Patent ID: 12213954

EXAMPLES

As colchicine may degrade under the influence of light, the process was performed shielded from direct sunlight. The process was otherwise performed using regular manufacturing equipment. The basic steps are as follows:

Materials and Methods

Preparation of Colchicine Solutions Compositions

The following ingredients were used in the preparations described below:Colchicine (Sarv Labs, India; Indena, Italy);Sweetener: Sucralose (Nutrilo, Germany)Orange flavour (Symrise, Germany)Methyl paraben sodium (Emprove®, Merck, US)Propyl paraben sodium (Clariant, Germany)Hydroxyethylcellulose (Natrosol 250 HX®, Ashland, US)Xanthan Gum (Jungbunzlauer, Switzerland)Citric acid anhydrous (Citrique Belge, Belgium)Trisodium citrate dihydrate (Jungbunzlauer, Switzerland)Propylene glycol (BASF, Germany)Glycerol (glycerine 4808, 99.5%, Oleon NV, Belgium).Metabisulphite sodium (Merck, Germany)Propyl gallate (Panreac Applichem, Germany)

Formulas 1-9.2 as given in table 1 are true solutions and were prepared as described below. A further formula 4.3 was prepared that differed from formula 4.2 only in the citric acid and sodium citrate content. sample 4.3 comprises 0.12 w/v % citric acid and 0.84 w/v % sodium citrate, whereas sample 4.2 has 0.20 and 0.60 w/v %, respectively. As a result, sample 4.3 has a pH of 6.5, whereas sample 4.2 has a pH of 5.5.

For a batch size of 100 ml, 0.026 g of colchicine is added to about 90 gr of purified water under light protected conditions (i.e. protected against artificial or sun light) at room temperature, and mixed until clear solution is obtained.

TABLE 1composition of samples (w/v %)Sample1234.14.256789.19.2grgrgrgrgrgrgrgrgrgrgrColchicine (dry substance)API0.0250.0250.0250.0250.0250.0250.0250.0250.0250.0120.012HydroxyethylcelluloseThickener0.20.20.2——0.2—Xanthan gumThickener0.15Methyl parabenAntimicrobial0.180.180.180.180.20.180.180.180.180.180.2Propyl parabenAntimicrobial0.0180.0180.0180.0180.0180.0180.0180.0180.0180Sodium metabisulphiteAntioxidant—————0.80.80.8Propyl gallateAntioxidant0.05GlycerolSolvent5—2.510105105101010Propylene glycolSolvent—52.5——————Citric acid anhydrousbuffer0.20.20.20.20.20.20.20.20.20.20.2Na citrate 2H2Obuffer0.50.50.50.50.50.50.60.50.60.50.5Sucralosesweetener0.1350.1350.1350.1350.1350.1350.1350.1350.1350.1350.135Orange flavourflavour0.10.10.10.10.10.10.10.10.10.10.1Purified watersolvent93.693.693.688.888.892.887.992.988.788.888.8Total100100100100100100100100100100100Final pH5.2-5.8

0.2 g of sodium methyl paraben, or 0.18 g of sodium methyl paraben together with 0.018 g propyl paraben (depending of the formula), are added to the above aqueous mixture and mixed well until a clear solution is obtained.

0.12 to 0.2 g of citric acid anhydrous and 0.5 to 0.83 g of trisodium citrate dihydrate (depending of the formula) are added to the previous mixture and mixed well until clear solution.

0.135 g of sucralose is added and mixed well until clear solution.

If present, 0.8 g of sodium bisulphite or 0.05 g propyl gallate was added and mixed well until clear solution. In case of propyl gallate usage, mild heated should be applied up to 45° C., whereafter the solution is cooled to room temperature (22-25° C.).

Glycerol is added as follows: 2.5 g for a final concentration of 2.5 w/v %, 5 g for a final concentration of 5 w/v %, and 10 g for a final concentration of 10 w/v %. If present, 2.5 g of propylene glycol was add to provide a 2.5 w/v % solution, and 5 g for a 5 w/v % solution. Solvents are mixed well at room temperature with the rest until a clear solution is obtained.

