Sitagliptin pharmaceutical composition with reduced amount of N-nitrosamine impurities

Abstract

A pharmaceutical composition comprising sitagliptin or a pharmaceutically acceptable salt thereof, wherein the composition comprises less than 5 ppm of 7-Nitroso-3-(trifluoromethyl)- 5,6,7,8-tetrahydr

Description

Field of the Invention The present invention relates to a pharmaceutical composition comprising sitagliptin or a pharmaceutically acceptable salt thereof, wherein the composition comprises less than 5 ppm of 7-Nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro[1,2,4]triazolo-[4,3- a]pyrazine. Furthermore, the present invention relates to a method for manufacturing a pharmaceutical composition comprising sitagliptin with reduced levels of 7-Nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro[1,2,4]triazolo-[4,3- a]pyrazine. Background Af-Nitrosamines are organic compounds with the chemical structure R2N-N=O, where R is usually an alkyl group. They feature a nitroso group (NO+) bonded to a deprotonated amine. A / -Nitrosamines are often referred to as nitroso compounds. / V-Nitrosamines have been found to be mutagenic and potentially carcinogenic in humans. The carcinogenic potential of A / -Nitrosamines has been found to be dependent on multiple factors: • the ability of the / V-nitrosamine to be metabolically activated by a-hydroxylation which leads to formation of diazonium ions; • the metabolic competence and capacity of the tissue to form diazonium ions; • the nature and stability of the diazonium ion and the related DNA-adducts formed; • the capacity, velocity and accuracy of the different cellular repair mechanisms responsible for the repair of the different DNA-adducts in tissues. The carcinogenic nature of A / -Nitrosamines means that exposure to them, even in small amounts, may have a significant risk to humans. Accordingly, acceptable daily limits of N-Nitrosamines such as A / -nitroso-dimethylamine (NDMA) and A / -Nitrosodiethylamine (NDEA) have been set at 96 ng / day and 26.5 ng / day respectively. Recently, / V-nitrosamine impurities have been detected in medicinal products for humans. Regulators first became aware of the presence of / V-nitrosamines in medicinal products in 2018, with reports of A / -nitroso-dimethylamine (NDMA) being detected in the angiotensin II receptor blocker valsartan. A subsequent EU review of all valsartan medicines was triggered by the European Medicines Agency (EMA) and was later extended to all sartans and other angiotensin receptor blockers. A separate review was also started for ranitidine medicines. This was followed by an EU-wide review of the risk of / V-nitrosamines being present in all medicines intended for humans. As part of this review, sitagliptin was found to be particularly impacted by nitrosamine contamination. Sitagliptin is an oral anti-diabetic medicine that helps control blood sugar levels in patients with type 2 diabetes mellitus. Recently, a new nitrosamine impurity, 7-Nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro[1,2,4]triazolo-[4,3- a]pyrazine (NTTP), has been detected in trace amounts in sitagliptin products. The maximum allowed daily intake of NTTP is limited to 37 ng / day, which is equivalent to 0.37 ppm of NTTP per 100 mg of active pharmaceutical ingredient (API). NTTP is formed from the M-nitrosation of trifluoromethyl triazolo pyrazine (TTP). TTP originates from crude sitagliptin as result of amide hydrolysis, or it can form through API decomposition over the shelf life of the pharmaceutical composition. The EU-review identified a number of root causes leading to the presence of A / -nitrosamines in medicines, all of which have in common the reaction of a nitrosable compound, such as a secondary or tertiary amine, with a nitrosating agent. Frequently, the nitrosating agent was found to be a nitrite or nitrate salt reacting under acidic or neutral conditions during manufacture of the API. It has been found that contaminated reagents or solvents may also comprise nitrosating agents. Nitrosating agents can also be found in many commonly used excipients at parts per million levels. Sodium starch glycolate, croscarmellose sodium, pregelatinized starch, polyvinylpyrrolidone (PVP), cross polyvinylpyrrolidone (cPVP), cellulosederived excipients such as microcrystalline cellulose (MCC), and lactose are just some examples of the excipients that could carry trace levels of nitrate or nitrite impurities. Improved testing methods enable the detection of trace amounts