PROCESS FOR THE PREPARATION OF FREEZE-DRIED 2-[(3-AMINOPROPYL)AMINO]ETHANETHIOL FORMULATION

Abstract

A process for the preparation of freeze-dried 2-[(3-aminopropyl)amino]ethanethiol including the following steps: a) the reaction of a solution of amifostine with a strong acid, to obtain a solution of 2-[(3-aminopropyl)amino]ethanethiol, and b) the freeze-drying of the solution of 2-[(3-aminopropyl)amino]ethanethiol, with or without addition of excipients.

Claims

1. A process for the preparation of freeze-dried 2-[(3-aminopropyl) amino]ethanethiol comprising the following steps: a) the reaction of a solution of amifostine with a strong acid, to obtain a solution of 2-[(3-aminopropyl)amino]ethanethiol, and b) the freeze-drying of the solution of 2-[(3-aminopropyl)amino]ethanethiol, with or without addition of excipients.

2. The process of claim 1, wherein the strong acid is chosen from the group consisting of: hydrochloric acid, phosphoric acid, sulfuric acid, and mixtures thereof.

3. The process of claim 1, wherein the strong acid is hydrochloric acid.

4. The process of claim 1, wherein step a) is carried out at a temperature comprised between 50 C. and 90 C.

5. The process of claim 1, wherein step a) is carried out for 30 minutes to 24 hours.

6. The process of claim 1, wherein the concentration of amifostine is comprised between 80 mg/ml and 500 mg/ml.

7. The process of claim 1, wherein the molarity of the acid is comprised between 1M and 4M.

8. The process of claim 1, wherein the molar ratio between the amount of amifostine and the amount of acid is comprised between 1:0.5 and 1:3.

9. The process of claim 1, further comprising a step a1), after step a) and before step b), for diluting the solution of 2-[(3-aminopropyl)amino]ethanethiol, with or without addition of excipients in the solution.

10. The process of claim 9, wherein step a1) consists in the addition of an aqueous solution comprising water or at least one poloxamer into the solution of 2-[(3-aminopropyl)amino]ethanethiol.

11. A lyophilisate of 2-[(3-aminopropyl)amino]ethanethiol susceptible to be obtained according to the process of claim 1.

12. A process for the preparation of a thermogel composition of 2-[(3-aminopropyl)amino]ethanethiol comprising the reconstitution of the lyophilisate of claim 11 with an aqueous solution, with or without addition of excipients.

13. The thermogel composition of 2-[(3-aminopropyl)amino]ethanethiol susceptible to be obtained according to the process of claim 12.

14. The thermogel composition of claim 13, for its use for treating or protecting mucosal or cutaneous tissue from damage associated with anticancer therapy.

15. The thermogel composition of claim 13, for its use for the treatment of radiation-induced oral mucositis.

16. The thermogel composition of claim 13, for its use for the treatment of radiation-induced cutaneous erythema.

Description

EXAMPLES

A-Conversion of Amifostine into Aminothiol in Solution

Example A-1: In Hydrochloric Acid or Phosphoric Acid 1M Condition

[0071] A solution of amifostine (drug substance purchased from Shangai Boylechem Co., Ltd) at 350 mg/ml is prepared in hydrochloric acid or phosphoric acid 1M, and heated in a water-bath at 50 C. during 24 hours. After 24 hours, the composition of the solution was analyzed by HPLC method. The area percentages (%) of amifostine, aminothiol and disulfides are given in table below.

TABLE-US-00001 % % % % Others Acid (1M) Amifostine Aminothiol Disulfides impurities hydrochloric acid 2.3% 96.0% 1.7% 0.0% phosphoric acid 0.0% 98.2% 1.8% 0.0%

[0072] The conversion of amifostine in phosphoric acid 1M condition is complete (and almost complete in hydrochloric acid 1M with few residual amifostine) without formation of unknown impurity and only few disulfides.

Example A-2: In Hydrochloric Acid 4M Condition

[0073] A solution of amifostine at 500 mg/ml is prepared in hydrochloric acid 4M, and heated in a water-bath at 50 C. or at 60 C. during 24 hours. At 1 h-3 h-6 h and 24 hours, the composition of the solution was analyzed by HPLC method. The area percentages (%) of amifostine, aminothiol and disulfides are given in table below.

TABLE-US-00002 Amifostine at 500 mg/ml HCl 4M 50 C. 60 C. 1 h % Amifostine 0.0 % Aminothiol 99.7 % Disulfides 0.3 3 h % Amifostine 2.9 0.0 % Aminothiol 96.7 99.6 % Disulfides 0.3 0.4 6 h % Amifostine 0.0 0.0 % Aminothiol 99.6 99.5 % Disulfides 0.4 0.5 24 h % Amifostine 0.0 0.0 % Aminothiol 99.0 99.1 % Disulfides 1.0 0.9

[0074] The conversion of amifostine in hydrochloric acid 4M condition is complete after 6 hours at 50 C. and only 1 hour at 60 C., without creating unknowing impurities.

Example A-3 (Comparative): In Citric Acid Condition

[0075] A solution of amifostine at 350 mg/ml is prepared in citric acid 1M (weak acid), and heated in a water-bath at 50 C. during 24 hours. After 24 hours, the composition of the solution was analyzed by HPLC method. The area percentages (%) of amifostine, aminothiol and disulfides (WR-33278) are given in table below.