If present, 0.15 g of xanthan gum or 0.2 g HEC was added and mixed, at room temperature, until complete dissolution. Depending the batch size this step could last up to 2 hours.

Flavour is added to the obtained solution in an amount of 0.1 w/v %, and purified water was added to a total volume of 100 ml.

The composition was filtered through a 10 μm sieve and filled in type III amber glass vials.

Analytical Procedures

Instrumentation:

Shimadzu (Duisburg, Germany) Prominence Series HPLC-DAD modular system consisting of: a DGU-20A5 mobile phase degasser, an LC-20 AD micro dual piston pump, an SIL-20ACHT autosampler, a CTO-20AC column oven, an SPD-M20 UV/Vis photodiode array detector, and a personal computer with Shimadzu LC Solutions software (v.1.11 SP1) installed for the system control, and the data record and process.

Reagents:

HPLC-grade water (H2O) (resistivity>18 MΩ cm) by deionization and distillation;Acetonitrile (ACN) (Fisher Chemical, Germany, HPLC grade);Methanol (MeOH) (Fisher Chemical, HPLC grade)Orthophosphoric acid (Fisher Chemical, Germany); PotassiumDihydrogen phosphate (LANH:NER)
DiluentMeOH/H2O: 50/50
Determination of Colchicine Content:Column: ZORBAX Eclipse XDB C18, 250×4.6 mm, 5 μm, (Lot: B20441-P/N: 990967-902)Reference: Colchicine Reference Standard, LGC, purity 93.0%, Lot. 1096857Standard solution of colchicine (0.01 mg/ml): 10 mg Colchicine reference standard was transferred into a 10 ml volumetric flask. Diluent was added to adjust the volume to 10 ml, followed by vortex mixing for homogenizing. Of this solution, 0.1 ml was transferred into a 10 ml volumetric flask, diluted to the envisaged volume with diluent and vortexed/mixed to homogenize. Standard solutions were prepared in duplo.Test solution (0.01 mg/ml): 0.2 ml sample oral solution was transferred into a 5.0 ml volumetric flask. Diluent was added followed by vortex mixing from homogenizing. Test solutions were prepared in duplo.
Chromatographic Parameters
Mobile Phase:Dissolved 6.8 g/l of potassium dihydrogen phosphate:MeOH (45:55). The pH was adjusted to 5.5 with ortho-phosphoric acid. The buffer was subjected to filtration through a 0.45-μm membrane filter.Injection Volume: 10 μlFlow rate: 1.0 ml/minColumn temperature: 20° C.Autosampler temperature: 25° C.Run time: 23 minutesQuantification wavelength: 350 nm
Procedures:

Six replicates of colchicine standard solution (before proceeding system suitability criteria should be met) and 2 replicates of verification standard solution were injected. The recovery against the mean areas of the two standard solutions were calculated. (The recovery should be between 98-102%). One replicate of each sample solution was injected.

System Suitability Criteria:

The 6 replicates of the standard solution were used:a) % RSD≤2.0%b) Tailing Factor≤1.5c) Plate Count>2000
Calculation of the % Content of Colchicine by the Equation:

%⁢Colchicine⁢=A⁢s⁢m⁢p*V⁢s⁢m⁢p*W⁢s⁢t⁢d*P⁢s⁢t⁢d*D⁢s⁢t⁢d*1⁢0⁢0A⁢s⁢t⁢d*W⁢s⁢m⁢p*L⁢C*D⁢s⁢m⁢p*V⁢s⁢t⁢d

wherein: Asmp is the area of the colchicine peak in the chromatogram of the test solution; Astd is the area of the Colchicine peak in the chromatogram of the standard solution; LC is the Label Claim of the formulation (=0.25 mg/ml); Wstd is the accurate weigh of the Colchicine reference standard used for the preparation of the standard solution in mg; Vsmp is the initial volume of the colchicine sample solution in ml; Vstd is the initial volume of the colchicine standard solution in ml; Pstd is the purity of the standard in decimal form (% assay as is from CoA); Dstd is the dilution of the standard; and Dsmp is the dilution of the sample.

