flocculating blade until complete homogeneous dispersion (40 minutes/speed range: 700 rpm - 1070 rpm).

Batch 100255

Batch 100257

Dissolution rate analysis

5-FC dissolution rates from batch 100255 and batch 100257 were evaluated using a

USP type II apparatus with a paddle rotating speed of 75 rpm. Tests were carried out in 900 ml of deionised water and in 900 ml of pH 1.2 and pH 6.8 buffers. All analyses were carried out at 37°C and on an equivalent of 3 grams of 5-FC. Comparisons were made to reference Ancotil^® tablets using the same dissolution test protocol.

Discussion

The results obtained for batch 100255 have shown that 5-FC dissolution out of the granules was too fast when compared to reference Ancotil^® tablets and this particularly when comparing the dissolution profiles in deionised water and in a pH 6.8 buffer. Figure 1 compares batch 100255 to reference Ancotil^® tablets analysed in similar conditions (mean values). Figure 1 also showed that, unlike reference Ancotil^® tablets, there was no influence of pH on the dissolution of 5-FC from batch 100255.

This difference in dissolution profiles (i.e. faster dissolution of 5-FC from batch 100255 than for reference Ancotil^® tablets) was attributed to the lower amount of stearic acid present in the Sepifilm^™ LP coating used for batch 100255 (i.e. 14% of stearic acid). When comparing the dissolution results obtained from batch 100257 and batch 100255, we in fact clearly notice the influence of an increased amount of stearic acid in the used Sepifilm^™ LP product (i.e. 30% for Sepifilm LP 030 vs. 14% for Sepifilm LP 014). It has been shown that 5-FC dissolution can be significantly reduced when using a higher load of stearic acid. Figure 2 compares batch 100257 to reference Ancotil^® tablets analysed in similar conditions. The comparisons made on Figure 2 (mean values) clearly show that batch 100257 was much closer to reference Ancotil^® tablets than batch 100255. The pH-dependence of the dissolution profiles for batch 100257 was more marked than for batch 100255 and presented moreover the same pattern than reference Ancotil^® tablets.

Example 3: Influence of the amount of Sepifilm^™ LP030 on 5-FC dissolution from coated granules

A 20%) w/w content of Sepifilm^™ LP 030 was evaluated (vs. 15%> w/w for batch 100257 - see above). In this example the 5-FC granules were also calibrated (forced sieving/before drying) on a larger screen size (i.e. 2 mm vs. 1.6 mm for batch 100257) to evaluate the influence of granule size on 5-FC dissolution.

Qualitative and quantitative composition batch 100283

Process

Preparation of granulating solution:

- Solubilize copovidone in 100 grams of deionised water under magnetic stirring (60 minutes at 800 rpm). Blending step: Add 5-fluorocytosine (sieved on 1 mm screen) and microcrystalline cellulose (sieved on 1 mm screen) and mix 4 minutes in a high shear mixer GLATT TMG (1 litter tank, impeller speed 200 rpm and chopper speed 300 rpm)

Granulation step (GLATT TMG 1 liter tank):

Calibration step: Granules were calibrated manually (forced sieving) on a 2 mm screen (time of operation: 10 minutes).

Drying step (GLATT GPCG1 2.4 liter Wurster tank):

Coating step (GLATT GPCG1 2.4 liter Wurster tank - 0.8 mm nozzle): - Preparation of Sepifilm^™ LP coating solution:

Progressively add the prescribed amount of Sepifilm LP 030 in the deionised water under stirring using a de flocculating blade and stir until complete homogeneous dispersion (1 hour and 30 minutes/790 rpm). Stir the dispersion overnight before use (stirring speed of deflocculating blade: 350 rpm). Heating Coating Drying

Air flow rate 50 m³/h 70 m³/h 70 m³/h

Air entrance temperature 60.0°C 45.0 °C 45.0 °C

Atomization pressure 2.6 bar

Product temperature 20.2 - 35.0°C 35.0 - 32.6°C 32.6 - 35.0°C

Coating solution rate 7.75 g/min

Time 4 min 120 min 1 min

Loss after drying 1.74%

Results

Dissolution rate analysis

5-FC dissolution rate from batch 100283 was evaluated using a USP type II apparatus with a paddle rotating speed of 75 rpm. Tests were carried out in 900 ml of deionised water, in 900 ml of a pH 1.2 buffer and in 900 ml of a pH 6.8 buffer. All analyses were carried out at 37°C and on an equivalent of 3 grams of 5-FC. Comparisons were made to reference Ancotil^® tablets using the same dissolution test protocol.

Discussion

Figure 3 compares batch 100283 to reference Ancotil^® tablets analysed in similar conditions (mean values).

The comparisons made on Figure 3 indicated that formulation of batch 100283 shows dissolution profiles almost similar to reference Ancotil^® tablets in the pH 6.8 buffer; drug dissolution having been found to be different at pH 1.2 and in deionised water with a slower dissolution rate observed for batch 100283 than for reference Ancotil^® tablets in these two media.

Figure 4 compares 5-FC dissolution profile from batch 100283 and 100257 (mean values). This Figure shows that 5-FC drug dissolution from the prepared granules was found to be slower for batch 100283 than for batch 100257 in deionised water and at pH 1.2. This phenomenon was attributed to the higher amount of Sepifilm^™ LP 030 present in formulation batch 100283 (i.e. 20% w/w) than in formulation batch 100257 (i.e. 15% w/w).

Example 4 : Influence of the presence of a viscosity increasing agent in the extra- granular phase on 5-FC dissolution from coated granules In an attempt to further decrease 5-FC dissolution rate from the prepared coated granules, it has been proposed to evaluate the impact of the presence of a viscosity increasing agent, such as xanthan gum, in the extra-granular phase of the developed formulation. This excipient would be mixed with the granules following the Sepifilm^™ LP 030 coating. Xanthan gum is a common pharmaceutical excipient used to increase formulation viscosity following its dispersion in water. In this trial, xanthan gum was mixed with the coated granules of batch 100257 in a 2:98 w/w ratio.

Qualitative and quantitative composition

Process

Blending step: Add coated granules of batch 100257 and xanthan gum and mix 1 minute in a Turbula blender (1 liter tank, 44 rpm).

Results

Dissolution rate analysis

5-FC dissolution rate from batch 100286 was evaluated using a USP type II apparatus with a paddle rotating speed of 75 rpm. Tests were carried out in 900 ml of deionised water, in 900 ml of a pH 1.2 buffer and in 900 ml of a pH 6.8 buffer. All analyses were carried out at 37°C and on an equivalent of 3 grams of 5-FC. Comparisons were made to reference Ancotil^® tablets using the same dissolution test protocol. Discussion

Figure 5 compares batch 100286 to reference Ancotil^® tablets analysed in similar conditions (mean values). The comparisons made on this figure clearly indicate that formulation of batch 100286 show dissolution profiles quite different than reference Ancotil^® tablets, especially in a pH 6.8 buffer and in deionised water. At pH 1.2, 5-FC dissolution from the prepared granules was however very close to drug dissolution observed for reference Ancotil^® tablets.