TABLE-US-00003 % % % % Others Amifostine Aminothiol Disulfides impurities 0.0% 65.1% 1.3% 33.6%

[0076] The conversion of amifostine in citric acid condition is complete, but not suitable due to formation of new unknowing impurities in large quantity.

[0077] This comparative example shows that the use of citric acid which is not a strong acid according to the invention is not suitable for the conversion of amifostine into aminothiol.

Example A-4 (Comparative): In Acetic Acid Condition

[0078] A solution of amifostine at 350 mg/ml is prepared in acetic acid 1M, and heated in a water-bath at 50 C. during 24 hours. After 24 hours, the composition of the solution was analyzed by HPLC method. The area percentages (%) of amifostine, aminothiol and disulfides are given in table below.

TABLE-US-00004 % Others % Amifostine % Aminothiol % Disulfides impurities 3.1% 79.3% 1.1% 16.4%

[0079] The conversion of amifostine in acetic acid condition is not complete, it remains unconverted residual amifostine. Moreover, new unknowing impurities in large quantity are formed.

[0080] This comparative example shows that the use of acetic acid which is not a strong acid according to the invention is not suitable for the conversion of amifostine into aminothiol.

B-Example of Freeze-Drying Conditions

Example B-1: Freeze-Drying Using Hydrochloric Acid 1M as Matrix

[0081] A solution of amifostine is firstly converted into aminothiol in hydrochloric acid 1M, based on Example A-1. After conversion, the solution is diluted in water to obtain a final solution at 50 mg/ml expressed in aminothiol, and 2 ml are distributed in glass vial for lyophilization.

Example of Freeze-Dry Recipe

[0082]

TABLE-US-00005 Step Temperature Time Pressure Shelf load 5 C. 300 min Freezing Rate 60 C. 40 min Hold 60 C. 360 min Primary Drying Hold 60 C. 30 min 600 bar Rate 50 C. 60 min 200 bar Hold 50 C. 600 min 200 bar Rate 40 C. 60 min 200 bar Hold 40 C. 600 min 200 bar Rate 10 C. 120 min 200 bar Hold 10 C. 600 min 200 bar Secondary Drying Hold +20 C. 240 min 30 bar Storage Hold +5 C. 900 bar

[0083] After freeze-drying, the appearance of cake obtained is good. For the reconstitution, add 2 ml of placebo gel containing Poloxamer 407/188 19/6 w/w, to obtain a final thermosensitive gel of aminothiol at 50 mg/ml, with a gelling temperature between 25 C. and 30 C.

Example B-2: Freeze-Drying Using Hydrochloric Acid 4M and Poloxamers 21/4 as Matrix

[0084] A solution of amifostine at 500 mg/ml is firstly converted totally into aminothiol in hydrochloric acid 4M during 1 hour at 60 C. (based on Example A-2). After conversion, the solution is diluted into Poloxamers P407/P188 21/4 w/w to obtain a final gel at 50 mg/ml expressed in aminothiol. Then 2 ml are distributed in glass vial for lyophilization.

Example of Freeze-Dry Recipe

[0085]

TABLE-US-00006 Step Temperature Time Pressure Shelf load 5 C. 600 min Freezing Rate 60 C. 40 min Hold 60 C. 360 min Primary Drying Hold 60 C. 30 min 600 bar Rate 40 C. 90 min 200 bar Hold 40 C. 600 min 200 bar Rate 10 C. 120 min 200 bar Hold 10 C. 600 min 200 bar Secondary Drying Hold +20 C. 240 min 30 bar Storage Hold +5 C. 900 bar

[0086] After freeze-drying, the cake obtained have good appearance.

[0087] For the reconstitution, add 1.4 ml of water, to obtain a final thermosensitive gel of WR-1065 at 50 mg/ml, with a gelling temperature between 28 C. and 30 C.

Example B-3: Freeze-Drying Using Hydrochloric Acid 4M and Poloxamers 18/6 as Matrix

[0088] A solution of amifostine at 500 mg/ml is firstly converted totally into aminothiol in hydrochloric acid 4M during 1 hour at 60 C. (based on Example A-2). After conversion, the solution is diluted into Poloxamers P407/P188 18/6 w/w to obtain a final gel at 50 mg/ml expressed in aminothiol. Then 2 ml are distributed in glass vial for lyophilization.

[0089] The freeze-dry recipe applied is the same as presented in above Example B-2.

[0090] After freeze-drying, the cake obtained have good appearance.

[0091] For the reconstitution, add 1.4 ml of water, to obtain a final thermosensitive gel of aminothiol at 50 mg/ml, with a gelling temperature between 65 C. and 70 C.

Example B-4: Freeze-Drying Using Hydrochloric Acid 1M and Poloxamers 20/4 as Matrix

[0092] A solution of amifostine at 350 mg/ml is firstly converted totally into aminothiol in hydrochloric acid 1M during 6 hours at 60 C. After conversion, the solution is diluted into Poloxamers P407/P188 20/4 w/w to obtain a final gel at 50 mg/ml expressed in aminothiol. Then 2 ml are distributed in glass vial for lyophilization.

[0093] The freeze-dry recipe applied is the same as presented above in Example B-2.

[0094] After freeze-drying, the cake obtained have good appearance.

[0095] For the reconstitution, add 1.5 ml of water, to obtain a final thermo sensible gel of WR-1065 at 50 mg/ml, with a gelling temperature between 30 C. and 35 C.

