NL2028937B1

NL2028937B1 - Colchicine solution

Info

Publication number: NL2028937B1
Application number: NL2028937A
Authority: NL
Inventors: Lioumis Konstantinos; Psarrakis Yannis
Original assignee: Pharma Data S A
Priority date: 2021-08-06
Filing date: 2021-08-06
Publication date: 2023-02-21

Abstract

Described is a stable buffered aqueous colchicine solution or suspension being free of benzyl alcohol, comprising colchicine, a water miscible solvent and a preservative, characterized in that the solution comprises 0.01 — 1.0 w/v% colchicine, 2.5 — 15.0 w/v% water miscible solvent, at least half ofthe weight thereof being glycerol and 0.05 — 1 w/v% preservative, the pH ofthe solution or suspension being 4.5 — 7.0. A method forthe preparation thereof is also disclosed.

Description

Colchicine solution

The invention relates to a stable buffered aqueous colchicine solution or suspension being free of benzylalcohol.

Colchicine or acetyltrimethyicolchicing (CAS 84-86-8) is a complex a-tropolong derivative. Pure colchicine has yellow needle-like crystals with a melting point of 157°C, It is well soluble in water, ethanol and chloroform, and has a bitter taste.

The systematic name of colchicine is {5)-N-15,6,7,S-tetlrahydro-1,2,3,10-telramethoxy-9-0x0- benzolalheplalene-7T-ylacetamide with the following chemical structure:

Colchicine is an alkaloid from the autumn crocus {Colchicum autumnale), which is effective against gout. Colchicine can inhibit mitosis, destroy the spindle, and arrest the chromosomes in the mid-division. The cell does not divide and resulting in doubling the chromosomes. For this reason, the substance is used in plant breeding to produce polyploid plants, In the past, twas also used in the treatment of cancer. At present, only the derivative demecolcine is used for this purpose, and only sporadically. Colchicine is also found in the climbing lily (Gloriosa superba), even in a higher concentration than in the autumn crocus, but the plant is difficult to propagate, rendering it impossible to use for colchicine production profitably.

In drug applications, colchicine inhibits the production of interleukin 6 in local cells, so as to control local pain, swelling and inflammation in the joints. The pharmacological effect of colchicine for gout symptoms is mainly to reduce the activity of white blood cells and phagocytosis and reduce the formation of lactic acid, thereby reducing the deposition of uric acid crystals, reducing the inflammatory response of gout inflammation in patients, and having an analgesic effect.

Colchicine preparations have been sold internationally since the 1960s and are currently in solid forms such as tablets and capsules. However, for gout patients, gout attacks and other causes of dysphagia make some patients unable to get immediate effects from solid preparations.

Aqueous oral liquid colchicine formulations are known. US9,907,751 describes such a formulation comprising benzyl alcohol, a 10 w/v% 1:1 mixture of glycerine and propylene glycol, xanthan gum, a buffer, a sweetener, and a flavour. Stability of the solution is obtained by the presence of xanthan gum. The product is marketed under the brand name Glopebra.

Benzyl alcohol is used as a bacteriostatic preservative. However, it is not suitable for young children. In addition, a number of toxic effects are known, including respiratory problems, vasodilatation, low blood pressure, cramps and paralysis.

A benzyl alcohol free oral colchicine solution is known from CN110448527, wherein the use of benzyl alcohol is avoided. Instead, a mixture of methylparaben and propylparaben is used. Instead of xanthan gum as thickener, hydroxypropyl cellulose is used. The colchicine solution of CN110448527 further comprises propylene glycol as solvent,

Propylene glycol is also not well tolerated by young children (European Medicines Agency assessment Report, EMA/175205/2014) and should therefore preferably be avoided. it has now been found that an aqueous oral colchicine solution or suspension can be stained, that is free of banzyi alcohol, with improved stability and that is also suitable for administration to small children. To this end, the invention provides a buffered aqueous colchicine solution or suspension being free of benzyl alcohol, comprising 0.01 — 1.0 w/v% colchicine, 2.5 - 15.0 w/v% of a water miscible solvent, at least half of the weight thereof being glycerol and 0.05 — 1 w/v% preservative, the pH of the solution or suspension being 3.5 — 7.0.

It has been found that the presence of glycerol in the composition results in a stable colchicine solution without the need to include benzyl alcohol. It has also surprisingly been found that the solution can be void of an antioxidant and of a thickener. The absence of a thickener allows sterilisation by filtration facilitating industrial production.