of impurities in drug products and allow the levels of A / -nitrosamines to be carefully monitored. However, whilst monitoring the amount of A / -nitrosamines in a pharmaceutical composition prevents non-compliant drug products entering the market, it does not represent an efficient way to manufacture compliant drug products. As the maximum allowed intake of / V-nitrosamine NTTP is limited to 37 ng per day for a sitagliptin daily dose of 100 mg (which corresponds to a limit of 0.37 ppm per day), there is a need to manufacture a sitagliptin pharmaceutical composition that has a level of M-nitrosamine impurities that, when taking into account the formation of NTTP during shelf life, does not exceed the maximum allowed intake. As such, there remains a need to effectively reduce the formation of A / -nitrosamine impurities during the manufacture of sitagliptin pharmaceutical compositions. Detailed Description The present invention relates to a pharmaceutical composition comprising sitagliptin or a pharmaceutically acceptable salt thereof. The pharmaceutical composition may comprise less than 5 ppm of 7-Nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro[1,2,4]triazolo-[4,3- ajpyrazine. In some embodiments of the present invention, the pharmaceutical composition may comprise less than 4.5 ppm, less than 4 ppm, less than 3.5 ppm, less than 3 ppm, less than 2.5 ppm, less than 2 ppm, less than 1.5 ppm, less than 1 ppm, less than 0.5 ppm, less than 0.45 ppm, less than 0.4 ppm, less than 0.35 ppm, less than 0.30 ppm, less than 0.25 ppm, less than 0.20 ppm, less than 0.15 ppm, less than 0.10 ppm, less than 0.095 ppm, less than 0.09 ppm, less than 0.085 ppm, less than 0.08 ppm, less than 0.075 ppm, less than 0.07 ppm, less than 0.065 ppm, less than 0.06 ppm, less than 0.055 ppm, less than 0.05 ppm, less than 0.045 ppm, less than 0.040 ppm, less than 0.035 ppm, less than 0.03 ppm, less than 0.025 ppm, less than 0.02 ppm, less than 0.015 ppm, less than 0.01 ppm, or less than 0.005 ppm of 7-Nitroso-3-(trifIuoromethyI)-5,6,7,8-tetrahydro[1,2,4]triazolo-[4,3- a]pyrazine. Preferably, the pharmaceutical composition according to the present invention may comprise less than 0.37 ppm of 7-Nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro[1,2,4]triazolo-[4,3- a]pyrazine. According to some preferred embodiments of the present invention, the pharmaceutical composition may comprise sitagliptin, wherein the sitagliptin may be non micronised. As used herein, the term “micronised” indicates particles having a D<»of less than 30 pm and / or a Dso of less than 15 pm. Surprisingly, it has been shown that the use of non micronised sitagliptin limits the amount of TTP which in turn reduces the levels of NTTP formation during the manufacture and shelf life of the pharmaceutical composition of the present invention. Advantageously, it has been found that the pharmaceutical composition of the present invention comprising non micronised sitagliptin comprises low levels of NTTP at the end of the manufacturing process (TO) and maintains a compliant level of NTTP during the shelf life of the pharmaceutical composition. The present invention relates also to a method for manufacturing a pharmaceutical composition comprising sitagliptin with reduced levels of 7-Nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro[1,2,4]triazolo-[4,3- a]pyrazine comprising: a. mixing non micronised sitagliptin and one or more pharmaceutically acceptable excipient; b. granulating the mixture; c. compressing the granulated mixture into tablets; d. optionally coating the tablets. According to the method of the present invention, the amount of 7-Nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro[1,2,4]triazolo-[4,3- a]pyrazine in the pharmaceutical composition comprising sitagliptin is less than 5 ppm. Preferably, according to the method of the present invention, the amount of 7-Nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro[1,2,4]triazolo-[4,3- a]pyrazine in the pharmaceutical composition comprising sitagliptin is less than 0.37 ppm. In some further preferred embodiments of the present invention, the non micronised sitagliptin of the pharmaceutical composition of the invention may have a median geometric diameter (D50) of more than 15 pm. In a further embodiment the non micronised sitagliptin may have a median geometric diameter of more than 50 pm. Preferably, the non micronised sitagliptin may have a median geometric diameter of more than 55 pm. As used herein, the term “median