Example 5 : Optimization of formulation redispersion characteristics

It has been observed during the analytical trials that the prepared granules could float on the top of the dissolution media and take a certain time to be properly wetted. This is attributed to the presence of stearic acid in the Sepifilm LP 030. Stearic acid is a hydrophobic molecule that can significantly decrease wettability characteristics. Based on said observation regarding the poor dispersion characteristics of the 5-FC coated granules and having in mind that the developed granule formulation needs to be dispersed in a glass of water prior to administration to the patient, it has been decided to try to optimise the extra-granular phase in order to increase the wettability of the granules.

It has thus been decided to identity and incorporate specific pharmaceutical excipients in the extra-granular phase of the 5-FC coated granules.

Formulation composition

The suggested pharmaceutical excipients that were chosen were the followings:

- Hydrophilic diluents: These excipients are freely water-soluble and can significantly enhance the water uptake by the formulation

- Surfactants: Enhancement of wettability characteristics (decrease of surface tension with dispersing medium)

- Others: Super-disintegrants

All formulations were prepared for an equivalent of 3 grams 5-FC and the mixing step with the specific pharmaceutical excipients was carried out manually. Visual controls upon redispersion were carried out in a first time.

First investigations consisted in evaluating the influence of mannitol as an hydrophilic diluent and/or of sodium lauryl sulphate as a surfactant on the formulation redispersion characteristics.

All investigated formulations are described in the following table:

These evaluations have shown that visual granule dispersion characteristics can be gnificantly improved when adding mannitol as an hydrophilic diluent in the extra-granular phase of the formulation (i.e. batch 100302 and batch 100303). Redispersion characteristics were found to be better for the batch 100303 (i.e. 20% w/w mannitol in the extra-granular phase) than for the batch 100302 (i.e. 10% w/w mannitol in the extra-granular phase). Increasing the amount of mannitol up to 30% w/w and adding sodium lauryl sulphate as surfactant (i.e. batch 100305) was shown to significantly enhance the granule dispersion characteristics as no more stirring was required to disperse the granules.

Redispersion characteristics of batches 100306 and 100307 were shown to be better than for batches 100283, 100302 and 100303 indicating that sodium lauryl sulphate alone can enhance granule dispersion characteristics. These characteristics were however shown to be less interesting than for batch 100305. This clearly indicates that the combination of sodium lauryl sulphate with mannitol should be the preferred choice to enhance granule dispersion.

Further trials were carried out to evaluate the influence of other specific pharmaceutical excipients on 5-FC granule redispersion characteristics. The other pharmaceutical excipients tested were:

- Xanthan gum: a viscosity increasing agent

- Xylitol: a hydrophilic diluent

- Sodium croscarmelose: a disintegrant

All investigated formulations can be found in the following Table :

All these tested formulations showed granule dispersion less interesting than the one obtained for batch 100305. Batch 100308 had granule dispersion characteristics even worse than the one of the batch 100283 due to the rapid swelling of the xanthan gum around the granules. All other tested excipients could not improve granule dispersion characteristics and were therefore not selected for further trials.

Two formulations were then prepared containing either mannitol and sodium lauryl sulphate (equivalent to batch 100305) or sodium lauryl sulphate alone (equivalent to batches 100306-7) to further evaluate the impact of these extra-granular excipients on 5-FC dissolution profile from Sepifilm^™ LP 030 coated granules. The qualitative and quantitative compositions of these formulations are described in the following table.

Dissolution rate analysis

5-FC dissolution rate from batches 100315 and 100317 were evaluated using a USP type II apparatus with a paddle rotating speed of 75 rpm. Tests were carried out in 900 ml of deionised water. All analyses were carried out at 37°C in deionised water and on an equivalent of 3 grams of 5-FC. Comparisons were made to reference Ancotil^® tablets using the same dissolution test protocol (see Figure 6).

It has been shown that introducing sodium lauryl sulphate as a surfactant alone or in combination with mannitol as an hydrophilic diluent can significantly enhance formulation wettability characteristics. However, introduction of such kind of pharmaceutical excipient in the extra-granular phase of the 5-FC coated granules has also been shown to strongly enhance 5- FC dissolution rate when compared to an equivalent formulation which did not contain such excipients (i.e. batch 100283). This increase of the dissolution rate was attributed to the enhanced granule redispersion characteristics and as well to the solubilizing effect of sodium lauryl sulphate.

Example 6 : Enhancement of the granules redispersion characteristics with a second coating using hydrophilic polymers Qualitative and quantitative composition

Batch 100333

Batch size (g) 460 g

Composition % Quantity mg/sachet Granulation

5-fluorocytosine 57.00 3000.00

Macrocrystalline cellulo se

(Vivapur^® 101) 12.00 631.58

Copovidone (Plasdone S-630^®) 5.00 263.16

Total (phase granulation) 74.00 3894.74

Coating

SepifimT LP 030 20.00 1052.63

Povidone K17/PEG4000 6.00 315.79

Total 100.00 5263.16

The povidone/polyethylene glycol ratio used for this trial is 10/3 w/w.

Process

Preparation of granulating solution: - Solubilize copovidone in 100 grams of deionised water under magnetic stirring (30 minutes at 300 rpm).

Blending step : - Add 5-fluorocytosine (sieved on 1 mm screen) and microcrystalline cellulose (sieved on 1 mm screen) and mix 4 minutes in a high shear mixer GLATT TMG (4 liter tank, impeller speed 200 rpm and chopper speed 300 rpm).

Granulation step (GLATT TMG 4 liter tank) (see note 1 in part 6.7.1):

Drying step (GLATT GPCG1 2.4 liter Wurster tank):

Airflow rate 100 m h

Time 20min OOsec

Inlet temperature 60°C

Product temperature 25.0 - 50.0°C

Loss on drying before operation 25.63%

Loss on drying after operation 0.85% Coating step (GLATT GPCG1 2.4 liter Wurster tank - 0.8 mm nozzle): - Preparation of Sepifilm^™ LP coating solution:

Progressively add the prescribed amount of Sepifilm LP 030 in the deionised water under stirring using a deflocculating blade and stir until complete homogeneous dispersion (950 rpm).

- Preparation of povidone/polyethylene glycol coating solution:

Progressively add the prescribed amount of povidone K17 in the deionised water under stirring using a deflocculating blade and stir until complete solubilisation (12 min 46 sec -410 rpm). Add PEG 4000 to this solution and stir until complete solubilisation (4 min 54 sec - 450 rpm).

Results

Dissolution rate analysis

5-FC dissolution rate from batch 100333 was evaluated using a USP type II apparatus with a paddle rotating speed of 75 rpm. Tests were carried out in 900 ml of deionised water, in 900 ml of a pH 1.2 buffer, in 900 ml of a pH 6.8 buffer, in 500 ml of a pH 4.5 buffer and in 500 ml of a pH 6.8 buffer. All analyses were carried out at 37°C and on an equivalent of 3 grams of 5-FC. Comparisons were made to reference Ancotil tablets using the same dissolution test protocol.

Redispersion analysis

Redispersion trials were carried out by dispersing the formulation (for an equivalent of 1.5 grams of 5-FC) in 120 ml of water. The granules were stirred until a homogeneous dispersion using a spatula (equivalent to a spoon) was obtained. Sample withdrawal was made at time 0 minute (i.e. just after dispersion) and after 5 minutes, 10 minutes and 15 minutes. The suspension was allowed to settle between each time points and was re-homogenized (spatula) before each sample withdrawal. This protocol was designed to mimic what would be done in the clinical trials or by the patient taking the 5-FC granule formulation.

