Patent ID: 12220403

EXAMPLE 1: PREPARATION OF COMPOSITIONS OF THE PRESENT INVENTION

The compositions shown in Table 1 were prepared by heating the carrier matrix to a temperature of between 60 to 70° C. with the aid of an oven. The temperature was held for approximately 2 hours to ensure that all the material is fully molten. The Agent was then gradually added and mechanically stirred into the carrier matrix using a magnetic stir bar or high-shear homogeniser. The system was maintained at a sufficiently high temperature to keep the mixture in a molten state during the stirring, which was continued until a visibly homogenous mixture was obtained. Stirring times varied depending on the particular composition but generally were in the range of 3 to 35 minutes. The resultant mixture was then filled into HPMC capsules and allowed to cool to ambient temperature. Capsules were sealed and generally stored under refrigerated conditions until use.

TABLE 1AgentCarrier MatrixExample(mg per capsule)(mg per capsule)1.112.10 mgVitamin E TPGS107.90 mg1.230.25 mgVitamin E TPGS119.75 mg1.36.05 mgVitamin E TPGS113.95 mg1.460.36 mgVitamin E TPGS (71.06 mg)Gelucire 44/14 (71.49 mg)1.530.25 mgVitamin E TPGS (107.78 mg)PEG 20000 (11.98 mg)1.630.25 mgVitamin E TPGS (107.78 mg)PEG 6000 (11.98 mg)1.730.25 mgGelucire 44/14119.75 mg

EXAMPLE 2: STABILITY OF COMPOSITIONS OF THE PRESENT INVENTION BY X-RAY POWDER DIFFRACTION (XRPD)

An indication of the stability of the Agent (i.e. hydrogen sulphate salt) in a formulation can be provided by XRPD. This technique is capable of simultaneously detecting the crystalline free-base form of the Agent and the crystalline hydrogen sulphate salt form of the Agent within the composition. Samples of the compositions were mounted on silicon wafer mounts and analysed using the Siemen's D5000 X-ray diffractometer. The samples were exposed for 4 seconds per 0.02° θ over the range 2° to 40° 2θ in continuous scan, theta-theta mode.

The approximate limit of detection of the crystalline free-base form of the Agent within a composition of the invention was determined by preparing formulations with varying relative amounts of the crystalline free-base form of the Agent to the crystalline Agent (i.e. hydrogen sulphate salt-form) and these compositions were analysed by XRPD.FIG.1shows that the free-base form of the Agent was detectable to a level of 2.5% w/w free-base in a Vitamin E TPGS based composition which also nominally contained 21.2% w/w of the Agent.

XRPD patterns were obtained for each of the compositions described in Examples 1.1, 1.2 and 1.3 immediately after their manufacture. These patterns (shown inFIG.2) demonstrate only the presence of the Agent (i.e. hydrogen sulphate form).

EXAMPLE 3: STABILITY OF COMPOSITIONS OF THE PRESENT INVENTION BY SOLID STATE NMR SPECTROSCOPY

An indication of the stability of the Agent in compositions of the invention can be provided by using19F Solid State NMR Spectroscopy (19F SS-NMR). This technique is capable of detecting both the crystalline free-base form of the Agent and the crystalline Agent (i.e. hydrogen sulphate salt form) within the composition. The free-base form of the Agent and the Agent (i.e. hydrogen sulphate salt) give distinct and characteristic fluorine peaks in the spectrum. These peaks can be integrated in the normal manner for NMR signals and the ratio of the peaks is proportional to the ratio of the two solid state forms present, i.e. the free-base form of the Agent and the Agent (i.e. hydrogen sulphate form). Analysis of compositions was carried out by placing sample material in a 4 mm MAS (Magic Angle Spinning) rotor.19F NMR [376 MHZ] spectra with1H composite pulse decoupling [TPPM15] was recorded on the Avance 400 spectrometer using the 4 mm HFX (Bruker Biospin) probe. All samples were spun at 12 kHz using the pulse program “aringdec” (anti-ring with decoupling). It should be noted that the frictional forces associated with the technique of magic angle spinning could result in sample heating, up to approximately 10° C.-20° C. above ambient temperature.