Determination of all Impurities (Except for Impurity A):

During manufacture and storage, impurities can be formed, mentioned in table 2 below.Column: Agilent Extend C18 250×4 mm, 5 μm (Lot: B09110)Reference solutions: Colchicine Reference Standard, LGC, purity 93.0%, Lot. 1096857, and Colchicine for system suitability CRS.

TABLE 2colchicine and impuritiesPh. Eur.USPProduct specsContent 97.0%-102.0% (anhydrous substanceColchicine is an alkaloid contained in various species of Colchicum and in other genera. It contains NLT 94.0% and NMT 101.0% of colchicine (C22H25NO6), calculated on the anhydrous, solvent-free basis.Reporting threshold: 0.1% Identification threshold: 0.5% Qualification threshold: 1.0%Impurity A NMT 3.0%Specified impurity Indena: Secondary plant metabolite and degradation product Sary: From starting materialNMT 3.0% (acc. to ph. eur)A. N-[(7S,12aM)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl] formamide(N-deacetyl-N-fomylcolchicine)Impurity BOther detectable impurity Conformational isomer of colchicine which is formed in situ in solution. Disregard peak due to impurity B Indena: Not to be considered as impurity Sary: From starting materialNMT 1.0%B. N-[(7S,12aP)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl] acetamide(Conformational isomer)Impurity COther detectable impurity Indena: Degradation product Sary: PhotodegradantNMT 1.0% Qualification thresholdC. N-[(7S,7bR,10aS)-1,2,3,9-tetramethoxy-8-oxo-5,6,7,7b,8,10a-hexahydrobenzo[a]cyclopenta-[3,4]cyclobuta[1,2-c]cyclohepten-7-yl] acetamide(β-lumicolchicine)Impurity DOther detectable impurity Indena: Secondary plant metabolite Sary: Starting material isolationNMT 0.5% (ICH Q3B R2, identification threshold)D. N-[(7S,12aM)-3-(β-D-glucopyranosyloxy)-1,2,10-trimethoxy-9-oxo-5,6,7,9-tetrahydro-benzo[a]heptalen-7-yl] acetamide (colchicoside)Impurity E NMT 0.2%Specified impurity Indena: Secondary plant metabolite Sary: From starting materialNMT 0.5% (ICH Q3B R2, identification threshold)E. N-[(7S,12aM)-3-hydroxy-1,2,10-trimethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl]acetamide (3-O-demethylcolchicine)Impurity F NMT 0.2% - Cas no: 477-27-0Specified impurity Acceptance criteria: No definite green color is produced. Indena: Degradation product Sary: From starting materialNMT 1.0% Qualification threshold (from Indena)F. N-[(7S,12aM)-10-hydroxy-1,2,3-trimethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl] acetamide(colchiceine)Imp G NMT 0.25%Specified impurity Indena: Degradation product Sary: Photo degradantNMT 1.0% Qualification thresholdG. N-[(7S,7bR,10aR)-1,2,3,9-tetramethoxy-8-oxo-5,6,7,7b,8,10a-hexahydrobenzo[a]cyclopenta-[3,4]cyclobuta[1,2-c]cyclohepten-7-yl] acetamide(γ-lumicolchicine)Unspecified impurities_NMT 0.10%CHROMATOGRAPHICUnspecifiedPURITYimpurities_NMT 0.5%Procedure: Proceed as(ICH Q3B R2,directed in the Assay.identification threshold)Total_NMT 4.0%Acceptance criteria: NMT5.0% other than that dueto colchicine, elutingwithin 1.5 times theretention time forcolchicine
Reference Colchicine Solution (0.000125 mg/ml):

10 mg colchicine was transferred into a 10 ml volumetric flask. The envisaged volume was obtained by dilution with diluent followed by vortex mixing to homogenize. Of this solution, 0.1 ml was transferred into a 20 ml volumetric flask, diluted to volume with diluent and vortex mixed to homogenize.