The followng table (see also Figure 7) shows the results obtained for batch 100333 :

Discussion

Figure 8 compares batch 100333 to reference Ancotil tablets analysed in similar conditions (500 ml volume dissolution medium, mean values). This experiment shows that 5-FC dissolution from granules of batch 100333 is still too rapid when compared to reference Ancotil^® tablets and especially in deionised water (not shown) and in the pH 6.8 buffer.

Example 7 : Influence on 5-FC dissolution from coated granules of the presence of an additional amount of stearic acid in the granule core and of the povidone grade used for the second coating

From the observations made in the previous examples, it has been shown that the use of a second coating composed of hydrophilic polymers such as povidone and polyethylene glycol is advantageous to significantly enhance granule redispersion characteristics upon contact with water. However, it has been shown, that enhancing these redispersion characteristics has led, although to a smaller extent than for sodium lauryl sulphate containing formulations, to a faster 5-FC dissolution rate than for granules coated only by Sepifilm^™ LP 030. It has previously been shown (not shown) that it was not possible to slow down 5-FC dissolution rate by increasing the stearic acid content in the Sepifilm^™ LP 030 coating layer. Thus, it was suggested to evaluate the influence of the presence of stearic acid in the granule core of the formulation on 5-FC dissolution rate. The stearic acid would be mixed with 5-FC prior to the granulation operation in an attempt to get a granule core more hydrophobic which would therefore slows down 5-FC dissolution out of the core.

The approach using a second coating step with hydrophilic polymers (i.e. povidone and polyethylene glycol) to enhance the Sepifilm^™ LP 030 coated granules redispersion characteristics was maintained for this example. Investigations were also made regarding the grade of povidone used for the second coating operation. Two grades of povidone (Povidone K17 and K90), with different molecular weights and consequently presenting two different aqueous solubilities, were used for this evaluation.

Qualitative and quantitative composition

Note: The granulation step and the Sepifilm LP 030 coating step were carried out in a single batch. This batch was then divided into two equal parts: one part for the povidone K17/PEG4000 coating (batch 100340) and the other part for the povidone K90/PEG4000 coating (batch 100341). Process

Preparation of granulating solution: - Solubilize copovidone in 100 grams of deionised water under magnetic stirring (45 minutes at 690 rpm).

Blending step: - Add 5-fluorocytosine and stearic acid (sieved on 1 mm screen) and mix during 2 minutes in a high shear mixer GLATT TMG (1 liter tank, impeller speed 200 rpm

- Add microcrystalline cellulose and mix 4 minutes in a high shear mixer GLATT TMG(1 liter tank, impeller speed 200 rpm and chopper speed 300 rpm).

Granulation step (GLATT TMG 4 liters tank):

Calibration step: Granules were calibrated manually (forced sieving) on a 2 mm screen

(time of operation: 10 minutes).

Drying step (GLATT GPCGl 2.4 liters Wurster tank):

Coating step (GLATT GPCGl 2.4 liters Wurster tank - 0.8 mm nozzle) (see note in part 6.8.1):

- Preparation of Sepifilm^™ LP coating solution:

SepifilnT LP030 10%

Deionised water 90%

Total 100% Progressively add the prescribed amount of Sepifilm LP 030 to the deionised water under stirring using a deflocculatmg blade and stir until complete homogeneous dispersion (185 minutes- 500 rpm).

- Preparation of povidone K17/polyethylene glycol coating solution:

Progressively add the prescribed amount of povidone K17 in the deionised water under stirring using a deflocculatmg blade and stir until complete solubilisation (12 min 46 sec -410 rpm). Add PEG 4000 to this solution and stir until complete solubilisation (4 min 54 sec - 450 rpm).

- Preparation of povidone K90/polyethylene glycol coating solution:

Progressively add the prescribed amount of povidone K90 in the deionised water under stirring using a deflocculatmg blade and stir until complete solubilisation (12 min 46 sec - 410 rpm). Add PEG 4000 to this solution and stir until complete solubilisation (4 min 54 sec - 450 rpm).

PVPK17/PEG4000 coating PVP 90/PEG4000 coating (Batch 100340) (Batch 100341)

Heating Coating Drying D ing Coating Drying

Air flow rate 50 m³/h 50 m³/h 50 m³/h 50 m³/h 50-70m³/h 70-120 m³/h

Air entrance 60.0°C 45.0 °C 45.0 °C 60.0°C 45.0 °C 45.0 °C

Results

Dissolution rate analysis

5-FC dissolution rates from batch 100340 and batch 100341 were evaluated using a USP type II apparatus with a paddle rotating speed of 75 rpm. Tests were carried out in 900 ml of deionised water for both batches. For batch 100341, additional dissolution rates were performed in 900 ml of a pH 1.2 buffer, in 900 ml of a pH 6.8 buffer, in 500 ml of a pH 4.5 buffer and in 500 ml of a pH 6.8 buffer. All analyses were carried out at 37°C and on an equivalent of 3 grams of 5-FC. Comparisons were made to reference Ancotil^® tablets using the same dissolution test protocol.

Redispersion analysis

Redispersion trials were carried out by dispersing the formulation (for an equivalent of 1.5 grams of 5-FC) in 120 ml of water. The granules were stirred until a homogeneous dispersion using a spatula (equivalent to a spoon) was obtained. Sample withdrawal was made at time 0 minute (i.e. just after dispersion) and after 5 minutes, 10 minutes and 15 minutes. The suspension was allowed to settle between each time points and was re-homogenized (spatula) before each withdrawal of sample. This protocol was designed to mimic what it would be done in the clinical trials or by the patient taking the 5-FC granule formulation.

The following Table (Figure 9) shows the results obtained for batch 100341. This analysis was not carried out on batch 100340.

Discussion

Figure 10 compares (mean values) batch 100340 and batch 100341 to reference Ancotil^® tablets and batch 100333 analysed in similar conditions (900 ml volume dissolution media - deionised water).

It was observed that 5-FC dissolution from granules of batches 100340 and 100341 was too fast when compared to reference Ancotil^® tablets. When comparing these data with those obtained for batch 100333, it was concluded that the presence of stearic acid, as a hydrophobic agent, in the granule core, at least at a 6% w/w ratio, has no influence on 5-FC dissolution out of the granules. The increased dissolution rate observed for batch 100340 versus batch 100333 could not be explained. However, the only difference between these formulations was the presence of stearic acid in the granule core (batch 100340). This could eventually decrease granule hardness and thus cause more rapid granule disintegration in the dissolution medium.

From the observations made, it was also possible to see the influence of the grade of povidone used for the second coating step. In fact, granules coated with Povidone K90 (i.e. batch 100341) showed a slower drug dissolution profile than the granules coated with Povidone K17 (i.e. batch 100340).

Example 8 : Influence of the presence and of the amount of Kollidon SR^® in the granule core on 5-FC dissolution from Sepifilm^™ LP 030/povidone/polyethylene glycol coated granules

Influence of the amount of Kollidon SR^® in the granule core on 5-FC dissolution from Sepifilm^™ LP 030/povidone/polvethylene glycol coated granules

The use of stearic acid as an hydrophobic agent inside the granules, at least at a 6% w/w ratio, having been shown to be unsuccessful with regards to slowing down 5-FC dissolution, it has been decided to evaluate the use of a specific pharmaceutical excipient aiming at sustaining the release of drugs. The pharmaceutical excipient to be tested was Kollidon SR^®. This excipient is a co-processed mixture of 80% (w/w) of polyvinylacetate and 20% (w/w) of povidone K30. Polyvinylacetate is an insoluble polymer used in the development of extended release formulations. litative and quantitative composition

The povidone/polyethylene glycol ratio used for these trials is 10/3 w/w.