The term ‘buffered’ means that the composition comprises a buffer system capable of buffering the pH of the solution in the envisaged range of 4.5 - 7.0. The skilled person is aware of suitable buffer systems. Herein, when it comes to colchicine, the term ‘solution’ also encompasses suspensions, unless otherwise indicated. The term ‘composition’ in this respect comprises both colchicine solutions and suspensions

Colchicine solutions, known in the art have a colchicine content of 12 mg/ml, i.e. 0.012 w/v%. It has attractively been found that the solution of the present invention can comprise up to 30 mg/ml while still being stable after several months at incubation conditions of 40°C or even higher. In an attractive embodiment, the solution therefore comprises 0.02 - 0.03 w/v% colchicine. Colchicine can be in the form of the colchicine base or a salt, solvate, derivative or solvate thereof.

The solution preferably comprises 5 — 10 wiv% water miscible solvent. At such concentration of the water miscible solvent, stable solution are ablained.

At least 50 w/v% of the water miscible solvent is glycerol. It was found that when at least half of the water miscible solvent is glycerol, improved stability is observed when compared with benzyl alcohol free colchicine solutions that do not have glycerol. Attractively, the amount of glycerol in the water miscible solvent is therefore more than 50%, preferably 80 w/v% of the water miscible solvent is glycerol, more preferably at least 90 w/v%, even more preferably at least 95 w/v% and most preferably the water miscible solvent is glycerol.

The solution comprises 0.05 — 1 w/v% preservative. Attractively, the solution comprises 0.10 — 0.40 w/v% preservative. Although may preservatives are suitable, the preservative is preferably chosen from the group, consisting of methyl paraben, ethyl paraben, propyl paraben, buiyl paraben, sorbic acid, potassium sorbate, and combinations thereof, the preservative preferably comprising methyl paraben or a combination of methyl and propyl paraben, in particular sodium methyl paraben. In order to provide a colchicine solution suitable for infants, the preservative preferably comprises methylparaben, is preferably void of propy! paraben, more preferably of any additional preservative. the preservative comprises in particular sodium methyl paraben.

The pH of the solution is in the acid to neutral range In order to be acceptable as a oral solution, e.g. for oral administration. The solution preferably has a pH of 4.5 — 6.5, more preferably 5.0 — 6.0, even more preferably 5.2 — 5.8, and most preferably 5.4 — 5.6.

In order to provide a stable pH, the solution is buffered, which means that the solution comprises a buffer system, in particular a pharmaceutically acceptable buffer system that comprises one or more pH buffering agents. The solution preferably comprises 0.05 — 2.0 w/v%, more preferably 0.5 — 1.0 w/v% buffering agent. Suitable buffer agents are known to the skilled person. Preferred buffer agents are chosen from the group, consisting of hydrochloric acid, acetic acid, ammonia solutions, monoethanolamine, diethanolamine, triethanolamine, meglumine, sodium citrate, citric acid, actie acid, phosphoric acid, propionic acid, sulphuric acid, tartaric acid, potassium bicarbonate, polassium citrate, polassium hydroxide, sodium bicarbonate, sodium borate, and sodium hydroxide, or a combination of two or more thereof. The buffering agent preferably comprises citric acid and a citrate salt, preferably sodium citrate.

Although the solution of the invention may comprise a thickener agent, it has been found that in the presence of glycerol in amounts as defined herein, the stability is not improved by the presence of a thickener. As the presence of a thickener results in cumbersome production on industrial scale as sterile fillration is cumbersome, it is advantageous to include less thickener. To this end, the solution preferably comprises 0.25 w/v% or less thickening agent. Any pharmaceutically acceptable thickening agent known to the skilled person can be chosen, and is in particular chosen from the group, consisting of hydroxyethyl cellulose, hydroxypropyl cellulose, sodium alginate, sodium carboxy methylcellulose, gellan gum, xanthan gum, acacia, guar gum, locust bean gum, gum tragacanth, starch, carbopois, methylceliuiose, polyvinylpyrrolidone, polyethylene oxide polymer and combinations thereof. The thickening agent preferably comprises hydroxyelhylceilulose or xanthan gum. The amount of the thickening agent, if present, is preferably 8.1 - 0.2 w/v%. However, it was found that the stability even increases if the solution is free of thickener. Therefore, in a very attractive embodiment, the solution of the invention is free of a thickening agent .

Although the solution of the invention may comprise an antioxidant, it was found that the presence of an antioxidant does not significantly improve the stability of the solution. If incorporation of an antioxidant is envisaged, the stability of the solution should be lesie as is was found that under rather harsh test conditions, solutions that comprises certain antioxidants had a limited stability. However, under normal storage conditions, the presence of an antioxddant does not hamper the stability at least not significantly. To this end, the solution of the invention can attractively comprise 1.0 w/v% or less antioxidant. Any pharmaceutically acceptable antioxidant known to the skilled person can be chosen. The antioxidant is preferably chosen from the group, consisting of butylated hydroxyl anisole, butylated hydroxyl toluene, tocopherol, ascorbyl palmitate, ascorbic acid, sodium metabisulfiie, sodium sulphite, sodium thiosulfate, propyl gallate, and combinations thereof, the antioxidant preferably comprising sodium metabisulphite. The amount of the antioxidant, If present in the solution, is preferably 0.01 — 0.25 w/v%, more preferably 3.01 — 8.1 w/v3:. It has surprisingly been found that the stability even increases if the solution is free of antioxidant. Therefore, in a very attractive embodiment, the solution of the invention is free of antioxidant.