Test solution (0.025 mg/ml): Of the sample preparation, 1.0 ml was transferred into a 10.0 ml volumetric flask. The envisaged volume was obtained by dilution with diluent followed by vortex mixing to homogenize. The obtained solution was filled in a HPLC vial.

System Suitability Solutions:

5 mg of Colchicine for system suitability CRS was transferred into a 5 ml volumetric flask and diluted to volume with diluent and vortex mixed to homogenize.

System Suitability Solution:

The 3 replicates of the standard solution for unknown impurities were used: % RSD≤2.0%, EP plate is NLT2000, Tailing factor is NMT 1.5. For the system suitability solution, the resolution between Impurity G and Impurity C should be >1.5.Mobile phase A: 6.8 g of potassium dihydrogen phosphate was dissolved in 1000 ml HPLC water.Mobile phase B: ACN: MeOH (60:40)Mobile phase C: H2OInjection volume: 30 μl; autosampler temperature: 25° C.; flow rate 1 ml/min, column temperature 15° C.; run time: 50 minutes; quantification wavelength: 254 nm.

Gradient Program is shown in table 3.

TABLE 3Gradient programTimeMobile phase AMobile phase BMobile phase C(min)(% v/v)(% v/v)(% v/v)0-4045154040-4545451045.0145451045.01-50451540

The colchicine content is calculated by the following equation:

%⁢Recovery=A⁢s⁢m⁢p×V⁢s⁢m⁢p×W⁢s⁢t⁢d×P⁢s⁢t⁢d×D⁢s⁢t⁢d×1⁢0⁢0A⁢s⁢t⁢d×L⁢C×D⁢s⁢m⁢p×V⁢s⁢t⁢d

wherein: Asmp is the peak area response of colchicine in the sample solution chromatograms; Astd is the average peak area response of colchicine in the standard solution chromatograms; LC is the Label Claim of the formulation (=50 mg/2 ml); Wstd is the weight of the Colchicine standard in mg; Vsmp is the initial volume of the test solution in ml; Vstd is the initial volume of the standard solution in ml; Pstd is the purity of the standard in decimal form; Dstd is the standard dilution; and Dsmp is the sample dilution.

Under the chromatography conditions as described above, the relative retention time RRT for impurity B is 0.99, for impurity C: 1.26, for impurity E: 0.72, for impurity F: 1.16, and for impurity G, 1.26. The response factor RF is 0.91 for impurity G, and 1.30 for impurity C.

The product specification of a colchicine solution of formula 1 is depicted in table 4.

Determination of Impurity A

Column: NUCLEOSIL EC 100-5 C8, 250×4 mm, 5 μm (Lot: 21000043-SN: E12060052)References: Colchicine Reference Standard, LGC, purity 93.0%, Lot. 1096857, and System Suitability for Impurity A, EP, CRS Y0001958, Batch 1.0.Reference colchicine solution (0.0005 mg/ml): 10 mg colchicine was transferred into a 10 ml volumetric flask. The envisaged volume was obtained by dilution with diluent followed by vortex mixing to homogenize. Of this solution, 0.05 ml was transferred into a 10 ml volumetric flask, diluted to volume with diluent and vortex mixed to homogenize. 0.1 ml of this solution was transferred to the HPLC vial, 0.9 ml diluent was added and vortex mixed to homogenize.Test solution (0.1 mg/ml): Of the sample preparation, 2.0 ml was transferred into a 5.0 ml volumetric flask. The envisaged volume was obtained by dilution with diluent followed by vortex mixing to homogenize. The obtained solution was filled in a HPLC vial.System Suitability solutions: 10 mg of Impurity A was diluted with diluent to volume in a 10 ml volumetric flask and vortex mixed to homogenize (Impurity A stock solution). 10 mg of colchicine was diluted with diluent in a 10 ml volumetric flask and vortex mixed to homogenize (Colchicine stock solution). 0.03 ml of Impurity A stock solution and 1 ml of Colchicine stock solution were diluted to volume with diluent in a 10 ml volumetric flask, and vortex mixed to homogenize.System suitability solution: The 3 replicates of the standard solution for unknown impurities were used: % RSD≤2.0%, EP plate is NLT2000, Tailing factor is NMT 1.5. For the system suitability solution, the resolution between Impurity A and colchicine should be >1.5.Mobile phase: 6.8 g of potassium dihydrogen phosphate was dissolved in 1000 ml HPLC water:MeOH (45:55), and the pH was adjusted to 5.5 with orthophosphoric acid. The solution was subjected to filtration through a 0.45 μm membrane.Injection volume: 10 μl; autosampler temperature: 25° C.; flow rate 1 ml/min, column temperature 25° C.; run time: 23 minutes; quantification wavelength: 254 nm.