Process

Preparation of granulating solution: - Solubilize copovidone into 100 g of deionised water under magnetic stirring (30 minutes at 540 rpm).

Blending step: - Add 5-fluorocytosine, Kollidon SR^® and microcrystallme cellulose and mix 4 minutes in a high shear mixer GLATT TMG (1 liter tank, impeller speed 200 rpm and chopper speed 300 rpm).

Granulation step (GLATT TMG 4 liters tank) (see note 1 in part 6.9.1.1):

Batch 100356/Batch 100357

Impeller speed Chopper speed

Quantity (g) Time

(rpm) (rpm)

Wetting (granulating 2min

200 300 solution) 125.00 OOsec

3min

200 300

Granulation OOsec

Wetting (additional lmin

200 300 deionised water) 40.00 OOsec

4min

200 300

Granulation OOsec

2min

300 300

Granulation OOsec Batch 100397

Drying step (GLATT GPCGl 2.4 liters Wurster tank) (see note 1 in part 6.9.1.1):

Coating step (GLATT GPCGl 2.4 liters Wurster tank - 0.8 mm nozzle) (see note 1 in

- Preparation of Sepifilm LP coating solution:

Progressively add the prescribed amount of Sepifilm LP 030 into the deionised water under stirring using a deflocculating blade and stir until a complete homogeneous dispersion (overnight stirring - 350 to 400 rpm) is obtained.

- Preparation of povidone/polyethylene glycol coating solution:

Povidone Kl 7 10%

PEG 4000 3%

Deionised water 87%

Total 100% Progressively add the prescribed amount of povidone K17 into the deionised water under stirring using a deflocculatmg blade and stir until complete solubilisation. Add PEG 4000 to this solution and stir until complete solubilisation.

- Preparation of povidone K90/polyethylene glycol coating solution:

Progressively add the prescribed amount of povidone K90 in the deionised water under stirring using a deflocculatmg blade and stir until complete solubilisation. Add PEG 4000 to this solution and stir until complete solubilisation. Batch 100356

ND: Not determined

Batch 100357

ND: Not determined Batch 100397

Results

Dissolution rate analysis

5 5-FC dissolution rates from batches 100356, 100357 and 100397 were evaluated using a

USP type II apparatus with a paddle rotating speed of 75 rpm. Tests were carried out in 900 ml of deionised water (not performed for batch 100397), in 900 ml of a pH 1 .2 buffer (not performed for batch 100397), in 900 ml of a pH 6.8 buffer (not performed for batch 100397), in 500 ml of a pH 4.5 buffer and in 500 ml of a pH 6.8 buffer. All analyses were carried out at 10 37°C and on an equivalent of 3 grams of 5-FC. Comparisons were made to reference Ancotil^® tablets using the same dissolution test protocol.

Redispersion analysis

Redispersion trials were carried out by dispersing the formulation (for an equivalent of 1.5 grams of 5-FC) in 120 ml of water. The granules were stirred until a homogeneous 15 dispersion using a spatula (equivalent to a spoon) was obtained. Sample withdrawal was made at time 0 minute (i.e. just after dispersion) and after 5, 10 and 15 minutes. The suspension was allowed to settle between each time points and was re-homogenized (spatula) before each withdrawal of sample. This protocol was designed to mimic what would be done in a clinical trial or by the patient taking the 5-FC granule formulation.

20 The following table (Figure 11) show the results obtained for batches 100356, 100357 and 100397. Batch 100356

Time (min) mg/dose dissolved (vs. 1.5 g 5-FC) % dissolved

0 41.6 2.8

5 269.3 18.0

10 433.3 28.9

15 606.7 40.5

Batch 100357

Time (min) mg/dose dissolved (vs. 1.5 g 5-FC) % dissolved

0 23.5 1.6

5 243.9 16.3

10 422.4 28.2

15 576.9 38.5

Batch 100397

Time (min) mg/dose dissolved (vs. 1.5 g 5-FC) % dissolved

0 24.1 1.6

5 200.4 13.4

10 358.2 23.9

15 515.4 34.4

Discussion

Figures 12 and 13 compares batch 100356 to reference Ancotil tablets (batch

80026952) analysed in similar conditions (900 ml volume dissolution media and 500 ml volume dissolution media, mean values).

From the observations made on Figure 13 for dissolution trials carried out in 900 ml of dissolution media, batch 100356 demonstrated an interesting dissolution profile very close to reference Ancotil^® tablets in water and in a pH 6.8 buffer. In these medium, drug dissolution in the first 20 minutes was still however a little too fast when compared to the reference Ancotil^® tablets and attempts should be made to slightly slow down this dissolution rate. 5-FC dissolution from batch 100356 was found to be slower than reference Ancotil^® tablets in a pH 1.2 buffer. For dissolution trials carried out in 500 ml of dissolution media (Figure 13), drug release from batch 100356 has been shown to be slower than the reference Ancotil^® tablets in the pH 6.8 buffer and faster in the pH 4.5 buffer. The dissolution profiles of batch 100356 have been shown in both test protocols to be greatly pH-dependent.

It was further observed for dissolution trials carried out in 900 ml of dissolution media, that batch 100357 exhibited dissolution profiles that were very different compared to the reference Ancotil^® tablets (not shown). Under these analytical conditions, drug release from batch 100357 was faster than reference Ancotil^® tablets in the pH 6.8 buffer and more particularly in water where a significant difference could be observed. This difference in deionised water was actually greater for the batch 100357 than for the batch 100356, where 5- FC dissolution was found to be very close to the reference Ancotil^® tablets. These results could not be explained as we would have expected to observe the inverse behaviour as batch 100357 was coated with povidone K90 whereas batch 100356 was coated with povidone K17 which dissolves more rapidly compared to povidone K90. These results were also opposite to the ones we obtained for batches 100340 and 100341, where granules coated with povidone K17 released in fact 5-FC faster than granules coated with provide K90. For dissolution trials carried out in 900 ml of buffer pH 1.2, 5-FC dissolution with batch 100357 was also found, as it was the case for batch 100356, to be slower compared to the reference Ancotil^® tablets.

It was shown for dissolution trials carried out in 500 ml of dissolution media that the batch 100357 displayed dissolution profiles that were closer to the reference Ancotil^® tablets than profiles observed for batch 100356. In fact, drug release from batch 100356 has been shown to be slower compared to the reference Ancotil^® tablets in the pH 6.8 buffer and faster in the pH 4.5 buffer, this difference being less pronounced than for batch 100357.

Batches 100356 and 100357 showed completely different behaviours by using the two dissolution protocols (i.e. either 900 ml or 500 ml of dissolution media), the first one being closer to the reference Ancotil^® tablets in tests carried out in 900 ml of dissolution media and the second being closer to reference Ancotil^® tablets in tests carried out in 500 ml of dissolution media.

Figure 14 compares batch 100397 to reference Ancotil^® tablets analysed in similar conditions (500 ml volume dissolution media, mean values).