The approximate limit of detection of the crystalline free-base form of the Agent within a composition of the invention was determined by preparing formulations with varying relative amounts of the crystalline free-base form of the Agent and the crystalline Agent (i.e. hydrogen sulphate salt-form). These formulations were then analysed by19F SS-NMR. The NMR spectra depicted inFIG.3show that the free-base form of the Agent was detectable to a level of 1% w/w free-base in a Vitamin E TPGS based composition that also contained 28.9% w/w of the Agent (i.e. hydrogen sulphate salt).

Formulations described in Examples 1.2 and 1.3 were tested by19F SS-NMR post manufacture and no evidence of the presence of free-base form of the Agent was found, seeFIG.4andFIG.5. Some sample heating was observed with the analysis of these samples, which may have led to the appearance of an isotropic peak at −129.5 ppm. Without wishing to be bound by any particular theory, the peak may be attributed to the presence of 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide dissolved in Vitamin E TPGS, which has become molten upon sample heating.

EXAMPLE 4. STABILITY OF COMPOSITIONS ON STORAGE

Stability studies on the compositions described in Examples 1.2 and 1.3 for up to 12 months have shown that they are stable at elevated temperatures and high humidities whilst being enclosed in white high-density polyethylene (HDPE) bottles (induction sealed and containing desiccant). No significant changes in the stability data for the compositions of Example 1.2 and 1.3 were observed after 12 months storage in the HDPE bottles at 25° C./60% Relative Humidity (RH) and 30° C./65% RH, see data in Table 1 and Table 2.

TABLE 1Composition from Example 1.3 stored in inductionsealed HDPE bottles containing desiccant at25° C./60% RH and 30° C./65% RH12 months12 monthsTestInitial25° C./60% RH30° C./65% RHDescriptionPlain, white,No changeNo changebanded capsulesDrug Contenta4.94.84.8Total organic0.64 (4)0.68 (4)0.67 (5)impuritiesbbyHPLC (% area)DissolutionCompliesCompliesComplieswith USPwith USPwith USPMean at 451079699minutes (%)RSD at 453.64.02.4minutes (%)Water content1.20.40.4(% w/w)PolymorphicNo free-baseNo free-baseNo free-baseidentityform of theform of theform of theby XRPDAgent detectedAgent detectedAgent detectedaExpressed as mg Free Base equivalent. Analysed using gradient reversed phase liquid chromatography with UV detection, using YMC-Pack ODS-AQ, 3 μm, 150 × 4.6 mm (id) column, Sample diluent 10% TH, 90% Methanol. Mobile Phase A: 0.01% HFBA/1% IPA/Water (v/v/v), Mobile Phase B: 0.01% HFBA/1% IPA/ACN (v/v/v). Gradient: 0 mins = 30% B, 7.5 min = 30% B, 10.5 min = 36% B, 16.5 min = 36% B, 30.5 min = 90% B, 33 min = 90% B, 34 min = 30% B, 40 min = 30% B. HPLC parameters: Flow rate = 1.2 ml/min, Column temperature = 40° C., Wavelength = 258 nm, Injection volume = 10 μl.bTotal organic impurities includes organic impurities at ≥0.05. The numbers in parentheses refer to the number of organic impurities detected at ≥0.05%.

TABLE 2Composition from Example 1.2 stored in inductionsealed HDPE bottles containing desiccant at25° C./60% RH and 30° C./65% RH12 months12 monthsTestInitial25° C./60% RH30° C./65% RHDescriptionPlain, white,No changeNo changebanded capsulesDrug contenta24.724.524.6Total organic0.66 (4)0.74 (6)0.72 (6)impuritiesbbyHPLC (% area)DissolutionCompliesCompliesComplieswith USPwith USPwith USPMean at 451039798minutes (%)RSD at 452.13.64.9minutes (%)Water content1.00.30.3(% w/w)PolymorphicNo free-baseNo free-baseNo free-baseidentityform of theform of theform of theby XRPDAgent detectedAgent detectedAgent detectedaExpressed as mg Free Base equivalent. Analysed using gradient reversed phase liquid chromatography with UV detection, using YMC-Pack ODS-AQ, 3 μm, 150 × 4.6 mm (id) column, Sample diluent 10% TH, 90% Methanol. Mobile Phase A: 0.01% HFBA/1% IPA/Water (v/v/v), Mobile Phase B: 0.01% HFBA/1% IPA/ACN (v/v/v). Gradient: 0 mins = 30% B, 7.5 min = 30% B, 10.5 min = 36% B, 16.5 min = 36% B, 30.5 min = 90% B, 33 min = 90% B, 34 min = 30% B, 40 min = 30% B. HPLC parameters: Flow rate = 1.2 ml/min, Column temperature = 40° C., Wavelength = 258 nm, Injection volume = 10 μl.bTotal organic impurities includes organic impurities at ≥0.05. The numbers in parentheses refer to the number of organic impurities detected at ≥0.05%.