The percentage of colchicine content was calculated with the same equation as presented in the previous section for all impurities.

Under the chromatography conditions as described above, the relative retention time RRT for impurity A is 0.9, and the response factor RF is 0.84.

TABLE 4Product specification Solution Formula 1 after productionTESTSMETHODSPECIFICATIONS1AppearanceVisual inspectionAmber glass bottle with cap2Clarity and degree ofPh. Eur. cur. ed. (2.2.1)Clear solutionopalescence of liquids3Degree of coloration of thePh. Eur. cur. ed. (2.2.2)Clear solutionliquids4pHPh. Eur. cur. ed. (2.2.3)5.6-5.95Relative DensityPh. Eur. cur. ed. (2.2.5)1.00-1.056Uniformity of mass ofPh. Eur. cur. ed. (2.9.27)Meets the requirementsdelivered doses7Uniformity of dosage unitsPh. Eur. cur. ed. (2.9.40)Meets the requirements (MV)8Deliverable VolumeUSP <698>Meets the requirements9Package integrityIn house methodPass10IdentificationHPLC (UV)Retention time complies with RSColchicineHPLC (Diode Array)UV spectrum complies with RS11IdentificationHPLC (UV)Retention time complies with RSmethylparaben14Assay of colchicineIn - house method95.0-105.0% of the stated amountPh. Eur. cur. ed. (2.2.29)of colchicine15Assay of methylparabenIn house methodEach 90.0-110.0%Ph. Eur. cur. ed. (2.2.29)18Related SubstancesIn house methodimp ANMT 3.0%Ph. Eur. cur. ed. (2.2.29)imp BNMT 1.0%imp CNMT 1.0%imp DNMT 0.5%imp ENMT 0.5%imp FNMT 0.5%imp GNMT 1.0%Any unspecifiedNMT 0.5%impurityTotal ImpuritiesNMT 5.0%19Microbial Limits TestingTotal Aerobic MicrobialPh. Eur. cur. ed. (5.1.4)100 CFU/mLCountTotal Combined Yeasts/10 CFU/mLMouldsE. ColiAbsent/1 mL20AntimicrobialPh. Eur. cur. ed. (5.1.3)PassEffectiveness Testing
Stability

Stability tests of samples 1-9.2 of table 1 are depicted in tables 5-18 and are discussed below. The samples were all placed in ICH stability chambers. The humidity was 60% in the tests at 5° C. and 25° C., 65% in the tests at 30° C. and 75% at the tests at 40° C. GLP are Glopebra samples.

TABLE 5stability testtime zero1234.14.256789.19.2GLPassay colchicine101.10100.20101.20101.00101.3097.5099.5098.30101.40101.0098.80105.60imp A0.400.410.360.300.310.450.410.350.440.350.330.48imp B0.200.220.240.270.260.220.240.120.100.060.42imp Cimp D0.110.13imp E0.100.10imp Fimp GUnspec. Imp1.020.720.41Total Impurities0.600.630.600.670.670.781.801.070.560.450.391.31