In order to provide a solution with an acceptable taste, the solution preferably comprises a sweetening agent. Sweeting agents are know in the art, such as commonly known saccharides, such as sucrose, fructose, glucose, lactose. However, it is preferred for the solution of the invention to comprise an artificial non-sugar alcohol sweetening agent.

Although sugar alcohols such as xylitol, mannitol, glycerol, erythritol, threitol, arabitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotritol, maltotetraitol, and polyglycitol can be used in the solution of the invenit ion, a sugar alcohol may also have a thickener function. As described above, the presence of a thickener is less preferred, and for that reason, sugar alcohols having a thickener function are less preferred. An artificial non-sugar alcohol sweetening agent is an additive that provides sweet taste like that of sugar but derived through manufacturing of plant extracts or processed by chemical synthesis, not belonging to the sugar alcohols as described above.

Such artificial sweeteners contain often far less energy than regular sugars used for sweetening. Such sweeteners can also be referred to as ‘non-nutritive sugar-based sweeleners, i.e. having no significant nutritional value and not sugar based. Herein, a chemically trealed sugar is an artificial sweetener. For example, sucralose is produced by controlled chlorination of the sugar saccharose, Sucralose is therefore an artificial sweetener, that is derived from a sugar, but the sweetener is defined herein as ‘non-sugar based’. Artificial non-sugar alcohol sweeteners do not have the thickening effect of sugar alcohol and have a significantly higher sweetening power.

Because of the high sweetening power of artificial non-sugar alcohol sweetening agents, it has become possible io provide for a composition wherein the ofi-tasie of the 5 colchicine is sufficiently masked, while retaining a workable volume of the composition, without the presence of a significant amount of sugar alcohol, while also achieving acceptable shelf stability.

The amount of sweetening agent is preferably chosen such, that H corresponds with the sweetening power in the solution of 20 - 500 w/v% saccharose. This means thal, e.g. in cass sucralose is used as the sole artificial sweetening agent, the amount of sucralose is 0.033 - 0.83 w/v%, as the sweetening power of sucralose is 800 times that of saccharose, More preferably, the amount of non-sugar alcohol sweetening agent in the solution has a sweetening power that corresponds with the sweetening power of 40 - 200 w/v% saccharose.

The solution of the invention comprises a water miscible solvent, at least 50 w/v% of which is glycerol. Other pharmaceutically acceptable water miscible solvents can be used together with glycerol, in particular selected from the group consisting of: polyols, such as alkane triols, alkane diols and polyethylene glycol; alcohols, such as ethanol, isopropyl alcohol; acetone, phthalates, such as dibutyl phthalate, diethyl phthalate, dimethyl phthalate; dimethyl sulfoxide, dimethylacetamide, glycofurol, isopropyl myristate, isopropyl palmitate, propylene carbonate, pyrrolidine, glycerine triacetate, triethyl citrate, triolein, or a combination of two or more thereof. If one or more additional water miscible solvents are present in the solution, the ratio thereof with glycerol is preferably 1 — 5: 10 glycerol. As indicated above, it is preferred to have a high relative glycerol content as compared to the one or more additional water miscible solvents.

In order to increase the attractivity of the solution even more, in parlicular for infants and young children, the solution comprises a flavouring agent. At acid or neutral pH as for the solution of the invention, the majority of commonly known flavouring agents can be used. The flavouring agent is preferably chosen from the group, consisting of forest fruits flavour, grapefruit, orange, lime, lemon, mandarin, pineapple, strawberry, raspberry, mango, passion fruit, kiwi, apple, pear, peach, apricot, cherry, grapes, banana, cranberry, blueberry, black currant, red currani, gooseberry, lingonberries, cumin, thyme, basil, chamomile, valerian, fennel, parsley, camomile, tarragon, lavender, dill, bergamot, salvia, aloe vera balsam, spearmint, peppermint, eucalyptus, and combinations of two or more thereof, the flavour preferably comprising orange or strawberry flavour. The solulion preferably comprises 0.01 ~ 1.0 wiv% more preferably 0.05 - 0.5 w/v% flavouring agent.

For practical and commercial use, the solulion of the invention will have at least 18 months, preferably at least 24 months shell life. Preferably, the colchicine solutions are stable at room temperature and do not require refrigeration. Aqueous colchicine solutions or suspensions of the invention therefore preferably comprise at least 95% colchicine after 24 months storage at 25°C at a relative humidity of 60%, preferably at least 96%, more preferably at least 97%, even more preferably at least 98% and most preferably at least 99% as compared to the amount of colchicine at the beginning of the storage.