Example B-5: Freeze-Drying Using Hydrochloric Acid 1M and Polysorbate 80 as Matrix

[0096] A solution of amifostine is firstly converted into aminothiol in hydrochloric acid 1M, based on Example A-1. After conversion, the solution is diluted in water containing 0.2% of Polysorbate 80 as cryoprotectant, to obtain a final solution at 50 mg/ml expressed in aminothiol. 2 ml are distributed in glass vial for lyophilization.

[0097] The freeze-dry recipe applied is the same as presented above in Example B-2.

[0098] During freeze-drying, the cake obtained have collapsed giving a melted aspect to the cake. For the reconstitution, add 2 ml of placebo gel containing Poloxamer 407/188 19/6 w/w, to obtain a final thermosensitive gel of aminothiol at 50 mg/ml, with a gelling temperature between 25 C. and 30 C.

Example B-6: Freeze-Drying Using Hydrochloric Acid 4M as Matrix

[0099] A solution of amifostine is firstly converted totally into aminothiol in hydrochloric acid 4M, based on Example A-2. After conversion, the solution is diluted in water to obtain a final solution at 50 mg/ml expressed in aminothiol, and 2 ml are distributed in glass vial for lyophilization.

Example of Freeze-Dry Recipe

[0100]

TABLE-US-00007 Step Temperature Time Pressure Shelf load 5 C. 120 min Freezing Rate 55 C. 120 min Hold 55 C. 720 min Primary Drying Hold 55 C. 30 min 200 bar Rate 40 C. 90 min 200 bar Hold 40 C. 180 min 200 bar Rate 30 C. 60 min 200 bar Hold 30 C. 300 min 200 bar Rate +5 C. 180 min 200 bar Hold +5 C. 360 min 200 bar Secondary Drying Hold +20 C. 240 min 30 bar Storage Hold +5 C. 900 bar

[0101] During freeze-drying, the cake obtained have collapsed giving a melted aspect to the cake.

[0102] For the reconstitution, add 2 ml of placebo gel containing Poloxamer 407/188 19/6 w/w, to obtain a final thermosensitive gel of aminothiol at 50 mg/ml, with a gelling temperature around 30 C.

Example B-7: Freeze-Drying Using Hydrochloric Acid 4M and Hydroxide Sodium as Matrix

[0103] A solution of amifostine is firstly converted totally into aminothiol in hydrochloric acid 4M, based on Example A-2. After conversion, the pH of solution is adjusted to pH>6.0 adding hydroxide sodium 10M. Then, the solution adjusted is diluted in water to obtain a final solution at 50 mg/ml expressed in aminothiol. 2 ml are distributed in glass vial for lyophilization.

[0104] The freeze-dry recipe applied is the same as presented above in Example B-1.

[0105] During freeze-drying, the cake obtained have considerably collapsed giving a melted appearance to the cake. For the reconstitution, add 2 ml of placebo gel containing Poloxamer 407/188 19/6 w/w, to obtain a final thermosensitive gel of aminothiol at 50 mg/ml, with a gelling temperature around 30 C.

C-Preparation of Gels of Aminothiol

Example C-1: Reconstitution of Freeze-Dried Powder with Poloxamers Gel to Obtain a Gel of Aminothiol at 50 mg/ml

[0106] In order to prepare a gel of aminothiol at 50 mg/ml, add 2 ml of P407/P188 19/6 w/w to the freeze-dried obtained as described on Example B-1 (HCl 1M in lyophilization matrix) or Example B-5 (HCl 1M+PS80 in lyophilization matrix). The final gel formulated has a gelling temperature between 25-30 C.

Example C-2: Reconstitution of Freeze-Dried Powder with Poloxamers Gel to Obtain a Gel of Aminothiol at 50 mg/ml

[0107] In order to prepare a gel of aminothiol at 50 mg/ml, add 2 ml of P407/P188 19/6 w/w to the freeze-dried obtained as described on Example B-6 (HCl 4M in lyophilization matrix) or Example B-7 (HCl 4M+NaOH in lyophilization matrix). The final gel formulated has a gelling temperature around 30 C.

Example C-3: Reconstitution of Freeze-Dried Powder with Aqueous Solution to Obtain a Gel of Aminothiol at 50 mg/ml

[0108] In order to prepare a gel of aminothiol at 50 mg/ml, add 1.4 ml of solution containing for example mint flavour/bitter masker/water 4%/0.05%/95.95% w/w/w to the freeze-dried powder obtained as described on Example B-2. The final gel formulated has a gelling temperature around 27-29 C.

Example C-4: Reconstitution of Freeze-Dried Powder with Aqueous Solution to Obtain a Gel of Aminothiol at 50 mg/ml

[0109] In order to prepare a gel of aminothiol at 50 mg/ml, add 1.5 ml of water to the freeze-dried powder obtained as described on Example B-4. The final gel formulated has a gelling temperature around 30-35 C.

Example C-5: Reconstitution of Freeze-Dried Powder with Aqueous Solution to Obtain a Gel of Aminothiol at 50 mg/ml

[0110] In order to prepare a gel of aminothiol at 50 mg/ml, add 1.4 ml of water to the freeze-dried powder obtained as described on Example B-3. The final gel formulated has a gelling temperature around 65-70 C.