geometric diameter” or “D50” indicates the particle size of a sample obtained by locating the diameter associated with the midpoint of the particle-size distribution. In some embodiments, the non micronised sitagliptin may have a median geometric diameter or D50 between 16 pm and 170 pm, between 21 pm and 165 pm, between 26 pm and 160 pm, between 31 pm and 155 pm, between 36 pm and 150 pm, between 41 pm and 145 pm, between 46 pm and 140 pm, between 51 pm and 135 pm, between 56 pm and 130 pm, between 61 pm and 125 pm, between 66 pm and 120 pm, between 71 pm and 115 pm, between 76 pm and 110 pm, between 81 pm and 105 pm, between 86 pm and 100 pm, or between 91 pm and 95 pm. In some further embodiments, the non micronised sitagliptin may have a median geometric diameter or D50 of more than 15 pm, more than 20 pm, more than 25 pm, more than 30 pm, more than 35 pm, more than 40 pm, more than 45 pm, more than 50 pm, more than 55 pm, more than 60 pm, more than 65 pm, or more than 70 pm. According to some embodiments of the present invention, the sitagliptin may have a particle size distribution with a D90 of 170 pm, a D50 of 60 pm, a D10 of 20 pm. Preferably, according to further embodiments of the present invention, the sitagliptin may have a particle size distribution with a D90 of 155 pm, a D50 of 55 pm, a D10 of 15 pm. As used herein, “particle size distribution” indicates the frequency of particles of a certain size in a sample. D90, D50, D10 are percentile values, that indicates the size below which 90%, 50% or 10% of all particles are found, respectively. According to some other embodiments of the present invention, the sitagliptin may have a particle size distribution with a D90 of 170 pm, D90 of 165 pm, D90 of 160 pm, D90 of 155 pm, D90 of 150 pm, Dgoof 145 pm, Dgoof 140 pm, Dgoof 135 pm, Dggof 130 pm, Dgoof 125 pm, Dgoof 120 pm, D90 of 115 pm, D90 of 110 pm, D90 of 105 pm, D90 of 100 pm, D90 of 95 pm, D90 of 90 pm, D90 of 85 pm, D90 of 80 pm, D90 of 75 pm, D90 of 70 pm, D90 of 65 pm, or D90 of 60 pm. According to some embodiments of the present invention, the sitagliptin may have a particle size distribution with a D10 of 60 pm, D10 of 55 pm, D10 of 50 pm, D10 of 45 pm, D10 of 40 pm, D10 of 35 pm, D10of 30 pm, D10of 25 pm, or D10 of 20 pm. In an aspect of the present invention, the sitagliptin may be at least 20% (w / w), at least 25% (w / w), at least 30% (w / w), at least 32% (w / w), at least 35% (w / w), at least 40% (w / w), at least 45% (w / w), or at least 50% (w / w) of the pharmaceutical composition. According to the present invention, the pharmaceutical composition may comprise one or more pharmaceutically acceptable excipient. In some preferred embodiments, the pharmaceutically acceptable excipient of the present invention may have a nitrite content of less than 2 ppm. In other preferred embodiments, the pharmaceutically acceptable excipient may have a nitrite content of less than 1.9 ppm, less than 1.8 ppm, less than 1.7 ppm, less than 1.6 ppm, less than 1.5 ppm, less than 1.4 ppm, less than 1.3 ppm, less than 1.2 ppm, less than 1.1 ppm, less than 1.0 ppm, less than 0.9 ppm, less than 0.8 ppm, less than 0.7 ppm, less than 0.6 ppm, less than 0.5 ppm, less than 0.4 ppm, less than 0.3 ppm, less than 0.2 ppm, less than 0.1 ppm, less than 0.09 ppm, less than 0.08, less than 0.07, less than 0.06 ppm, less than 0.05 ppm, less than 0.04 ppm, less than 0.03 ppm, less than 0.02 ppm, less than 0.01 ppm, less than 0.009 ppm, less than 0.008 ppm, less than 0.007 ppm, less than 0.006 ppm, less than 0.005 ppm, less than 0.004 ppm, less than 0.003 ppm, less than 0.002 ppm, or less than 0.001 ppm. According to alternative embodiments of the present invention, the one or more pharmaceutically acceptable excipient may be treated with a reducing agent. In some embodiments of the present invention, the reducing agent may be selected from at least one of sodium sulphite, sodium metabisulphite, sodium bisulphite, sodium thiobisulphate, ascorbic acid, acetylcysteine, L-cysteine hydrochloride, dithiothreitol, or a combination thereof. The reducing agent may be added during direct mixing of the one or more pharmaceutically acceptable excipient with the sitagliptin. Alternatively, it may be added to the one or more pharmaceutically acceptable excipient prior to further mixing with the sitagliptin, such that the one or more pharmaceutically acceptable excipient undergoes a pre-treatment step. In some embodiments of the present invention, the pre-treatment of the one or