Preferably, the colchicine solution comprises: a. less than 1.2%, preferably less than 1.1 total impurities after 6 months storage at 5°C at a relative humidity of 60%, and/or b. less than 1.2%, preferably less than 1.1% total impurities after 6 months storage at 25°C at a relative humidity of 60%, and/or c. less than 1.2%, preferably less than 1.1% total impurities after 6 months storage at 30°C at a relative humidity of 65%, and/or d. less than 1.4%, preferably less than 1.3%, more preferably less than 1.2% and most preferably less than 1.1% total impurities after 5 months storage at 40°C at a relative humidity of 75% and/or e. less than 1.4%, preferably less than 1.3% total impurities after 6 months storage at 40°C at a relative humidity of 75%.

The invention also relates to a method for the preparation of a solution of the invention, comprising the steps of: (i) mixing colchicine and purified water, (ii) admixing the preservative agent, (iii) admixing the pH buffering agents (iv) admixing the water miscible solvent, (Vv) if necessary, adjust the pH by addition of a pH buffering agent, (vi) if necessary, adjust the final volume by adding from the rest of the water of step (i), (vid optionally, filter through 1 to 10 um pore sieve, and (viii) filling in an appropriate light protected container.

As colchicine is light sensitive, at least step (i) of the method is preferably performed under light protected conditions. This can be accomplished by preforming the step or method in the dark, or by using a container that protect the contents from light. Additional components, such as those as described above can be added as part of, during or between one or more of the above steps (i) — (viii).

For examples, the sweetener, if present, is preferably added in step (iii) or between steps (iii) and (iv), and, if present, flavour, thickening agent and/or antioxidant are added in step (iii) or between steps (iii) and (iv) or after step (iv). The flavouring agent is preferably added after the water miscible solvent is added.

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, 992.5%, 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. 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.

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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%, 5g 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 um sieve and filled in type lll 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 (H20) (resistivity > 18 MQ cm) by deionization and distillation; — Acetonitrile (ACN) (Fisher Chemical, Germany, HPLC grade); — Methanol (MeOH) (Fisher Chemical, HPLC grade) — Orthophosphoric acid (Fisher Chemical, Germany); Potassium — Dihydrogen phosphate (LANH:NER)

Diluent

MeOH/H20: 50/50

Determination of Colchicine content:

Column: ZORBAX Eclipse XDB C18, 250 x 4.6 mm, 5 um, (Lot: B20441- P/N: 980967- 902)

Reference: Colchicine Reference Standard, LGC, purity 93.0%, Lot. 1096857

Standard 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-um membrane filter.

Injection volume: 10 HI

Flow rate: 1.0 ml/min

Column temperature: 20°C

Autosampler temperature: 25°C

Run time: 23 minutes

Quantification 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 < 20% b) Tailing Factor < 1.5 c) Plate Count >2000

Calculation of the %content of Colchicine by the equation:

YColchicine = Asmp * Vsmp = Wstd * Pstd * Dstd * 100

Astd * Wsmp * LC * Dsmp = Vstd 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 x 4mm, 5 um (Lot: B09110)

Reference solutions: Colchicine Reference Standard, LGC, purity 93.0%, Lot. 1096857, and Colchicine for system suitability CRS

Table 2 colchicine and impurities

Ph, Eur. USP Product specs

Colchicine is an alkaloid

Content 97.0% - 102.0% (anhydrous substance contained in various Reporting threshold:

HEN a ae species of Colchicum 0.1%

Ig oy x and in other genera. It Identification threshold: so . Spe, Ta contains NLT 94.0% and | 0.5% woe | NMT 101.0% of Qualification threshold:

Se colchicine (C22H25NO6), | 1.0%

Sw calculated on the colchicine anhydrous, solvent-free basis.

Impurity A NMT 3.0% Specified impurity NMT 3.0%

LENE | (acc. to ph.eur) ree a > Indena: wee Secondary plant

Iai metabolite and

A RHE ERAEM EEA Seton elu roe S47 8- degradation product

From starting material

Impurity B Other detectable impurity | NMT 1.0%

Son oe : wv Conformational isomer of

RE { 3 Í colchicine which is en formed in situ in solution. 5 TEE ILL tr aaybeote SAT Disregard peak due to

Indena: Not to be considered as impurity

Sary:

From starting material

Impurity C Other detectable impurity | NMT 1.0%

Qualification threshold

SER eH / Indena: ood og Mg SH son Sed PG Degradation product and on

FEA pee

WT es

A Sary: a See me Ee En . . Photodegradant

To REETE VRAD EN

EAT A 8 bab pdolenedsivalopuse

Seidel LI fovclnhepten-Tr plastid {i hunviosichicine},

Impurity D Other detectable impurity | NMT 0.5%

WW Indena: (ICH Q3B R2,

TTR RG ee TH Secondary plant identification threshold)

IRN Set 3 3 NLS hey

NE A Sed .