According to the observations made on Figure 15, batch 100397 presented dissolution profiles that were very close to reference Ancotil^® tablets. 5-FC dissolution from batch 100397 in the pH 4.5 was slightly slower than the one observed with the reference Ancotil^® tablets but dissolution in the pH 6.8 was almost similar.

With regards to the results obtained, batch 100397 was actually the batch showing the closest dissolution profile to the reference Ancotil^® tablets.

Example 9: Optimization of the granulation process: introduction of Kollidon SR^® in the granulating solution

Although the dissolution results obtained for batch 100397 were very interesting and found to be very close to those obtained for the reference Ancotil^® tablets, one of the drawback of this formulation is that the total load, for an equivalent dose of 1.5 g or 3 g of 5-FC, was very high. With regards to this issue, it has been suggested to try to optimize the granulation process by using a part of the Kollidon SR^® in the granulation solution and by reducing the amount of microcrystalline cellulose and of copovidone. The use of Kollidon SR in the granulating solution should allow enhancing the binding of 5-FC granules.

Qualitative and quantitative composition

Process

Preparation of granulating solution: - Solubilize copovidone into 100 g of deionised water under magnetic stirring (5 minutes at 390 rpm)

- Add ½ Kollidon SR^® into the copovidone solution under magnetic stirring and stir until homogeneous dispersion (32 minutes at 800 rpm)

Blending step: - Add 5-fluorocytosine, ½ Kollidon SR^® (sieved on 1mm screen) and microcrystalline cellulose and mix 4 minutes in a high shear mixer GLATT TMG (1 liter tank, impeller speed 200 rpm and chopper speed 300 rpm)

Granulation step (GLATT TMG 4 liters tank):

Batch 100412

Impeller speed Chopper

Quantity (g) Time

(rpm) speed (rpm)

Wetting (granulating 3min

200 300 solution) 125.00 OOsec

2min

300 300

Granulation OOsec

Wetting (additional 10.00 Omin 200 300 deionised water) 30sec

lmin

200 300

Granulation 30sec

lmin

300 300

Granulation OOsec

Batch 100413

Impeller speed Chopper

Quantity (g) Time

(rpm) speed (rpm)

Wetting (granulating 3min

200 300 solution) 125.00 OOsec

2min

200 300

Granulation OOsec

Wetting (additional Omin

200 300 deionised water) 10.00 30sec

lmin

200 300

Granulation 30sec

2min

300 300

Granulation OOsec

Calibration step: Granules were calibrated manually (forced sieving) on a 2 mm screen (time of operation: 10 minutes for batch 100412 and 15 minutes for batch 100413).

Drying step (GLATT GPCG1 2.4 liters Wurster tank):

Coating step (GLATT GPCG1 2.4 liters Wurster tank - 0.8mm nozzle) :

- Preparation of Sepifilm^™ LP coating solution:

Progressively add the prescribed amount of Sepifilm LP 030 into the deionised water under stirring using a deflocculating blade and stir until complete homogeneous dispersion (overnight stirring - 350 to 400 rpm).

- Preparation of povidone/polyethylene glycol coating solution: Povidone Kl 7 10%

PEG 4000 3%

Deionised water 87%

Total 100%

Progressively add the prescribed amount of povidone K17 into the deionised water under stirring using a deflocculating blade and stir until complete solubilisation. Add PEG 4000 to this solution and stir until complete solubilisation.

Batch 100412

Batch 100413

Sepil^™ lm LP 030 coating PVP K17/PEG coating

Heating Coating Drying Heating Coating Drying

Air flow

60 m³/h 40 - 60 m³/h 50 m³/h 50 m³/h 50 m³/h 50 m³/h rate

Air entrance

55.0°C 55.0 - 60.0 °C 60.0 °C 50.0°C 50.0 °C 50.0 C temperature

Atomization

2.6 bar 2.6 bar

pressure

Product

35.0°C 31.2 - 27.7°C 42.0°C 37.2°C 33.2°C 41.0°C temperature

Coating

12.00 g/min 8.00 g/min

solution rate

Time 2 min 83 min 3 min 2 min 20 min 4 min Results

Particle size distribution analysis: Particle size distribution, determined by sieving, for batches 100412 and 100413 (after the coating step) are given in Figures 16 and 17.

Dissolution rate analysis: 5-FC dissolution rate from batches 100412 and 100413 was evaluated using a USP type II apparatus with a paddle rotating at a speed of 75 rpm in 500 ml of a pH 4.5 buffer and in 500 ml of a pH 6.8 buffer. All analyses were carried out at 37°C and on an equivalent of 3 g of 5-FC. Comparisons were made to reference Ancotil^® tablets (batch 80026952) using the same dissolution test protocol.

Redispersion analysis: Redispersion trials were carried out by dispersing the formulation (for an equivalent of 1.5 g of 5-FC) in 120 ml of water. The granules were stirred until a homogeneous dispersion using a spatula (equivalent to a spoon) was obtained. Sample withdrawal was made at time 0 minute (i.e. just after dispersion) and after 5, 10 and 15 minutes. The suspension was allowed to settle between each time points and was re-homogenized (spatula) before each withdrawal of samples. This protocol was designed to mimic what it would be done in a clinical trial or by the patient taking the 5-FC granule formulation.

The following Table and Figure 18 show the results obtained for batches 100412 and

100413.

Discussion

The following Figure 19 compares batch 100412 to the reference Ancotil^® tablets (batch 80026952) analysed in similar conditions (500 ml volume dissolution medium, mean values). The following Figure 20 compares batch 100413 to reference Ancotil tablets (batch 80026952) analysed in similar conditions (500 ml volume dissolution media, mean values).

As it can be seen on the Figures 19 and 20, the granulation protocol using a part of the Kollidon SR^® in the granulating solution led to an acceleration of 5-FC dissolution out of the granules when compared to the dissolution profiles obtained for batch 100397 (see previous example) in similar conditions. It should be noted that, in contrast to what was done for batch 100397, the batches 100412 and 100413 were not over-granulated.

The observation of the dissolution results obtained for batches 100412 and 100413 led to the conclusion that over-granulation, and thus final granule size, was probably preferable in order to achieve the desired dissolution profile similar to the one of the reference Ancotil^® tablets.

Example 10 : Influence of the over-granulation on 5-FC dissolution

10.1. Evaluation of the influence of over- granulation on the composition of batch 100397

Qualitative and quantitative composition

Batch 100397 Batch 100426 Batch 100428

(over-granulation) (over-granulation) (no over-granulation)

Batch size (g) 500 g 500 g 500 g

Quantity

Quantity mg/sache Quantity

Composition % mg/sachet % t % mg/sachet

Granulation

5-fluorocytosine 32.00 3000.00 32.00 3000.00 32.00 3000.00

Macrocrystalline

cellulose (Vivapur^®

101) 12.00 1125.00 12.00 1125.00 12.00 1125.00

Kollidon SR^® 25.00 2343.75 25.00 2343.75 25.00 2343.75

Copovidone

(Plasdone S-630^®) 5.00 468.75 5.00 468.75 5.00 468.75

Total (phase

granulation) 74.00 6937.50 74.00 6937.50 74.00 6937.50

Coating

Sepifilm^™ LP 030 20.00 1875.00 20.00 1875.00 20.00 1875.00

Povidone

K17/PEG4000 6.00 562.50 6.00 562.50 6.00 562.50

Total 100.00 9375.00 100.00 9375.00 100.00 9375.00 Process

Preparation of granulating solution: - Solubilize copovidone in 100 g of deionised water under magnetic stirring (30 minutes at 300 rpm)

Blending step: - Add 5-fluorocytosine (sieved on 1 mm screen), Kollidon SR^® (sieved on 1mm screen) and microcrystalline cellulose (sieved on 1 mm screen) and mix 4 minutes in a high shear mixer GLATT TMG (1 liter tank, impeller speed 200 rpm and chopper speed 300 rpm)

Granulation step (GLATT TMG 4 liter tank):

Batch 100397 (over-granulation)

Batch 100426 (over-granulation)

Batch 100428 (no over-granulation)

Drying step (GLATT GPCG1 2.4 liters Wurster tank):

Coating step (GLATT GPCG1 2.4 liters Wurster tank - 0.8 mm nozzle):

- Preparation of Sepifilm^™ LP coating solution:

Progressively add the prescribed amount of Sepifilm LP 030 into the deionised water under stirring using a deflocculatmg blade and stir until complete homogeneous dispersion (overnight stirring - 350 to 400 rpm).