TABLE 6Stability test2 months at 25° C.1234.14.256789.19.2assay colchicine101.0099.80101.00100.80100.8096.1099.0097.50101.00101.0098.80imp A0.410.430.440.300.320.500.400.410.460.350.35imp B0.420.480.440.450.450.430.460.430.380.150.12imp Cimp Dimp Eimp Fimp G0.100.10Unspec. Imp1.160.650.91Total Impurities0.831.010.980.750.772.091.511.750.840.500.47

TABLE 7Stability test5 months at 25° C.1234.14.256789.19.2GLPassay colchicine100.8099.80100.20100.00100.0095.5099.1096.00101.00100.8098.50105.30imp A0.420.480.480.300.380.520.540.470.500.360.370.57imp B0.480.570.500.510.490.490.590.480.450.170.150.57imp Cimp Dimp Eimp F0.100.100.11imp G0.110.100.100.100.11Unspec. Imp1.250.871.120.44Total Impurities1.011.251.180.910.982.262.112.070.950.530.521.58

TABLE 8Stability test6 months at 25° C.1234.14.256789.19.2assay colchicine100.5099.50100.0099.5099.60100.20100.1098.40imp A0.510.50.350.300.340.370.360.38imp B0.570.600.580.520.540.410.250.22imp Cimp Dimp Eimp F0.12imp G0.100.100.100.130.12Unspec. ImpTotal Impurities1.181.201.150.951.000.780.610.60

TABLE 9Stability test2 months at 30° C.1234.14.256789.19.2assay colchicine101.0099.70100.50100.60100.0095.1098.8097.50100.90100.9098.70imp A0.450.500.490.310.320.500.540.470.530.350.35imp B0.420.550.440.450.460.430.460.430.380.180.16imp Cimp Dimp Eimp Fimp G0.100.10Unspecified impurities1.160.650.91Total Impurities0.871.151.030.760.782.091.651.810.910.530.51

TABLE 10Stability test5 months at 30° C.1234.14.256789.19.2GLPassay colchicine100.1099.6099.80100.2099.5094.8097.8097.00100.80100.8098.50105.70imp A0.470.500.500.320.380.530.550.430.520.360.370.60imp B0.500.570.560.510.480.490.60.490.460.340.320.50imp Cimp Dimp Eimp F0.100.12imp G0.100.100.120.110.10Unspec. Imp1.320.931.460.44Total Impurities1.071.171.180.940.962.342.182.500.980.700.691.54

TABLE 11Stability test6 months at 30° C.1234.14.256789.19.2assay colchicine99.6099.0099.6099.7099.20101.0099.3098.10imp A0.520.520.500.340.380.360.350.34imp B0.570.600.600.470.490.560.370.35imp Cimp Dimp Eimp F0.10imp G0.120.100.130.120.11Unspec. ImpTotal Impurities1.311.221.230.930.980.920.720.69

TABLE 12Stability test2 months at 40° C.1234.14.256789.19.2assay colchicine100.8099.7099.80100.4099.5094.8098.1097.40100.70100.8098.60imp A0.460.500.510.300.310.500.540.470.520.450.44imp B0.440.550.470.450.520.430.460.430.380.190.18imp Cimp Dimp Eimp Fimp G0.100.10Unspec. Imp1.160.650.91Total Impurities0.901.151.080.750.832.091.651.810.900.640.62

TABLE 13Stability test5 months at 40° C.1234.14.256789.19.2GLPassay colchicine99.8099.5099.0099.8099.2094.1097.5096.8097.50100.5098.50104.40imp A0.520.500.500.340.320.430.440.400.460.450.460.58imp B0.510.600.600.560.600.440.570.511.090.250.260.53imp C0.12imp Dimp E0.12imp Fimp G0.120.150.160.12Unspec. Imp0.002.141.851.883.200.55Total Impurities1.151.251.261.021.043.012.862.794.870.700.721.66