more pharmaceutically acceptable excipient with a reducing agent may be performed by a process selected from the group consisting of dry homogenisation, co-sifting, compaction, high-shear, wet granulation, dry granulation, fluid-bed granulation, hot-melt extrusion, and spray drying. According to a further embodiment of the present invention, the pre-treatment may be performed via fluidbed granulation. When considering treating the pharmaceutically acceptable excipient with a reducing agent, particular attention must be given to the reducing agent as not all reducing agents comply with regulatory requirements. Whilst reducing agents have their benefits, not having to use a reducing agent reduces manufacturing steps, lowers costs and avoids any risk of non-compliance with national regulatory requirements. More advantageously, the use of excipients with a low nitrite content has been shown to control the formation of nitroso compounds over the shelf life of the pharmaceutical composition. Surprisingly, it has been found that the combined effect of having excipients with low levels of nitrite and non-micronised sitagliptin significantly reduces the formation of nitroso compounds. Specifically, it has been found that combining non-micronised sitagliptin with excipients having low nitrite content resulted in a reduced formation of NTTP. According to specific embodiments of the present invention, the pharmaceutically acceptable excipient may be a cellulose-derived excipient. Preferably, the pharmaceutically acceptable excipient may be microcrystalline cellulose (MCC). According to the present invention, the pharmaceutical composition may be a solid dosage form, such as a tablet, capsule, powder or topical formulation. Preferably, the pharmaceutical composition may be a tablet. In a further embodiment, the tablet may be film-coated. The present invention is now described in more detail by, but is not limited to, the following Examples. Example 1: Nitrite content in excipients used for sitagliptin tablets Tablets were prepared using the following ingredients • 32% of micronised sitagliptin (active pharmaceutical ingredient - API); • 15% of microcrystalline cellulose (MCC); • 43% of calcium hydrogen phosphate anhydrous (CaP); • 5% of croscarmellose sodium (CC); • 1% of silica colloidal anhydrous (SCA); • 3% of sodium stearyl fumarate (SSF); • 1 % of magnesium stearate (MgS). The blend of API, MCC and parts of CaP, CC, SCA, SSF and MgS was prepared, followed by a dry granulation step and granulate sizing. Prepared granulate was admixed with the remaining parts of CaP, CC, SCA, SSF and MgS to prepare a final mixture for compression. Finally, tablet cores were prepared on a conventional rotary tablet press, coated in a conventional perforated drum tablet coater and packed in PVC / PVDC blisters lidded with Al foil. Prior to tabletting, the nitrite content of each excipient was evaluated and the results are summarised in Table 1. Table 1 Nitrite content Sitagliptin N.A. Cellulose Microcrystalline 112 High Calcium hydrogen phosphate anhydrous Low Croscarmellose Sodium Low Aerosil 200 (Silicium Dioxidum Colloidal) Low Sodium Stearyl Fumarate Low Magnesium stearate High • High nitrite content >0.4 ppm • Low nitrite content <0.4 ppm. Individual changes to this manufacturing process, including fluid bed pre-treating of excipients, are indicated in the following examples. Further sitagliptin tablets were contemplated having the following composition: A B Micronised sitagliptin (active pharmaceutical ingredient - API) 22% 42% Microcrystalline cellulose (MCC) 20% 10% Calcium hydrogen phosphate anhydrous (CaP) 48% 38% Croscarmellose sodium (CC) 5% 5% Silica colloidal anhydrous (SCA) 1% 1% Sodium stearyl fumarate (SSF) 3% 3% Magnesium stearate (MgS) 1% 1% Example 2: TTP and NTTP content of sitagliptin tablets under stressed conditions The level of TTP and NTTP in sitagliptin tablets according to Example 1 was assessed under stressed conditions. Tablet samples were kept for 15 months at 25aC and 60% R.H. and for 18 months at 30sC and 75% R.H.. TTP and NTTP content in each tested sample at the end of the test period is summarised in Table 2. Table 2 TTP content in ppm NTTP content in ppm Strength in mg 15 months @ 25 -C / 60% R.H. 15 months @ 25 -C / 60% R.H. 18 months @ 30 eC / 75% R.H. 25 62.7 0.82 1.27 50 56.5 0.66 1.18 100 57.5 1.00 1.51 25 58.1 1.13 1.63 50 52.9 0.81 1.39 100 54.3 1.40 1.99 Example 3: TTP and NTTP content in micronised and non-micronised sitagliptin API under