Net * Loi EN ĳ = a metabolite

Og Fd wee oe o

Sumy Sary: nan HFS Zed Afi ghasoppranosyhoy LEW Starting material isolation kasar Powe 5.6.7 8 etedvpdo dementia theptadin- 7. whiacetamdde frokolinesidad,

Impurity E NMT 0.2% Specified impurity NMT 0.5% iL EN indena: (ICH Q3B R2, { i Ne ON

Na Sonn . ‚ ‚

Mar ON Fn, 8 Secondary plant identification threshold)

Ag metabolite

Ge,

ENTS 3 hed LAYS rime Senor Sore .

STe in thepraden 7 glaceteonids Sary: {3d demetbyboddinmel From starting material

Impurity F NMT 0.2% - Cas no: 477-27-0 Specified impurity NMT 1.0%

Acceptance criteria: No definite green color is Qualification threshold

MOE ee oS produced. (from Indena) wT UN Tey TG

Hea i I. indena: iy ATG

Ses Degradation product

EL MNS 2 1 Sadar Lineker an S858 wetvahpdedaneaiaihopialen “Fa 3 ketens ochtens),

Sary:

From starting material

Imp G NMT 0.25% Specified impurity NMT 1.0%

GEL a ee Indena: Qualification threshold

Loi AS Degradation product

METH hee OR oe “TN = Sary: es Photo degradant

NEELIE treme thang Sam

ES SENS Ae IKJEESNPIN RSS

Dy humndussohing

CHROMATOGRAPHIC Unspecified -

Procedure; Proceed as (ICH Q3B R2, directed in the Assay. identification threshold)

Total _ NMT 4.0% Acceptance criteria: NMT 5.0% other than that due to colchicine , eluting within 1.5 times the retention time for colchicine =

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: H2O

Injection volume: 30 pl; 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 3: Gradient program © Mobile phase A Mobile phase B Mobile phase C

Time (min) (%oviv) (%oviv) (Soviv) 0-40 45 15 40 40 - 45 45 45 10 45.01 45 45 10 45.01 - 50 45 15 40

The colchicine content is calculated by the following equation:

Y%Recovery = Asmp x Vsmp x Wstd x Pstd x Dstd x 100

Astd x LC xX Dsmp x Vstd 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; Vsfd 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 x 4mm, 5 um (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 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 10 (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 um membrane.

Injection volume: 10 ul; 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.

RRT for impurity A is 0.9, and the response factor RF is 0.84.

Table 4: Product specification Solution Formula 1 after production (Jom | weer eeen al 2 Ph. Eur. cur. ed. (2.2.1) |Clear solution opalescence of liquids liquids

Uniformity of mass of

Ph. Eur. cur. ed. (2.9.27) | Meets the requirements delivered doses

Uniformity of dosage units | Ph. Eur. cur. ed. (2.9.40) | Meets the requirements (MV) © Deliverable Volume USP < 698> Meets the requirements 9 Package integrity In house method

Identification -HPLC (UV) -Retention time complies with RS

Colchicine -HPLC (Diode Array) -UV spectrum complies with RS

Identification 11 HPLC (UV) Retention time complies with RS methylparaben

In — house method 95.0-105.0% of the stated amount 14 | Assay of colchicine ==

Ph. Eur. cur. ed. (2.2.29) [of colchicine

In house method | Assay of methylparaben Each 80.0 - 110.0%

Ph. Eur. cur. ed. (2.2.29)

NMT3.0%

NMT 1.0%

NMT 1.0% [

NMT 0.5%

In house method NMT 0.5% 18 | Related Substances NMT 0.5%

Ph. Eur. cur. ed. (2.2.29) NMT 1.0%

Any unspecified impurity NMT 0.5%

Impurities

Total Aerobic Microbial 100 CFU/mL

Count

Total Combined Yeasts/| Ph. Eur. cur. ed. (5.1.4) 10 CFU/mL

Moulds

Antimicrobial

Ph. Eur. cur. ed. (5.1.3) |Pass

Effectiveness 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 5:stability test

TTT RE EG mp8 lose bose tose doav tose tos toast owt} oses | sa met ddd hdd] poo 4 4 bbb tem tend Ff 4 ob] mee Lago 4 4 fv 4 1 eG TT vsspee wp 4 FY bf 4 tamsiewet 4

Table 6; Stability test

ES a2 Fs 4 6 FF 1B OS 1 87 poet 444i] pn RN NN NN NN NN NN NN NN NS Mn

RBE ee posp® ohhh] eps Is 4 4 bbb]