D-Stability of Lyophilized Powder of Aminothiol (WR-1065)

[0111] A stability study is performed to compare stability of: [0112] WR-1065 obtained by conversion of amifostine (350 mg/ml or 500 mg/ml) in acidic condition (respectively HCl 1M or HCl 4M), diluted in water or in P407/P188 mixture and then lyophilized (based on examples described in parts A & B), and [0113] WR-1065 hydrochloride salt crystalline powder.

[0114] Stability is assessed by analysis of samples stored under argon atmosphere at 25 C./60% RH. The aim of argon is to provide two materials under same stability condition.

TABLE-US-00008 Amifostine Aminothiol Disulfides Unknow WR-10652HCl native T0 0.0 99.7 0.3 <LOQ under argon atm. T15d-25 C./60% RH 0.0 99.6 0.4 <LOQ T1m-25 C./60% RH 0.0 99.3 0.5 0.2 Freeze-dried T0 3.8 94.6 1.6 ND (350 mg/ml amifostine T15d-25 C./60% RH 3.4 95.5 1.1 <LOQ conversion in HCl 1M, T1m-25 C./60% RH 3.1 95.8 1.1 ND diluted in water) under argon atm. Freeze-dried T0 0.0 99.1 0.9 ND (500 mg/ml amifostine T15d-25 C./60% RH 0.2 99.2 0.6 <LOQ conversion in HCl 4M, T1m-25 C./60% RH 0.0 98.9 0.6 0.5 diluted in P407/P188) under argon atm

[0115] According to the stability results obtained: [0116] after 1 month at 25 C./60% RH, less than 2% of impurities have been detected in all tested aminothiol formulations and this value remained stable; and [0117] freeze-dried WR-1065 obtained from amifostine conversion in acidic conditions and WR-1065 hydrochloride salt have the same stability.

E-Attempts to Develop Stable Amifostine Active Metabolite Gel Treatment

Example E-1: Stabilization of Aminothiol

[0118] Initially, the possibility to develop a thermogel of aminothiol was evaluated, to obtain a stable ready-to-use formulation with at least 6 months stability in refrigerated conditions.

[0119] Aminothiol gel formulations are not stable when stored at 5 C. or at room temperature. During storage, aminothiol degrades in oxidation impurity (disulfide WR-33278) significantly.

[0120] For example, an aminothiol gel at 50 mg/ml presented an increase of disulfide impurity from 3.6 to 13.8% (area %) after 15 days storage at 5 C.

##STR00003##

[0121] Different parameters were tested to improve aminothiol stability in solution: [0122] additives of preservative agent like an antioxidant and/or chelating agent (metabisulfide, EDTA, tartaric acid, citric acid, N-acetylcysteine, alpha lipoc acid, hydrochloric cysteine, ascorbic acid, Dithiothreitol DTT), [0123] nitrogen inerting, or [0124] drug concentration (50 mg/ml, 220 mg/ml).

[0125] For example, a stability study was performed after one month at 5 C.3 C. on aminothiol gel at 50 mg/ml. Firstly, aminothiol solution is prepared by conversion of 350 mg/ml amifostine solution heated in HCl 1M (based on example A-1). Then the solution is diluted at 50 mg/ml (expressed in aminothiol) in different antioxidant solution and put at 5 C.3 C. stability.

TABLE-US-00009 Disulfides + Antioxidant reagent Time Amifostine Aminothiol Unknown 1 None Initial 2.2% 96.2% 1.6% 7 days 2.2% 92.4% 5.4% 1 2.0% 84.2% 13.8% month 2 Tartaric acid Initial 2.3% 96.6% 1.1% 0.5% 7 days 2.2% 93.2% 4.6% 1 1.8% 84.7% 13.5% month 3 Citric acid Initial 2.3% 96.5% 1.1% 0.5% 7 days 2.2% 93.2% 4.6% 1 1.8% 85.0% 13.2% month 4 N-acetylcysteine Initial 2.0% 83.9% 14.1% 1.6% 7 days 1.5% 69.3% 29.2% 1 0.9% 39.8% 59.3% month 5 -Lipoic acid Initial 2.3% 92.4% 5.3% 0.04% 7 days 2.1% 93.1% 4.8% 1 2.0% 83.6% 14.4% month 6 Cysteine Initial 2.3% 96.2% 1.5% hydrochloride 7 days 2.1% 93.2% 4.7% 0.5% 1 1.9% 79.6% 18.5% month 7 Ascorbic acid Initial 2.3% 94.1% 3.7% 0.5% 7 days 2.1% 85.6% 12.2% 1 1.9% 76.9% 21.2% month 8 DTT Initial 2.3% 96.7% 1.1% 0.05% 7 days 2.2% 94.5% 3.3% 1 2.0% 83.4% 14.6% month 9 DTT Initial 2.3% 97.0% 0.7% 0.5% 7 days 2.2% 95.5% 2.3% 1 2.0% 97.5% 0.5% month

[0126] Whatever the additives tested (antioxidant, chelating agent), aminothiol solution is not stable enough at 5 C., and the disulfide bridge formation is not prevented. Moreover, N-acetylcysteine accelerates the degradation of aminothiol, creating an important new impurity. So, this option of aminothiol stabilization is not suitable.

[0127] Among them, the best stability was obtained by the formulation with DTT at 0.5%. It is stable for at least one month at 5 C., but the amount of DTT at 0.5% is not appropriate for intended use.