- Preparation of povidone/polyethylene glycol coating solution:

Progressively add the prescribed amount of povidone K17 into the deionised water under stirring using a deflocculatmg blade and stir until complete solubilisation. Add PEG 4000 to this solution and stir until complete solubilisation Batch 100397

Sepifilm^™ LP 030 coating PVP K17/PEG coating

Heating Coating Drying Coating Drying

Air flow rate 60 m³/h 80 m³/h 80 m³/h 70 m³/h 70 m³/h

A i r e n t r a n c e 60.0°C 50.0 °C 50.0 °C 50.0 °C 50.0 °C temperature

Atomization

2.6 bar 2.6 bar

pressure

Product

33.7°C 33.0°C 43.0°C 39.6 - 38.2°C 43.0°C temperature

Coating solution

10.00 g/min 5.00 g/min

rate

Time 3 min 86 min 3 min 19 min 3 min

Batch 100426

Results

Particle size distribution analysis : Particle size distribution, determined by sieving, for batches 100397, 100426 and 100428 (after the coating step) are given in Figures 21, 22 and 23.

The data presented Figures 22 and 23 clearly show the difference between granules that were over-granulated and those for which it was not the case. As it can be seen, over 72% (in weight) of particles above 710 μιη could be obtained for a batch that was over-granulated (i.e. batches 100397 and 100426) versus less than 25% for a batch, of equivalent qualitative and quantitative composition, that was not over-granulated (i.e. batch 100428). Results for batches 100397 and 100426 were found to be nearly similar.

Dissolution rate analysis : 5-FC dissolution rate from batches 100397, 100426 and 100428 was evaluated using a USP type II apparatus with a paddle rotating speed of 75 rpm. Trials were carried out in 500 ml of a pH 4.5 buffer and in 500 ml of a pH 6.8 buffer. All analyses were carried out at 37°C and on an equivalent of 3 grams of 5-FC. Comparisons were made to the reference Ancotil^® tablets using the same dissolution test protocol.

Redispersion analysis : Redispersion trials were carried out by dispersing the formulations (for an equivalent of 1.5 g of 5-FC) in 120 ml of tap water. The granules were stirred until a homogeneous dispersion using a spatula (equivalent to a spoon) was obtained. Sample withdrawal was made at time 0 minute (i.e. just after dispersion) and after 5, 10 and 15 minutes. The suspension was allowed to settle between each time points and was re-homogenized (spatula) before each withdrawal of sample. This protocol was designed to mimic what it would be done in a clinical trial or by the patient taking the 5-FC granule formulation.

The following Table and Figure 24 show the results obtained for batches 100426, 100428 and 100397.

Batch 100426 (over-granulation)

Time (min) mg/dose dissolved (vs. 1.5 g 5-FC) % dissolved

0 32.0 2.1

5 126.1 8.4

10 209.4 14.0

15 328.7 21.9

Batch 100428 (no over-granulation)

Time (min) mg/dose dissolved (vs. 1.5 g 5-FC) % dissolved

0 32.6 2.2

5 208.5 13.9

10 395.3 26.4

15 531.9 35.5

Batch 100397 (over-granulation)

Time (min) mg/dose dissolved (vs. 1.5 g 5-FC) % dissolved

0 24.1 1.6

5 200.4 13.4

10 358.2 23.9

15 515.4 34.4 The release profile of 5-FC from granules upon redispersion in water was found to be slower for a granule that was over-granulated as it can be seen when comparing the results obtained for batch 100428 (over-granulation) and batch 100426 (no over-granulation).

Discussion

The following Figure 25 compares batches 100426 and 100397 analysed in similar conditions (500 ml volume dissolution media, mean values). This trial was carried out in order to evaluate if the over-granulation process was repeatable.

As it can be seen in the Figure 25, similar dissolution profiles were obtained for the batches 100426 and 100397 and this, at both tested pHs. Both batches had the same qualitative and quantitative compositions and were manufactured using the exact same parameters for granulation and coating. These observations indicated that the over-granulation process was perfectly repeatable with regards to 5-FC dissolution.

The next trial consisted in repeating the manufacturing of batch 100397 without over- granulating the powder during the granulation step. This trial was carried out in order to evaluate if the over-granulation process was necessary to achieve the desired dissolution profile or if this was only formulation- dependent.

The following Figure 26 compares batches 100428 and 100397 analysed in similar conditions (500 ml volume dissolution media, mean values).

As it can be noticed in the Figure 26, 5-FC dissolution from batch 100428 was found to be faster than from batch 100397 (and 100426 - data not shown) and this in both tested media. These data clearly indicated that the over-granulation of the 5-FC is preferable to achieve the desired dissolution profile (i.e. similar to the reference Ancotil^® tablets).

Figure 27 compares batch 100426 to reference Ancotil^® tablets analysed in similar conditions (500 ml volume dissolution media, mean values).

Formulation batch 100426 has been shown to present, the closest dissolution profiles to the reference Ancotil^® tablets.

10.2. Evaluation of the influence of the over- granulation on the composition of batch 100333

This step was carried out to evaluate the over-granulation process for a batch composition that does not contain Kollidon SR^® as a sustained release agent in the granule core. Qualitative and quantitative composition

Process

Preparation of granulating solution: Solubilize copovidone in 100 g of deionised water under magnetic stirring (30 minutes at 300 rpm).

Blending step: Add 5-fluorocytosine (sieved on 1 mm screen) and microcrystalline cellulose (sieved on 1mm screen) and mix 4 minutes in a high shear mixer GLATT TMG (4 liters tank, impeller speed 200 rpm and chopper speed 300 rpm).

Granulation step (GLATT TMG 4 liters tank):

Batch 100333

Batch 100432

Calibration step:

Granules were calibrated manually (forced sieving) on a 2 mm screen.

Drying step (GLATT GPCGl 2.4 liters Wurster tank):

Coating step (GLATT GPCGl 2.4 liters Wurster tank - 0.8 mm nozzle):

- Preparation of Sepifilm^™ LP coating solution:

Progressively add the prescribed amount of Sepifilm LP 030 into the deionised water under stirring using a deflocculating blade and stir until complete homogeneous dispersion (950rpm). - Preparation of povidone/polyethylene glycol coating solution:

Progressively add the prescribed amount of povidone K17 into the deionised water under stirring using a de flocculating blade and stir until complete solubilisation (12 min 46 sec - 5 410 rpm). Add PEG 4000 to this solution and stir until complete solubilisation (4 min 54 sec - 450 rpm)

Batch 100333

Batch 100432

Results

Particle size distribution analysis :Particle size distribution, determined by sieving, for batches 100333 and 100432 (after the coating step) are given in Figures 28 and 29.