Unsper dos eee eee bis dass baat EE

Table 7: Stability test px bor bos bax aa os fos 3 1 B 121 1 82 | aw pepe, dba] pope ob] pope TD

OF dng SD Se pops LD LSD Da tow Dn iT dd sper mp TS Se 0

Table 8: Stability test

A EE 2 OEE 91 92 assay colchicine 1100301 0850 [10000] 3950 49 TT 1300301 M0010] DEAN pps tose boos tase base dom dF 4 1887 i038 § 6]

EE lose dessa les db 1 boatload doo

ETC CO OE NO OO OE NO VON WON OOR NOR mgb hd] poops 4b hh] ppp BL pps toss Lam tgn i an ant id espe bnp ee eee]

Total impurities EE EE ES EN

Table 9: Stability test eb Af 2 403 ax oa] 0S B 1 7 LB Bij 92

Bep Eee eee ee eeen pop ob bb] npt ee ee bed py OS ON OE VO SO OO OE WE pope U U OO OO OO

VospestBed wwporites FF 1 1 DS tase db 4]

Table 10: Stability test

Ee doar Os JB Fv 1 BOF 2 | 82 we pt 4 byt dd] noe LDD oee ee ee de met 9 bd ob] npe jose na an lenen l TL 4 4 bb] asper we Je as JOO

Table 11: Stability test

EEE ET B assageokhieine | 3939 | 9300 joren ora 9920 JT NON] 9936 § 8810

CT ES ims imsc dors joy 0 Fb 0 1 0351 03 1 0% og teow les ioasstoee EEN peop odd] mat LL impt SE dd eb] me BOE RA spe imp V4 4 4 ob bd] rotattmpuities | 13} ver} sox foaex domes dd 4 boas oad ines

Table 12: Stability test

ED a2 3 5 3 6 Fv 08 1 83 | 82 wet odd] bop 4 ohhh] poof ddd dh npt 4 tr rr d pope} dese oss DL spee]

Table 13: Stability test ep dp bs 2 15 6 7 LE | 93 | ose | Gir me eee do 2 Zdee doseome] pops LD pe 4 4 4 4 teas db Vd bd] ipod bab] oS. Ga Rie LG

Wespen tmp twee fb 1b SO De 1 ass]

Table 14: Stability test

DE ZO B 7 8 St 92 ] assayoolehieine | 95,69 | 9900 | 3870 | sxe MeT TL SRS {IORI KOO

EL ES ES SE laa]

EE EE as met ob bbb hte] me 4 odd]

DE [NNN ANNE FAT [NUNN ANN FUNUN NUN FE UN —_—_—_—_—_" pps wa lamiawient of 0 4 0 fb pswi pps leu lew less Da aa 4 1 lewd oF onspec, mg 1 4 be bbs

Fosatimpuries | 138 EE | ues | 30 Jaa fa Be | 69%

Table 15: Stability test

EB faa OS 8 Fv 1B 1 91} 82 peg ob] mee bh] wen ddd] ee] ucspec mp EE en dt 1 1]

Table 15: Stability test ee 4 1 8 3 dT fos JS 82 | air] met nes Lb yb] mee ot oo br ry bbb] wss iid selon sn LD uospee. wp |L ST

Table 17: Stability test & athe at ST ee ee p23 3 tax baz bos LB} 7 108 101} 92 assay colchicion | xvod sao trooanlanvoet sensed 4 1 130040] 100001 9980

CTY SE SCE CEC SS ACEI A A EN op | SS [Ds Den tess beset Fb 1 83 1 08 | 459 met ibibo bb] pop V4 4b hh pt Vf 4 oo bbb

BoE dodo ee pope bestow tase TD spec mp JD

Votaltoparities | vas fo var jan jam jin] LJ 1 ows | ods for

N3. 85] stress B 38 poo ISN pope Fd] bet b 1 1830] mee popes] ee TN NU unspee, tmp SE} 1 O58

Total purities

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 33 (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 IA: Fracevation officay cools 4.2 re eren

RED

PRATEN EN ANDER zat ES xd SW

Se]

Table TRE Prosevation efficacy sample 4.3 Qd wijs methyipamden ml . on nusuizhog . .