Example E-2: Rapid Conversion from Amifostine into Aminothiol

[0128] As aminothiol is not stable, the inventors evaluated the possibility to develop a thermogel of amifostine which will be converted into aminothiol (WR-1065) just before administration. To develop a commercially suitable product, the aim is to find suitable conditions to obtain total conversion (>90%) in less than 30 minutes.

[0129] In the following experiments, amifostine crystalline powder was added to different placebo gels, and conversion rate and yield into Aminothiol WR-1065 was measured.

[0130] Different parameters were tested: [0131] acid (hydrochloric acid, acetic acid, phosphoric acid, citric acid, sulfuric acid, perchloric acid), [0132] amifostine concentration (20-80 mg/ml), or [0133] temperature (37 C. or room temperature).

[0134] For example, amifostine crystalline powder was directly incorporated at a concentration of 40 mg/ml in placebo gels containing a mixture of 14% w/w of Poloxamer P407 and 26% w/w of P188 and 15% or 30% v/v of HCl 1M. Conversion was monitored after 30 min at 37 C. and from 30 min up to 3 hours at room temperature.

TABLE-US-00010 % HCl Condition Amifostine Aminothiol Disulfide Unknown HCl RT/T = 0 98.9% 1.1% 0.0% 0.0% 30% RT/T = 35 min 94.2% 5.3% 0.3% 0.2% RT/T = 70 min 89.9% 9.3% 0.4% 0.3% RT/T = 3 H 73.1% 25.3% 0.9% 0.7% 37 C./T = 0 99.1% 0.8% 0.1% 0.1% 37 C./T = 68.7% 30.0% 0.7% 0.6% 35 min HCl RT/T = 0 96.9% 1.4% 1.1% 0.6% 15% RT/T = 35 min 93.5% 3.7% 1.8% 0.9% RT/T = 70 min 87.3% 9.2% 2.4% 1.1% RT/T = 3 H 71.1% 25.1% 2.6% 1.2% 37 C./T = 0 98.4% 1.2% 0.2% 0.2% 37 C./T = 63.1% 33.6% 2.2% 1.1% 35 min

[0135] After 30 min at room temperature or at 37 C., the conversion into aminothiol is very low, whatever the parameters tested (the maximum obtained is 30%).

Example E-3: Rapid Conversion from Disulfide into Aminothiol

[0136] As the conversion of amifostine into aminothiol within 30 minutes is difficult, another option of formulation was evaluated, converting disulfide WR-33278 into aminothiol WR-1065 just before administration.

[0137] Conversion of disulfide to aminothiol was tested on a gel formulation containing high disulfide amount (37.7% area). To reduce disulfide bonds, a strong reducing agent, dithiothreitol (DTT), was tested at different concentrations

[0138] Firstly, aminothiol solution is prepared by conversion of 350 mg/ml amifostine solution heated in HCl 1M (based on example A-1). Then the solution is diluted at 50 mg/ml (expressed in aminothiol) in water and stored for a few days at room temperature to obtain high formation of disulfides by degradation. After a few days, DTT is finally added to the gel and analyses were performed after 30 minutes of incubation with DTT at room temperature.

TABLE-US-00011 Purity profile 30 min after addition of DTT % Area without 5 mM 41 mM 81 mM RRT DTT DTT DTT DTT Amifostine 1 5.6 5.0 6.2 7.0 Aminothiol 3.6 56.8 50.7 93.4 90.5 Disulfide 3.7 37.7 44.3 0.4 2.6

[0139] Using DTT, disulfides can be reduced considerably to aminothiol in 30 minutes at room temperature, but the concentration needed in DTT is higher than the amount authorized for toxicology reactions.

F-Preclinical Evaluation of a Thermogel According to the Invention Containing Aminothiol for the Prevention of Radiation-Related Oral Mucositis

Example F-1: Preparation of Gel Formulations for Efficacy Study

[0140] The following formulations were used for an efficacy study as explained hereafter. Formulations were prepared the day before the efficacy study, stored at 5 C. and analyzed on dosing day.

[0141] Purity of amifostine batch used was taken into account. For aminothiol formulation, the concentration in mg/ml is expressed as equivalent amifostine concentration. Using molecular ratio, 80 mg/ml amifostine is equivalent to 50 mg/ml aminothiol.

[0142] All gel formulations were colored by brilliant blue for a better visualization of the application.

TABLE-US-00012 Final composition Formulation Preparation method % w/w Gel n1 Prepared by mixing 22.9% v/v of a Amifostine 80 mg/ml aminothiol 350 mg/ml concentrated solution of converted in thiol (50 amifostine previously converted mg/ml) into thiol (heating 24 h at 50 C. in Kleptose HPB 3.4% 15% Kleptose HPB in HCl 1M) HCl 1M 19.5% with 77.1% v/v of a placebo gel Kolliphor P407 11.6% Kolliphor P188 20.8% Water milliQ 44.7% Gel n2 Prepared by mixing amifostine Amifostine 80 mg/ml amifostine powder with a placebo gel Kleptose HPB 10% alkalinized with NaOH. NaOH 1M 25% Kolliphor P407 11.5% Kolliphor P188 20% Water milliQ 33.5% Gel n3 Placebo gel preparation Kolliphor P407 15% placebo Kolliphor P188 27% Water milliQ 58% Solution IV Prepared by mixing amifostine in Amifostine 20 mg/ml NaCl 0.9% in NaCl 0.9%

[0143] Gel n1 has the same composition as a thermogel composition according to the invention (corresponding to a reconstituted lyophilisate according to the invention).