TABLE 14Stability test6 months at 40° C.1234.14.256789.19.2assay colchicine99.6099.0098.7099.5099.0098.00100.1098.00imp A0.530.550.510.370.390.360.390.41imp B0.580.620.680.630.710.940.370.37imp C0.10imp Dimp Eimp F0.120.110.160.110.10limp G0.110.120.100.110.100.15Unspec. Imp3.25Total Impurities1.341.401.451.221.204.800.860.78

TABLE 15Stability test2 months at 5° C.1234.14.256789.19.2assay colchicine101.0099.70100.50100.70100.6095.4098.1096.40100.90100.8098.40imp A0.450.500.510.300.300.500.540.470.520.380.33imp B0.420.480.440.450.480.430.460.430.380.110.10imp Cimp Dimp Eimp FimpG0.100.10Unspec. Imp1.160.650.91Total Impurities0.871.081.050.750.782.091.651.810.900.490.43

TABLE 16Stability test5 months at 5° C.1234.14.256789.19.2GLPassay colchicine100.5098.70100.20100.4099.8095.2097.8096.80100.80100.7098.70105.70imp A0.480.490.540.320.320.480.480.490.510.380.350.53imp B0.500.600.560.470.500.480.600.490.490.120.110.50imp Cimp Dimp Eimp Fimp G0.110.100.100.00Unspec. Imp1.690.660.780.41Total Impurities1.091.191.200.790.822.651.741.761.000.500.461.44

TABLE 17Stability test6 months at 5° C.1234.14.256789.19.2assay colchicine100.2098.20100.00100.0099.50100.40100.2098.50imp A0.500.50.360.350.340.340.290.28imp B0.530.610.560.550.570.40.190.59imp Cimp Dimp Eimp F0.100.100.10imp G0.100.110.10Unspec. ImpTotal Impurities1.131.321.021.001.010.740.480.87

TABLE 18Stability teststress studies14.16(pH 8.5)light stress(pH 3.5)assay colchicine99.1074.20100.50imp A0.400.080.40imp B0.200.150.29imp C26.26imp Dimp E0.10imp F2.36imp GUnspec. Imp5.980.58Total Impurities6.6028.851.37

From the stability data, it can clearly be observed that samples 4.1 and 4.2 as well as samples 9 and 9.1, all without thickener and without antioxidant have the overall highest stability as can be seen by the percentage of the impurities as compared to the same sample at time zero (lowest row in tables 3-9). The stability is better than from the known product Glopebra. The solution comprising the antioxidant propyl gallate (sample 8) is stable at 5° C., content of 2.5% seem to have slightly less long term stability as compared to the formulas with a lower colchicine content of 1.25%.

Solutions that comprise glycerol as sole water miscible solvent (samples 1, 4 and 9) have a higher stability than a mixture of 50:50 (on weight basis) glycerol and propylene glycol (sample 3). Also, sample 1 seems to be more stable than solutions that have propylene glycol as sole water miscible solvent.

The low stability of sample 5 indicates that the combination of thickener and sodium metabisulphite as antioxidant do not seem to be compatible.

From the stress data in table 18 it can be seen that a basic pH, but in particular light results in severe loss of stability. A low pH of 3.5 does not seem to have a significant impact on the stability.

Microbial Efficacy

Formulas 4.2 and 4.3 were tested for efficacy of antimicrobial preservation according to the teaching of the European Pharmacopeia 9.0, section 5.1.3, pp 577 ﬀ. by Quality Assurance & Control Systems Ltd., Athens, Greece. Further, two formulas as formula 4.2, wherein only 70% of the paraben (i.e. 0.14 w/v %) or no paraben was present were tested as well. The test consists of challenging the sample solution with a prescribed inoculum of suitable micro-organisms as shown in the tables 19A-D, storing the inoculated solution at ambient temperature, avoiding sunlight, withdrawing samples from the container at specified intervals of time and counting the micro-organisms in the samples so removed. The preservative properties of the solution are adequate if, in the conditions of the test, there is a significant fall or no increase, as appropriate, in the number of micro-organisms in the inoculated solution after 14 and 28 days. ATCC stands for the deposit number of the micro-organism at the American Type Culture Collection ATCC.