EXAMPLE 5. DISSOLUTION OF COMPOSITIONS OF THE PRESENT INVENTION

An in-vitro dissolution method was developed to test the performance of formulations contained within HPMC capsules. Dissolution in duplicate or triplicate was carried out on the formulations listed below in Table 3.

Dissolution of capsules is performed according to the general procedure of the United States Pharmacopoeia Apparatus II (paddle). Samples of the dissolution medium are withdrawn at various time points after capsule addition and the 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide concentration is quantified by comparison of its HPLC response peak area to that of a standard solution prepared at a level equivalent to 100% release of the compound. The method uses clear glass peak vessel dissolution pots, and Spiral Stainless Steel Capsule Sinkers are used to hold the capsules. 900 ml pH2.0 735 mOsmol\L phosphate buffer solution is used at 27° C. and a paddle speed of 100 rpm is used.

TABLE 3Dissolution resultsDissolution atDissolution atFormulation of50 minutes (%)Formulation of50 minutes (%)Example 1.1100Comparator 173 (45 min)(2% w/w Free Base,21.9% w/w Agent(i.e. hydrogensulphate salt))Example 1.299Comparator 256 (40 min)(5% w/w Free Base,18.2% w/w Agent(i.e. hydrogensulphate salt))Example 1.399Comparator 341(batch 1)(20% w/w Free Base)Example 1.395(batch 2)Example 1.596Example 1.6101Example 1.795

In addition to the compositions described in Examples 1.1-1.3 and 1.5-1.7, some further comparator formulations were manufactured using mixtures of crystalline free-base and crystalline Agent (i.e. hydrogen sulphate salt). The mixture of the two forms were dispersed in Vitamin E TPGS according to analogous methods to those described in Example 1 and filled into HPMC capsules.

The dissolution data for the comparator formulations show that dissolution decreased as the amount of free base form of the Agent in the composition increased. A 17% drop in dissolution at 50 minutes is observed for a formulation containing 2% w/w Free Base form of the Agent. The data generated by analysing the free-base containing comparator formulations show that the dissolution method gives an indication of the level of free-base form of the Agent present within the compositions. The dissolution results for the compositions described in Examples 1.1-1.3 and 1.5-1.7 show that 95% or greater dissolution is achieved indicating that the compound is substantially present in it's hydrogen sulphate salt-form (i.e. as the Agent).

EXAMPLE 6: PREPARATION OF FURTHER COMPOSITIONS OF THE PRESENT INVENTION

The compositions shown in Table 4 were prepared by heating the carrier matrix in an oven set at 70° C. for at least one hour. The Agent was then gradually added and mechanically stirred into the carrier matrix using a magnetic stir bar or a high-shear homogeniser. The system was maintained at sufficiently high temperature to keep the mixture in a molten state during stirring. Stirring was performed until a visibly homogenous mixture was obtained. The time taken for this to be achieved varied depending on the composition but was at least 10 minutes and could have been up to 60 minutes. The systems ranged in total weight from 3.75 g to 75 g (as indicated in Table 4). The resultant mixture was filled into HPMC capsules and allowed to cool to ambient temperature and solidify. Capsules were stored at either room temperature or under refrigerated conditions until use.

TABLE 4AgentTotal weight(mg perCarrier Matrixof batchExamplecapsule)(mg per capsule)prepared (g)6.130.25 mgVitamin E TPGS (89.75 mg)3.75Tween 80 (30.00 mg)6.230.25 mgVitamin E TPGS (89.75 mg)3.75Cremophor EL (30.00 mg)6.330.25 mgVitamin E TPGS (89.75 mg)3.75Pluronic F-68 (30.00 mg)6.430.25 mgVitamin E TPGS (89.75 mg)3.75PEG 1000 (30.00 mg)6.530.25 mgVitamin E TPGS (97.25 mg)3.75PEG 1000 (22.50 mg)6.630.25 mgVitamin E TPGS (104.75 mg)3.75PEG 1000 (15.00 mg)6.730.25 mgVitamin E TPGS (112.25 mg)3.75PEG 1000 (7.50 mg)6.830.25 mgVitamin E TPGS (119.75 mg)3.75(API batch 1)6.930.25 mgVitamin E TPGS (119.75 mg)3.75(API batch 2)6.1030.25 mgVitamin E TPGS (119.75 mg)3.75(API batch 3)6.1130.25 mgVitamin E TPGS (269.75 mg)7.56.1230.25 mgVitamin E TPGS (119.75 mg)756.1315.12 mgVitamin E TPGS (134.88 mg)756.1460.5 mgVitamin E TPGS (239.5 mg)156.1590.75 mgVitamin E TPGS (359.25 mg)15