stressed conditions The level of TTP and NTTP content in micronised and non-micronised API sitagliptin was assessed post processing but pre-formulation. TTP and NTTP content is summarised in Table 3. Table 3 API Micronised TTP content in ppm NTTP content in ppm Sitagliptin No 3.2 0.03 Sitagliptin No <1 <0.03 Sitagliptin No <1 <0.03 Sitagliptin Yes 36.6 0.11 Sitagliptin Yes 37.4 0.12 Sitagliptin Yes 33.2 0.19 Sitagliptin Yes 23.7 0.22 Example 4: Dry granulation of micronised sitaqliptin with a reducing agent Samples were prepared according to the process of Example 1 with the inclusion of a reducing agent during the dry-granulation step. The reducing agents were selected from: • 0.5% ascorbic acid; • 0.5% dithiothreitol; • 0.5% sodium metabisulphite. For comparison, a reducing agent was not added to a fourth sample. The NTTP content of all samples was tested at TO and under stressed condition for 14 days at 40°C / 75% R.H.. The complete list of results is summarised in Table 4. Table 4 NTTP content In ppm Strength in mg Composition TO 14 days @ 40 -C / 75% R.H. 100 No reducing agent 1.31 2.08 100 0.5% Ascorbic acid 1.24 1.34 100 0.5% dithiothreitol 0.20 0.18 100 0.5% Sodium metabisulphite 0.21 0.22 Example 5: Pre-treatment of excipients with a reducing agent via fluid-bed granulation Samples were prepared according to the process of Example 1 with the inclusion of a pretreatment step of the excipients with reducing agent via fluid-bed granulation. A reducing agent solution was prepared comprising: • 0.5 % (w / w) of reducing agent; • 2% solution of agent in purified water. The reducing agent was selected from ascorbic acid; dithiothreitol; L-cysteine hydrochloride and added in amount of 0.5% respective to tablet core composition as a 2% water solution to the complete composition (apart from API and silica colloidal anhydrous (SCA)). The reducing agent solution was applied to the excipients in a fluid-bed granulator for 30 minutes and subsequently cooled. The treated excipients were then mixed with sitagliptin and silica colloidal anhydrous (SCA) before undergoing dry granulation and being compressed into tablet cores. A comparison sample excluding the pre-treatment step was also prepared. All samples were tested for NTTP content at TO and under stressed condition for 14 days at 40°C 175% R.H. and the results are summarised in Table 5. Table 5 NTTP content in ppm Strength in mg Composition TO 14 days @ 40 -C / 75% R.H. 100 No reducing agent 0.29 0.40 100 0.5% Ascorbic acid 0.19 0.19 100 0.5% dithiothreitol 0.20 0.19 100 0.5% L-cysteine hydrochloride 0.19 0.19 Example 6: Comparison of compositions involving pre-treatment of excipients with a reducing agent via dry granulation and comprising micronised vs non-micronised sitagliptin The samples were prepared according to Example 1 with the inclusion of either a pre-treatment step of the excipients with reducing agent via dry granulation or a pre-treatment step of the excipients with reducing agent via fluid-bed granulation. Table 6 NTTP content in ppm Composition TO 14 days @ 40 eC / 75% R.H. 1 month @ 30 -C / 75% R.H. pAPI; no-preT, low nitrite MCC, no reducing agent 0.24 0.36 0.40 pAPI; no-preT, low nitrite MCC, ascorbic acid 0.23 0.26 0.27 pAPI; no-preT, high nitrite MCC, sodium metabisulfite 0.21 0.22 0.20 pAPI; preT, high nitrite MCC, ascorbic acid 0.19 0.19 0.18 Non pAPI; no-preT, high nitrite MCC, no reducing agent 0.04 0.10 0.09 Non pAPI; no-preT, low nitrite MCC, no reducing agent 0.06 0.13 0.12 pAPI = micronised sitagliptin non pAPI = non micronised sitagliptin no-preT = no pre treatment of excipient with a reducing agent preT = pre treatment of excipient with a reducing agent Example 7: Evaluation of the combined effect of using low nitrite containing excipients or high nitrite containing excipients with micronised or non-micronised sitagliptin in reducing the content of NTTP in a sitagliptin pharmaceutical composition Four samples were prepared based on the process of Example 1 with the following adaptations: • low nitrite content MCC (< 0.4 ppm); • high nitrite content MCC (> 0.4 ppm); • micronised sitagliptin (D90 <30 pm); • non-micronised sitagliptin (D90157 pm). All samples were tested for NTTP content and the results are summarised in Table 7. Table 7 NTTP content in ppm Strength (mg) Sitagliptin mlcronisation status MCC content of nitrite TO 14 days @ 40 -C / 75% R.H. 100 No Low nitrite <0.03 0.06 100 No High nitrite 0.15 0.76 100 Yes Low nitrite 0.21 0.31 100 Yes High nitrite 0.53 1.19