Miro ergs ATCC ety Day 14 Day 28

NrWNDOSS

Fine Wed ane NEV De eed yb Sloe 2 In SN cas dhe GF Sen sR vg

Tais 1800 Procovation efficacy sample 8.2 INZ wiv ® mettybarasben inosuiation . re i a

NUTS ATOS Lod Chui Ting zes Dey 14 Day 25 od BOXY SOLER PO FEN =H Td

ASTEN : a

AEpaius easangs SE a Da ce AE da vanden 8 CY TEE aod o OR Ke x AYR war Hr A >. FPS

Table 10; Prasevativn aff, seis SIL AID wiv NN pethyinaraben, DIL wiv XN pronvnasben x . mocniziion . Co

Micro-prganism ATOR Let } i Tine one Thay 48 Day 38 hag y 2 3

Faaiurionss ‚ . . : ; i

NAIR RF SRAVEASE Sg) AR <= zw

BOLING

Sandee RUE

Daris siens ANAL LEARN av XO Das and a ¢ a

LRTRON JN0ang WEE AFAR Ax ZS <3 “id

BE Nt. A A RE nn ~ ab 9 Nao EN ass ~ SL SNC aaa wo x ni 3 .

Fable 190: Prosevation office snie 11 ~ DUIS wiv 8 motiwyiparaban, S018 wiv SN ropyiharshan ier ; i Prarouivtivg

Micra-peganism ATQE fat Sa Tine 3910 Day 13 Day 38

ARTUNINGES RS IHSIFSSE FARA RIX > < 49

SDE DS

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 8.5. 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).

Claims

CONCLUSIONS

1. A buffered aqueous solution or suspension of colchicine, which solution or suspension is free of benzyl alcohol, and comprises colchicine, a water-miscible solvent and a preservative, characterized in that the solution or suspension contains 0.01 - 1.0 w/v % colchicine, 2.5 - 15.0 w/v% water-miscible solvent, of which at least half the weight is glycerol, and includes 0.05 - 1 w/v% preservative, where the pH of the solution or suspension is 3 .5 - 7.0.

A buffered aqueous solution or suspension of colchicine according to claim 1 comprising 0.02 - 0.03 w/v% colchicine.

A buffered aqueous solution or suspension of colchicine according to claim 1 or 2 comprising 5-10 w/iv% water-miscible solvent.

Buffered aqueous solution or suspension of colchicine according to any one of the preceding claims, wherein at least 80 w/v% of the water-miscible solvent is glycerol, preferably at least 90 w/v%, more preferably at least 95 w/v% and wherein the most preferred water-miscible solvent is glycerol.

A buffered aqueous solution or suspension of colchicine according to any one of the preceding claims comprising 0.10 - 0.40 w/v% preservative.

A buffered aqueous solution or suspension of colchicine according to any one of the preceding claims, wherein the preservative is selected from the group consisting of methyl paraben, ethyl paraben, propyl paraben, butyl paraben, sorbic acid, potassium sorbate and combinations thereof, preferably wherein the preservative is methyl paraben or a combination of methyl and propyl paraben, especially sodium methyl paraben.

A buffered aqueous solution or suspension of colchicine according to any one of the preceding claims, having a pH of 4.5 - 6.5, preferably 5.0 - 6.0, preferably 5.2 - 5.8, and preferably 5.4 - 5.6.

A buffered aqueous solution or suspension of colchicine according to any one of the preceding claims, comprising 0.05 - 2.0 w/v%, preferably 0.5 - 1.0 w/v% buffering agent.

A buffered aqueous solution or suspension of colchicine according to any one of the preceding claims, wherein the buffering agent is selected from the group consisting of hydrochloric acid, acetic acid, ammonia solutions, monoethanolamine, diethanolamine, triethanolamine, meglumine, sodium citrate, citric acid, lactic acid, phosphoric acid, propionic acid , sulfuric acid, tartaric acid, potassium bicarbonate, potassium citrate, potassium hydroxide, sodium bicarbonate, sodium borate and sodium hydroxide, wherein the buffering agent preferably consists of citric acid and a citrate salt, preferably sodium citrate.

A buffered aqueous solution or suspension of colchicine according to any one of the preceding claims comprising 0.25 w/v% or less of thickener.

The buffered aqueous colchicine solution or suspension of claim 10, wherein the thickening agent, if present, is selected from the group consisting of hydroxyethyl cellulose, hydroxypropyl cellulose, sodium alginate, sodium carboxymethyl cellulose, gellan gum, xanthan gum, acacia, guar gum, locust bean gum, tragacanth gum, starch, carbopols , methylcellulose, polyvinylpyrrolidone, polyethylene oxide polymer and combinations thereof, wherein the thickening agent is preferably hydroxyethylcellulose or xanthan gum and the amount of thickening agent, if present, is preferably 0.1 - 9.2 w/v%.

A buffered aqueous solution or suspension of colchicine according to any one of claims 1 to 9, which is free from thickener.

A buffered aqueous solution or suspension of colchicine according to any one of the preceding claims comprising 1.0 w/v% or less antioxidant.