The data presented in Figures 28 and 29 clearly show the difference between granules that were over-granulated and those which were not. Over 43% (in weight) of particles above 710μιη can be obtained for a batch that was over-granulated (i.e. batches 100432 and 100426) versus less than 5% for a batch, of equivalent qualitative and quantitative composition, that was not over-granulated (i.e. batch 100333). It should be emphasized that, compared to formulation of batches 100397/100426/100428, formulation of batches 100333/100432 did not allow over- granulation. This can be observed by comparing the particle size distribution profiles of batches 100432 and 100397/100426. The fact that it was found impossible to over-granulate batch 100432 was attributed to the absence of Kollidon SR^® in the formulation which is preferred for obtaining over-granulation.

Dissolution rate analysis: 5-FC dissolution rate from batches 100333 and 100432 was evaluated using a USP type II apparatus with a paddle rotating speed of 75 rpm in 500 ml of a pH 4.5 buffer and in 500 ml of a pH 6.8 buffer. All analyses were carried out at 37°C and on an equivalent of 3 grams of 5-FC. Comparisons were made to reference Ancotil^® tablets using the same dissolution test protocol.

Redispersion analysis: Redispersion trials were carried out by dispersing the formulation (for an equivalent of 1.5 grams of 5-FC) in 120 ml of tap water. The granules were stirred until a homogeneous dispersion using a spatula (equivalent to a spoon) was obtained. Sample withdrawal was made at time 0 minute (i.e. just after dispersion) and after 5, 10 and 15 minutes. The suspension was allowed to settle between each time points and was re-homogenized (spatula) before each withdrawal of the samples. This protocol was designed to mimic what it would be done in a clinical trial or by the patient taking the 5-FC granule formulation.

The following Table and Figure 30 show the results obtained for batches 100432 and

100333.

Batch 100432 (over-granulation)

Time (min) mg/dose dissolved (vs. 1.5 g 5-FC) % dissolved

0 23.6 1.6

5 124.1 8.3

10 232.2 15.5

15 319.7 21.3 Batch 100333 (no over-granulation)

Time (min) mg/dose dissolved (vs. 1.5 g 5-FC) % dissolved

0 40.2 2.7

5 253.3 16.9

10 472.5 31.5

15 635.2 42.3

5-FC release from the formulation upon reconstitution was found to be slower for batch 100432 than for batch 100333. This might most probably be linked to the fact that granule size was found to be greater for batch 100432 than for batch 100333.

Discussion

Figure 31 compares batch 100432 to batch 100333 analysed in similar conditions (500 ml volume dissolution media, mean values). Both batches have the same qualitative and quantitative compositions but, on the contrary to the batch 100333, the batch 100432 was over- granulated.

5-FC dissolution is slightly slowed down for batch 100432 compared to batch 100333. As previously observed for batches 100397/100426/100428, this was attributed to the over- granulation of 5-FC.

For indicative purposes, the following Figure 32 compares batch 100432 to the reference Ancotil^® tablets (batch 80026952) analysed in similar conditions (500 ml volume dissolution medium, mean values).

10.3. Evaluation of the influence of the over- granulation on the composition of batch

100412

Qualitative and quantitative composition

Batch 100412 Batch 100434

(no over-granulation) (over-granulation)

Batch size (g) 500 g 500 g

Quantity Quantity

Composition % mg/sachet % mg/sachet

Granulation

5-fluorocytosine 39.00 3000.00 39.00 3000.00

Macrocrystalline cellulose

(Vivapur^® 101) 8.00 615.38 8.00 615.38

ollidon SR^® 25.00 1923.08 25.00 1923.08

Copovidone (Plasdone S-

630^®) 2.00 153.85 2.00 153.85

Total (phase granulation) 74.00 5692.30 74.00 5692.30 Coating

SepifimT LP 030 20.00 1538.46 20.00 1538.46

Povidone K17/PEG4000 6.00 461.54 6.00 461.54

Total 100.00 7692.30 100.00 7692.30

Process

Preparation of granulating solution: Solubilize copovidone in 100 g of deionised water under magnetic stirring (5 minutes at 390 rpm).

- Add ½ Kollidon SR^® in the copovidone solution under magnetic stirring and stir until a homogeneous dispersion (32 minutes at 800 rpm) is obtained.

Blending step: Add 5-fluorocytosine (sieved on 1 mm screen), ½ Kollidon SR^® (sieved on 1 mm screen) and microcrystalline cellulose (sieved on 1 mm screen) and mix 4 minutes in a high shear mixer GLATT TMG (1 liter tank, impeller speed 200 rpm and chopper speed 300 rpm).

Granulation step (GLATT TMG 4 liters tank):

Batch 100412

Batch 100434

Calibration step:

Granules were calibrated manually (forced sieving) on a 2 mm screen (time of operation: 10 minutes for batch 100412 and 15 minutes for batch 100434).

Drying step (GLATT GPCG1 2.4 liters Wurster tank):

Coating step (GLATT GPCG1 2.4 liters Wurster tank - 0.8 mm nozzle):

- Preparation of Sepifilm LP coating solution:

- Preparation of povidone/polyethylene glycol coating solution:

Progressively add the prescribed amount of povidone Kl 7 in the deionised water under stirring using a deflocculatmg blade and stir until complete solubilisation. Add PEG 4000 to this solution and stir until complete solubilisation. Batch 100412 (reminder of process parameters)

Results

Particle size distribution analysis: Particle size distribution, determined by sieving, for batch 100412 and batch 100434 (after the coating step) are given in Figures 33 and 34.

These data clearly show the difference between granules that were over-granulated and those which were not over-granulated. Over 73% (in weight) of particles above 710 μιη can be obtained for a batch that was over-granulated (i.e. batch 100434) versus less than 30%> for a batch, of equivalent qualitative and quantitative composition, that was not over-granulated (i.e. batch 100412).

Dissolution rate analysis : 5-FC dissolution rate from batches 100434 and 100412 was evaluated using a USP type II apparatus with a paddle rotating speed of 75 rpm in 500 ml of a pH 4.5 buffer and in 500 ml of a pH 6.8 buffer. All analyses were carried out at 37°C and on an equivalent of 3 g of 5-FC. Comparisons were made to the reference Ancotil^® tablets using the same dissolution test protocol. Redispersion analysis : Redispersion trials were carried out by dispersing the formulation (for an equivalent of 1.5 g of 5-FC) in 120 ml of water. The granules were stirred until a homogeneous dispersion using a spatula (equivalent to a spoon) was obtained. Sample withdrawal was made at time 0 minute (i.e. just after dispersion) and after 5, 10 and 15 minutes. The suspension was allowed to settle between each time points and was re-homogenized (spatula) before each sample withdrawal. This protocol was designed to mimic what would be done in a clinical trial or by the patient taking the 5-FC granule formulation.

The following Table and Figure 35 show the results obtained for batches 100434 and

100412.

5-FC release from the formulation upon reconstitution was found to be slower for batch 100434 than for batch 100412. This was most probably due to the fact that granule size was found to be greater for batch 100434 than for batch 100412.