Example F-2: Efficacy Study of the Gel Formulations of Example F-1

[0144] The aim of this example was to evaluate the efficacy of the thermogel compositions of example F-1 containing amifostine against radiation-induced oral mucositis in vivo.

[0145] Female C57BL/6 mice (12 weeks old) purchased from Janvier CERT (Le Genest St. Isle, France) were used after an acclimation time of 7 days. They had free access to food (SAFE reference R0340, Augy, France) and water. They were housed on a 12 hours light/dark cycle at a room temperature of 22 C.2 C. and a relative humidity of 55%15%.

[0146] Irradiation was carried out locally with a X-ray Varian NDI 226 tube (200 kV, 15 mA, 0.2 mm Cu) at a dose rate of 1.33 Gy/min, the rest of the body being protected by a lead shield. Mice were euthanized when body weight loss exceeded 20% of the initial weight for more than 24 hours or in case of severe clinical signs (ethical endpoints).

[0147] The following experiment was carried out by using the model of oral mucositis as described in Mangoni, M., Vozenin, M. C., Biti, G., and Deutsch, E. (2012). Normal tissues toxicities triggered by combined anti-angiogenic and radiation therapies: hurdles might be ahead. British journal of cancer, 107, 308-314.

[0148] Two parameters were monitored: the body weight and the Parkins score. This represents an arbitrary score system devised by Parkins and co-workers with separate scores for edema and erythema evaluated by macroscopic observation (Parkins, C. S., Fowler, J. F., and Yu, S. (1983). A murine model of lip epidermal/mucosal reactions to X-irradiation. Radiotherapy and oncology: Journal of the European Society for Therapeutic Radiology and Oncology 1, 159-165). Indeed, at high doses of irradiation, lip epidermal/mucosal reactions are observed, beginning approximately 5 days after irradiation and reaching a peak about 10 to 13 days after irradiation. They consist mainly of an edema and an erythema. The Parkins score represents the sum of the score for edema and the score for erythema.

[0149] The Parkins score is calculated as follows: Parkins score=edema score+erythema score.

TABLE-US-00013 Erythema or exudation score 50-50 doubtful if any abnormally pink 0.5 Slight but definite redding 1 Severe redding 2 Focal desquamation 3 Exudate or crusting involving about half lip 4 area Exudate or crusting involving more than 5 half lip area

TABLE-US-00014 Edema score 50-50 doubtful if any swelling 0.5 Slight but definite swelling 1 Severe swelling 2

Experiment

[0150] A single dose irradiation was delivered to the snout of mice. This induced a mucosal/epidermal lip reaction that was monitored through macroscopic observation and scoring.

[0151] A blind experiment was conducted to determine the effect of gels containing or not an active substance on the severity and duration of oral mucositis. Three gels were evaluated: a placebo gel (Gel n3), a gel containing 80 mg/ml Amifostine (Gel n2) and a gel containing 50 mg/ml Amifostine thiol (Gel n1) (corresponding to 80 mg/ml Amifostine). Mice were anesthetized (intraperitoneal injection of Ketamine 75 mg/kg+Domitor 1 mg/kg) 10 minutes before gel application. The gel was applied into the mouth (5 l on each cheek) and onto the lips (40 l). So, a total volume of 50 l covered the mouse mucosa, which corresponds to 4 mg Amifostine per mouse. Mice snouts were irradiated 30 minutes after the application of the gel using a 20 Gy single fraction (dose rate 1.33 Gy/min). Antisedan was injected 1 hour after the injection of the anesthetic mix. These mice were compared with mice receiving an intravenous (IV) injection of Amifostine (4 mg per mouse, i.e. corresponding to an intravenous injection of 200 l of a 20 mg/ml Amifostine solution) 30 minutes before irradiation and to a control group of mice receiving only a 20 Gy localized irradiation.

[0152] To sum up, 30 mice were allocated to 5 groups and then irradiated: [0153] Control [0154] Placebo gel [0155] Amifostine gel (80 mg/ml, 4 mg amifostine/mouse) [0156] Aminothiol gel (50 mg/ml, corresponding to 80 mg/ml amifostine or 4 mg amifostine/mouse) [0157] Amifostine intravenous (IV) (20 mg/ml, 4 mg amifostine/mouse)

[0158] Graphs depicting the evolution of the Parkins score and the body weight over time are presented in FIG. 1. In FIG. 1, the curve (*) corresponds to the control, the curve (.square-solid.) corresponds to the placebo gel, the curve (.diamond-solid.) corresponds to the amifostine gel, the curve () corresponds to the amifostine thiol gel, and the curve (x) corresponds to the amifostine IV.