The buffered aqueous solution or suspension of colchicine according to claim 13, wherein the antioxidant is selected from the group consisting of butylated hydroxyl anisole, butylated hydroxyl toluene, tocopherol, ascorbyl palmitate, ascorbic acid, sodium metabisulfite, sodium sulfite, sodium thiosulfate, propyl gallate, and combinations thereof, wherein the antioxidant preferably comprises sodium metabisulphite and the amount of antioxidant, if present, is preferably 0.01 - 0.25 w/v%, more preferably 0.01 - 0.1 w/v%.

The buffered aqueous solution or suspension of colchicine according to any one of the preceding claims 1-12, is free of antioxidant.

A buffered aqueous solution or suspension of colchicine according to any one of the preceding claims, comprising a sweetener.

A buffered aqueous solution or suspension of colchicine according to claim 15, wherein the amount of sweetener in the solution or suspension has a sweetening power corresponding to the sweetening power of 20-500 w/v% sucrose, the sweetener preferably being an artificial sweetener. and is preferably selected from the group consisting of sucralose, sodium saccharin, aspartame, alitame, acesulfame-K, cyclamate, stevioside, glycyrrhizin, neohesperidin, dihydrochalcone, thaumatin, and combinations thereof.

18. Buffered aqueous solution or suspension of colchicine according to claim 15 or 16, wherein the solution contains 0.05 - 0.3 w/v% sweetener.

A buffered aqueous solution or suspension of colchicine according to any one of the preceding claims, comprising, in addition to glycerol, a water-miscible organic solvent selected from the group consisting of: polyols, such as alkane triols, alkanediols and polyethylene glycol; alcohols such as ethanol, isopropyl alcohol; acetone, phthalates, such as dibutyl phthalate, diethyl phthalate, dimethyl phthalate, dimethyl sulfoxide, dimethylacetamide, glycofurol, isopropyl myristate, isopropyl palmitate, propylene carbonate, pyrrolidine, glycerine triacetate, triethyl citrate, triolein, or a combination of two or more of these substances, the ratio of which to glycerol at preferably 1-5:10 glycerol.

A buffered aqueous solution or suspension of colchicine according to any one of the preceding claims, further comprising a flavoring agent.

The buffered aqueous solution or suspension of colchicine according to claim 20, wherein the solution contains 0.05 - 0.2 w/v% flavor.

The buffered aqueous solution or suspension of colchicine according to claim 20 or 21, wherein the flavor is selected from the group consisting of forest fruit flavour, grapefruit, orange, lime, lemon, tangerine, pineapple, strawberry, raspberry, mango, passion fruit, kiwi, apple , pear, peach, apricot, cherry, grapes, banana, cranberry, blueberry, black currant, red currant, gooseberry, lingonberry, cumin, thyme, basil, chamomile, valerian, fennel, parsley, chamomile, tarragon, lavender, dill, bergamot , salvia, aloe vera balm, spearmint, peppermint, eucalyptus, and combinations of two or more thereof, wherein the flavoring preferably comprises orange or strawberry flavor.

A buffered aqueous solution or suspension of colchicine according to any one of the preceding claims, wherein the solution or suspension contains at least 95% colchicine after 24 months storage at 25°C and a relative humidity of 60%, preferably at least 96, more preferably at least 97%, still more preferably at least 98 and most preferably at least 99%, relative to the amount of colchicine at the start of storage.

A buffered aqueous solution or suspension of colchicine according to any of the preceding claims, further comprising one or more pharmaceutically acceptable excipients selected from the group consisting of chelating agents, surfactants, wetting agents, coloring agents, and combinations thereof.

A buffered aqueous solution or suspension of colchicine according to any one of the preceding claims, wherein the solution or suspension contains less than 3% total impurities after 24 months storage at 25°C and 60% relative humidity.

A buffered aqueous solution or suspension of colchicine according to any one of the preceding claims, comprising:

a. less than 1.2%, preferably less than 1.1% total impurities after 6 months storage at 5°C and 60% relative humidity, and/or b. less than 1.2%, preferably less than 1.1% total impurities after 6 months storage at 25°C and 60% relative humidity, and/or c. less than 1.2%, preferably less than 1.1% total impurities after 6 months storage at 30°C and 65% relative humidity, and/or d. less than 1.4%, preferably less than 1.1%, total impurities after 5 months storage at 40°C and 75% relative humidity, and/or e. less than 1.4%, preferably less than 1.3% total impurities after 6 months storage at 40°C and 75% relative humidity.

A method of preparing a buffered aqueous solution or suspension of colchicine according to any one of the preceding claims, which method comprises the steps of: (i) mixing colchicine and purified water, (iy adding the preservative, (iii) adding of the pH buffering agents, (ivy adding the water-miscible solvent, (v) if present, adding the flavouring, (vi if necessary, adjusting the pH by adding a pH buffering agent, (vii) if necessary, adjusting the final volume by adding the remaining water from step i), (viii) optionally filtering through a sieve with pores of 1 to 10 µm, and (ix) filling into a suitable container protected from light.