Claims

1. A pharmaceutical composition comprising sitagliptin or a pharmaceutically acceptable salt thereof, wherein the composition comprises less than 5 ppm of 7-Nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro[1,2,4]triazolo-[4,3- a]pyrazine.

2. The pharmaceutical composition according to claim 1 comprising less than 0.5 ppm of 7-Nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro[1,2,4]triazolo-[4,3- a]pyrazine.

3. The pharmaceutical composition according to claims 1 and 2, wherein the sitagliptin is non micronised.

4. The pharmaceutical composition according to any preceding claim, wherein the sitagliptin has a median geometric diameter (Dso) of more than 50 pm.

5. The pharmaceutical composition according to any preceding claim, wherein the sitagliptin has a particle size distribution with a Dm of 170 pm, a Dso of 60 pm, a Dw of 20 pm.

6. The pharmaceutical composition according to any preceding claim, wherein the sitagliptin is more than 20% (w / w), more than 30% (w / w), or more than 50% (w / w) of the pharmaceutical composition.

7. The pharmaceutical composition according to any preceding claim, wherein the composition comprises one or more pharmaceutically acceptable excipient.

8. The pharmaceutical composition according to any preceding claim, wherein the pharmaceutically acceptable excipient has a nitrite content of less than 2 ppm.

9. The pharmaceutical composition according to any preceding claim, wherein the pharmaceutical composition is a tablet.

10. A method for manufacturing a pharmaceutical composition comprising sitagliptin with reduced levels of 7-Nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro[1,2,4]triazolo-[4,3-a]pyrazine comprising:a. mixing non micronised sitagliptin and one or more pharmaceutically acceptable excipient;b. granulating the mixture;c. compressing the granulated mixture into tablets;d. optionally coating the tablets.

11. The method according to claim 10, wherein the pharmaceutically acceptable excipient has a nitrite content of less than 2 ppm.

12. The method according to claim 10 or 11, wherein mixing step (a) comprises a reducing agent.

13. The method according to claims 10 or 11, comprising the step of pre-treating one or more pharmaceutically acceptable excipient with a reducing agent.

14. The method according to claims 12 or 13, wherein the reducing agent is selected from sodium sulphite, sodium metabisulphite, sodium bisulphite, sodium thiobisulphate, ascorbic acid, acetylcysteine, L-cysteine hydrochloride, dithiothreitol, or a combination thereof.

15. The method according to claims 10 to 14 where the pharmaceutical composition is according to claim 1 to 9.