[0159] The irradiation of mice snout at a dose of 20 Gy in a ventral position induced a severe phenotype characterized by a significant body weight loss and a worsening of the general health status. All the control mice had to be sacrificed between day 14 and day 20. In the placebo gel group, mice exhibited a trend towards an even worse phenotype, as they had to be sacrificed a little bit earlier. The median survival was of 16 days versus 19 days for the placebo gel and the control groups respectively. However, it has to be noticed that the difference was not statistically significant (Log-rank test). Surprisingly, the presence of Amifostine in the gel had no visible impact on the development and the intensity of the mucositis, as mice presented a similar phenotype over time to those of the control group mice. On the contrary, the intravenous injection of Amifostine resulted in a mucosal reaction of moderate intensity. Edema and erythema completely disappeared as soon as day 20 after irradiation and no second peak (healing period) was observed for this group (FIG. 1). Interestingly, with the Amifostine thiol gel, mice showed an intermediate phenotype. They did not lose weight during the acute reaction (days 10 to 15), even if an established mucositis was detected. These mice did not reach an endpoint leading to their sacrifice and all the mice were still alive at the end of the experiment, in clear contrast to the fate of mice of the Amifostine gel group.

[0160] The opposite phenotype observed for the Amifostine gel and the Amifostine thiol gel groups and the clear absence of efficacy of the Amifostine gel suggest that Amifostine is not or not efficiently converted into Amifostine thiol when it is included in the gel and applied at the snout. Thus, the free active compound amifostine thiol has to be delivered directly to prevent at least partly the development of the radiation-induced mucositis.

Example F-3: Preparation of Gel Formulations for a Second Efficacy Study

[0161] The following formulations were used for an efficacy study as explained hereafter. Formulations were prepared the day before the efficacy study, stored at 5 C. and analyzed on dosing day.

[0162] Purity of amifostine batch used was taken into account and concentration in mg/ml is expressed as equivalent amifostine concentration.

TABLE-US-00015 Final composition Formulation Preparation method % w/w Gel n4 Prepared by mixing 8% v/v of a Amifostine 20 mg/ml aminothiol 250 mg/mIL concentrated solution converted in thiol at 12.5 mg/ of amifostine previously converted (12.5 mg/ml) ml to thiol (heating 25 h at 50 C. in Kleptose HPB 1.2% 15% Kleptose HPB in HCl 1M) HCl 1M 6.8% with 92% v/v of a placebo gel Kolliphor P407 13.8% Kolliphor P188 24.8% Water milliQ 53.4% Gel n5 Prepared by mixing 11.4% v/v of a Amifostine 40 mg/ml aminothiol 350 mg/ml concentrated solution of converted in thiol (25 at 25 mg/ amifostine previously converted to mg/ml) ml thiol (heating 25 h at 50 C. in 15% Kleptose HPB 1.7% Kleptose HPB in HCl 1M) with HCl 1M 9.7% 88.6% v/v of a placebo gel Kolliphor P407 13.8% Kolliphor P188 24.8% Water milliQ 53.4% Gel n6 Prepared by mixing 22.9% v/v of a Amifostine 80 mg/ml aminothiol 350 mg/ml concentrated solution of converted in thiol (50 at 50 mg/ amifostine previously converted to mg/ml) ml thiol (heating 25 h at 50 C. in Kleptose HPB 3.4% 15% Kleptose HPB in HCl 1M) HCl 1M 19.5% with 77.1% v/v of a placebo gel Kolliphor P407 11.6% Kolliphor P188 20.8% Water milliQ 44.7% Gel n7 Placebo gel preparation Kolliphor P407 14.5% Placebo Kolliphor P188 26.2% Kleptose HPB 0.4% Water milliQ 58.9% Solution IV Prepared by mixing amifostine in Amifostine 20 mg/ml NaCl 0.9% in NaCl 0.9%

[0163] Gels n4, 5, and 6 have the same composition as a thermogel composition according to the invention (corresponding to a reconstituted lyophilisate according to the invention).

Example F-4: Efficacy Study of the Gel Formulations of Example F-3

[0164] The purpose of this second efficacy experiment was to confirm the results obtained in the first experiment regarding the impact of Amifostine thiol on radiation-induced mucositis and to establish data about a dose-response relationship. As an intermediate effect was observed with the gel containing 80 mg/ml Amifostine (corresponding to 50 mg/ml amifostine thiol), it would have been interesting to test a more concentrated gel to determine if we could improve more radically oral mucosa protection against ionizing radiation.

[0165] The experiment was conducted with gels containing 20, 40 or 80 mg/ml Amifostine (corresponding to 12.5, 25 or 50 mg/ml amifostine thiol respectively). A lower dose of irradiation (a single fraction irradiation of 18 Gy) was delivered to the snout, so that all the mice could be kept alive until the end of the experiment. Different amounts of compound were administered to mice (1; 2 or 4 mg per mouse).

[0166] The same experimental procedure as for in example F-2 was used. Briefly, mice were anesthetized, Amifostine or Amifostine thiol was delivered 10 minutes later or not depending on the group (gel application or intravenous injection), mice were irradiated dorsally at an 18 Gy dose 30 minutes after the administration of Amifostine or Amifostine thiol and awakened 20 minutes later.

[0167] Forty mice were allocated to 6 groups: [0168] Control (n=6 mice); [0169] Placebo gel (n=6 mice); [0170] Amifostine thiol 20 mg/ml gel (1 mg amifostine/mouse, n=6 mice); [0171] Amifostine thiol 40 mg/ml gel (2 mg amifostine/mouse, n=6 mice); [0172] Amifostine thiol 80 mg/ml gel (4 mg amifostine/mouse, n=6 mice); and [0173] Amifostine intravenous (IV) (20 mg/ml) (4 mg amifostine/mouse, n=5 mice).