Discussion

Figure 36 compares batches 100434 and 100412 analysed in similar conditions (500 ml volume dissolution media, mean values). Both batches have the same qualitative and quantitative compositions but, contrarily to the batch 100412, batch 100434 was over- granulated.

5-FC dissolution could be significantly slowed down for batch 100434 compared to the batch 100412. As previously stated for batches 100397 and 100426/100428, this was attributed to the over-granulation of 5-FC. These results clearly showed that batch 100434 has a pH-independent dissolution profile. Figure 37 compares batch 100434 to the reference Ancotil tablets analysed in similar conditions (500 ml volume dissolution media, mean values).

As it can be noticed in Figure 37, 5-FC dissolution from batch 100434 was similar to the dissolution profile of the reference Ancotil^® tablets at pH 6.8. At pH 4.5, 5-FC dissolution from batch 100434 was found to be slightly slower than for the reference Ancotil^® tablets.

With regards to the results obtained for batch 100434, it has been decided to select this formulation and this specific over-granulation manufacturing process for the pre-pilot phase of this project (Phase 4). Results observed with batches 100397/100426 were also found to be very interesting, as drug dissolution profiles were also close to the reference Ancotil^® tablets. It was nevertheless decided to choose batch 100434 as the total load of the formulation, for an equivalent dose of 1.5 or 3 g of 5-FC, was lower than for batches 100397/100426 (7692.3 mg versus 9375.0 mg, respectively).

Conclusion

A granule formulation containing 5-fluorocytosine and exhibiting similar dissolution profile to the reference Ancotil^® tablets was successfully developed according to the Invention.

The qualitative and quantitative compositions of the developed formulation are given in the following table :

The dissolution results of the selected formulation are given in figure 38.

A statistical analysis aiming at evaluating the similarity of the dissolution curves presented in Figure 38 has been carried out. One of the most common statistical tests for evaluating the similarity of in vitro dissolution curves is the F1/F2 test (Shah et al., In vitro dissolution profile comparison - statistics and analysis of the similarity factor, f2. Pharm. Res. 15: 889-896, 1998) which concluded that batch 100397 was similar, dissolution-wise, to the reference Ancotil^® tablets. BIBLIOGRAPHIC REFERENCES

Dias et al, Clin Cancer Res (2010), 16,2540-9;

Erbs et al., 2008, Cancer Gene Therapy, 15, 18-28;

Foloppe et al., 2008, Gene Ther., 15, 1361-1371;

Lipps (1993) J. Pharm. Sci. 83: 937-947;

Portsmouth et al., 2007, Molecular Aspects of Medicine, 28, 4-41

Shah et al., Pharm. Res. 15: 889-896, 1998;

WO05/07857;

WO2009/065547;

WO2009/065546;

WO9531105;

W099/54481.

Claims

1. A granule formulation comprising 5-fluorocytosine wherein said granule comprises :

(i) a core matrix, and

(ii) a coating

2. The granule formulation of claim 1, wherein the core matrix comprises 5- fluorocytosine (5-FC) and at least one pharmaceutically acceptable carrier.

3. The granule formulation of claim 2, wherein the pharmaceutically acceptable carrier included in the core matrix is a granulating agent.

4. The granule formulation of claims 2 or 3, wherein the carrier amount in the core matrix ranges from about 5% to about 40% by weight of the core matrix.

5. The granule formulation of anyone of claims 2 to 4, wherein said carrier comprises microcrystalline cellulose and polyvinyl pyrrolidone.

6. The granule formulation of anyone of claims 2 to 5, wherein said carrier comprises microcrystalline cellulose, polyvinyl pyrrolidone and polyvinyl acetate.

7. The granule formulation of anyone of claims 2 to 4, wherein said carrier comprises microcrystalline cellulose and copovidone.

8. The granule formulation of claim 7, wherein the weight ratio microcrystalline cellulose / copovidone is from about 2 to about 6.

9. The granule formulation of anyone of claims 1 to 8, wherein the core matrix of the granule formulation comprises 5-fluorocytosine (5-FC), microcrystalline cellulose and polyvinyl pyrrolidone.

10. The granule formulation of claim 9, wherein the core matrix further contains polyvinyl acetate.

11. The granule formulation of anyone of claims 1 to 10, wherein the core matrix further comprises a sustained release agent.

12. The granule formulation of claim 11, wherein the sustained release agent is in an amount above 10 % by weight of the core matrix.

13. The granule formulation of claim 11 or claim 12, wherein said sustained release agent is a mixture of polyvinyl acetate and polyvinyl pyrrolidone.

14. The granule formulation of claim 13, wherein the weight ratio polyvinyl acetate / polyvinyl pyrrolidone is 80 %/20 %.

15. The granule formulation of anyone of claims 11 to 14, wherein the sustained release agent is a spray dried, non-hygroscopic powder consisting of polyvinyl acetate (8 parts w/w) and polyvinyl pyrrolidone (2 parts w/w).

16. The granule formulation of anyone of claims 1-15 wherein said granule comprises one first coating and wherein said first coating comprises hydrophilic polymer.

17. The granule formulation of anyone of claims 1-16 wherein the first coating comprises hydroxypropylmethylcellulose (HPMC).

18. The granule formulation of anyone of claims 1-17 wherein the first coating further comprises at least one lubricant.

19. The granule formulation of anyone of claims 1-17 wherein the first coating further comprises stearic acid.

20. The granule formulation of anyone of claims 1-17 wherein the first coating comprises hydroxypropylmethylcellulose (HPMC) and stearic acid.

21. The granule formulation of claim 20 wherein the first coating composition is a mixture of hydroxypropylmethylcellulose, microcrystalline cellulose, stearic acid, pigments and lakes.

22. The granule formulation of anyone of claims 1 -21 wherein said granule further comprises one second coating and wherein the second coating composition comprises at least one water soluble polymer.

23. The granule formulation of claim 22 wherein said water-soluble polymer is selected in the group consisting of polyvinylpyrrolidone (PVP), copolymers of vinylpyrrolidone and vinyl acetate (copovidone), hydroxypropylmethyl cellulose (HPMC) and polyethylene glycol (PEG).

24. The granule formulation of claim 22 wherein said water-soluble polymer comprises polyvinylpyrrolidone and polyethylene glycol.

25. The granule formulation of claim 22 or 24 wherein the polyvinylpyrrolidone is PVP K17 or K90.

26. The granule formulation of claim 22 or 24 the polyethylene glycol is PEG4000.

27. A granule formulation comprising 5-fluorocytosine wherein said granule comprises :

(i) a core matrix, and

(ii) a coating,

and wherein the granules exhibit a 5-FC dissolution rate defined as follows:

a) the dissolution is carried out by using USP type II apparatus with a paddle rotating speed of 75 rpm in 500 ml NaCl pH 6.8 and the time percent 5-FC release of the granule formulation is :

5 min: at least 25%,

15 min: at least 70%;

and/or

b) the dissolution is carried out by using eq. 3g of 5-FC, USP type II apparatus with a paddle rotating speed of 75 rpm, 37°C, in 500 ml NaCl pH 4.5 and the time percent 5-FC release of the granule formulation is :

5 min: at least 25%,

15 min: at least 80%.

28. The granule formulation of anyone of claims 1-27, which comprises at least 1000 mg, preferably at least 1500 mg, of 5-FC.