EXAMPLE 7. DISSOLUTION OF COMPOSITIONS IN PH 6.5 DISSOLUTION MEDIA

An in-vitro dissolution method employing pH 6.5 dissolution media was used to test the performance of compositions contained within HPMC capsules. The pH 6.5 dissolution method provided improved discrimination of the presence of free base form of the Agent in compositions when compared with the dissolution method described in Example 5. Dissolution in duplicate or triplicate was carried out on the formulations listed in Table 4 and also on the formulation of Example 1.7.

Dissolution of capsules was performed according to the general procedure of the United States Pharmacopoeia Apparatus II (paddle). Samples of the dissolution medium are withdrawn at various time points after capsule addition and the 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide concentration is quantified by comparison of its HPLC response peak area to that of a standard solution prepared at a level equivalent to 100% release of the compound. The method uses clear glass peak vessel dissolution pots, and Spiral Stainless Steel Capsule Sinkers are used to hold the capsules. 1000 ml of pH 6.5 dissolution media is used at 37° C. and a paddle speed of 50 rpm is used.

The pH 6.5 dissolution media is prepared by the addition of 1.74 g sodium hydroxide pellets, 19.77 g sodium dihydrogenphosphate hydrous (or 17.19 g sodium dihydrogenphosphate anhydrous) and 30.93 g sodium chloride to 5 litres of deionised water. The pH is then adjusted to 6.5 with 1M hydrochloric acid or 1M sodium hydroxide.

In addition to the compositions described in Table 4, some further comparator formulations were manufactured using mixtures of crystalline free-base of the Agent and crystalline Agent (i.e. hydrogen sulphate salt-form). The mixture of the two forms were dispersed in Vitamin E TPGS according to analogous methods to those described in Example 6 and filled into HPMC capsules. The specific compositions of the comparator formulations are shown in Table 5.

TABLE 5Comparator compositionsAgent, free baseAgent (i.e. hydrogenform/mgsulphate salt)/mgCarrier MatrixExample(% w/w)(% w/w)(mg)C10.605 mg29.645 mgVitamin E TPGS(0.4% w/w)(19.76% w/w)(119.75 mg)C20.15 mg30.09 mgVitamin E TPGS(0.1% w/w)(20.06% w/w)(119.76 mg)C30.075 mg30.165 mgVitamin E TPGS(0.05% w/w)(20.11% w/w)(119.76 mg)C40.03 mg30.21 mgVitamin E TPGS(0.02% w/w)(20.14% w/w)(119.76 mg)

The dissolution data for the comparator formulations (Table 6) show that dissolution decreased as the amount of free base form of the Agent in the composition increased. A 90% drop in dissolution at 60 minutes is observed for a formulation containing 0.4% w/w free base form of the Agent. Furthermore, the presence of 0.02% w/w free base of the Agent caused a 13% drop in dissolution at 60 minutes. The data generated by analysing the free-base containing comparator formulations show that the pH 6.5 dissolution method provides a good indication of the level of free-base form of the Agent present within the compositions.

TABLE 6Dissolution results for comparator compositionsin pH 6.5 dissolution mediaFormulation ofDissolution at 60 minutes (%)C110C243C378C487

The dissolution results for the compositions described in Example 6 and also for the formulation of Example 1.7 are shown in Table 7. Greater than 96% dissolution at 60 minutes is achieved for all of the formulations, indicating that the Agent is substantially present in it's hydrogen sulphate salt-form in these compositions.

TABLE 7Dissolution results in pH 6.5 dissolution mediaFormulation ofDissolution atDissolution atExample60 minutes (%)Formulation of60 minutes (%)1.7996.81016.1986.91016.2986.101006.3966.11986.4976.12996.51026.13996.61006.14976.71016.1597