TABLE 19APresevation efficacy sample 4.2 - no parabenInoculationMicro-organismATCCLotCfu/gTime zeroDay 14Day 28Pseudomonas aeruginosa9027484124527.0 × 1056.3 × 105<10<10Staphylococcus aureus653848548215.3 × 1056.0 × 105<10<10Escherichia coli873948356648.7 × 1057.9 × 1052.5 × 1055.2 × 104Candida albicans1023144359033.8 × 1053.3 × 1052.9 × 1053.3 × 106Aspergillus brasiliensis1640439295522.0 × 1058.8 × 1042.0 × 1051.7 × 105

TABLE 19BPresevation efficacy sample 4.2 - 0.14 w/v % methylparabenInoculationMicro-organismATCCLotCfu/gTime zeroDay 14Day 28Pseudomonas aeruginosa9027484124527.1 × 1057.1 × 105<10<10Staphylococcus aureus653848548215.3 × 1056.5 × 105<10<10Escherichia coli873948356648.7 × 1058.4 × 105<10<10Candida albicans1023144359033.8 × 1053.5 × 105<∥10<10Aspergillus brasiliensis1640439295522.0 × 1051.5 × 1052.4 × 1031.2 × 102

TABLE 19CPresevation efficacy sample 4.2 - 0.2 w/v % methylparabenInoculationMicro-organismATCCLotCfu/gTime zeroDay 14Day 28Pseudomonas aeruginosa9027484124527.0 × 1057.3 × 105<10<10Staphylococcus aureus653848548215.3 × 1055.3 × 105<10<10Escherichia coli873948356648.7 × 1058.4 × 105<10<10Candida albicans1023144359033.8 × 1054.4 × 105<10<10Aspergillus brasiliensis1640439295522.0 × 1052.8 × 105<10<10

TABLE 19DPresevation eff. sample 4.1 - 0.126 w/v % methylparaben, 0.013 w/v % propylparabenInoculationMicro-organismATCCLotCfu/gTime zeroDay 14Day 28Pseudomonas aeruginosa9027484124528.9 × 1051.5 × 104<10<10Staphylococcus aureus653848548217.2 × 1057.2 × 105<10<10Escherichia coli873948356649.7 × 1058.0 × 105<10<10Candida albicans1023144359034.9 × 1055.5 × 105<10<10Aspergillus brasiliensis1640439295522.5 × 1051.1 × 1052.2 × 1037.6 × 102

TABLE 19EPresevation efficacy sample 4.1 - 0.18 w/v % methylparaben, 0.018 w/v % propylparabenInoculationMicro-organismATCCLotCfu/gTime zeroDay 14Day 28Pseudomonas aeruginosa9027484124527.0 × 1056.3 × 105<10<10Staphylococcus aureus653848548215.3 × 1056.7 × 105<10<10Escherichia coli873948356648.7 × 1058.8 × 105<10<10Candida albicans1023144359033.8 × 1054.0 × 105<10<10Aspergillus brasiliensis1640439295522.0 × 1051.4 × 1055.5 × 101<10

TABLE 19FPresevation efficacy sample 4.3 - 0.2 w/v % methylparabenInoculationMicro-organismATCCLotCfu/gTime zeroDay 14Day 28Pseudomonas aeruginosa9027484124527.0 × 1057.5 × 105<10<10Staphylococcus aureus653848548215.3 × 1056.9 × 105<10<10Escherichia coli873948356648.7 × 1058.4 × 105<10<10Candida albicans1023144359033.8 × 1054.5 × 1051.8 × 1011.8 × 101Aspergillus brasiliensis1640439295522.0 × 1052.1 × 1056.4 × 101<10

From the stability tests it can be observed that formula 4.2, having a pH of 5.5, appeared to be more stable than formula 4.3, having a pH of 6.5. Further, without paraben, the microbial efficacy was below acceptable level. Similar results were obtained when instead of methylparaben, a combination of methylparaben and propylparaben in a weight ratio of 10:1 was used (formula 4.1).