[0174] For each group, the development of oral mucositis was evaluated on 5 or 6 mice depending on the group. An 18 Gy dose irradiation induced a less severe phenotype than the 20 Gy dose irradiation that was used in the first efficacy experiment. The body weight loss was manageable and all the mice were still alive at the end of the experiment.

[0175] Graphs depicting the evolution of the Parkins score and the body weight over time are presented in FIG. 2. In FIG. 2, the curve (.diamond-solid.) corresponds to the control, the curve (*) corresponds to the placebo gel, the curve () corresponds to the gel n4, the curve (.square-solid.) corresponds to the gel n5, the curve (x) corresponds to the gel n6, and the curve (.circle-solid.) corresponds to the amifostine IV.

[0176] As expected, the irradiation induced a peak of mucosal reaction in the control group at day 13, as shown in FIG. 2. This was followed by a healing period extending from day 18 to day 30 as already observed in example F-2 for the Amifostine thiol gel group. For the Amifostine IV group, the acute peak of mucosal reaction was also observed at day 12, but it was of lower intensity than what is observed for the control gel (score of 2.2 versus 3.6 respectively). The healing period was nearly absent in this group as it was already noticed in example F-2. For the other groups, the score at the peak was of 4.1; 3.2; 2.8 and 2.4 for the placebo gel and the 20 mg/ml; 40 mg/ml and 80 mg/ml Amifostine (corresponding to 12.5, 25 or 50 mg/ml amifostine thiol) gels respectively. During the healing period, the curves for the control, the placebo gel, the 20 mg/kg and the 40 mg/kg Amifostine thiol gels were barely distinguishable from one another. The application of the 20 mg/kg Amifostine thiol gel did not alter the intensity of the peak of mucositis and the score recorded during the healing period was comparable to those of the control group. With the 40 mg/ml Amifostine thiol gel, a decrease of the intensity of the mucositis peak was observed. This was further enhanced with the 80 mg/ml Amifostine thiol gel, which also induced a reduction of the score recorded during the healing period.

Example F-5: Preparation of Gel Formulations by Reconstitution of Freeze-Dried Powder with Aqueous Solution for a Third Efficacy Study

[0177] The following formulations were used for an efficacy study with fractionated irradiation as explained hereafter. The freeze-dried thermogel were reconstituted the day before the efficacy study, stored at 5 C. and analyzed on dosing day.

[0178] Purity of amifostine batch used was taken into account and concentration in mg/ml is expressed as equivalent amifostine concentration.

TABLE-US-00016 Formu- Final composition lation Preparation method % w/w Gel n8 Prepare the Placebo gel Kolliphor P407: 21% Placebo Freeze-drying the placebo gel Kolliphor P188: 4% Gel Reconstitute freeze-dried powder Water 75% using water to volume Gel n9 Prepare the gel by mixing 8% v/v of Amifostine 40 mg/ml amino- a 500 mg/ml concentrated solution converted in thiol thiol of amifostine previously converted (25 mg/ml) at 25 into thiol (heating 1 h at 60 C. in HCl 4M: ~8% mg/ml HCl 4M) with 92% v/v of a Kolliphor P407: placebo gel (P407/P188: 20/4) 18.4% Freeze-drying the gel Kolliphor Reconstitution freeze-dried powder P188: 3.7% using water to volume Water: ~69.9% Solution Prepared by mixing amifostine in Amifostine 20 mg/ml IV NaCl 0.9% in NaCl 0.9%

[0179] After reconstitution, final formulation Gels n8 and 9 have the same composition as that before lyophilization, according to the invention.

Example F-6: Efficacy Study of the Gel Formulations of Example F-5

[0180] The purpose of this third efficacy experiment was to confirm the results obtained in the first experiment regarding the impact of Amifostine thiol on radiation-induced mucositis in a setting of a fractionated irradiation regimen and using reconstituted freeze-dried thermogels.

[0181] The experiment was conducted with a placebo gel and a gel containing 40 mg/ml Amifostine (corresponding to 25 mg/ml amifostine thiol). Four fractions of 8 Gy were delivered for four consecutive days (everyday from day 0 to day 3; day 0 corresponding to the first day of treatment) to the snout. The fractionation scheme of four fractions of 8 Gy was chosen, because it is biologically equivalent to 1 fraction of 18 Gy (Biologically Effective Dose: BED120 Gy). The same amount of compound (corresponding to 2 mg amifostine per mouse) was administered to mice before each fraction of irradiation. Thus, the compound was applied 4 times for four consecutive days.

[0182] For each day, a similar experimental procedure as for in example F-2 was used. Briefly, mice were anesthetized, Amifostine or Amifostine thiol was delivered 10 minutes later or not depending on the group (gel application or intravenous injection), mice were irradiated dorsally at an 8 Gy dose 30 minutes after the administration of Amifostine or Amifostine thiol and awakened 20 minutes later. The same experimental procedure was repeated for 4 consecutive days.

[0183] Twenty-four mice were allocated to 4 groups: [0184] Irradiation without gel (n=6 mice); [0185] Irradiation with Amifostine thiol gel (25 mg/ml, corresponding to 2 mg amifostine or 1.25 mg amifostine thiol/mouse/day) (n=6 mice); [0186] Irradiation with Placebo gel (n=4 mice); [0187] Irradiation with intravenous injection (IV) of amifostine (2 mg amifostine/mice) (n=6 mice).

[0188] For each group, the development of oral mucositis and body weight were analyzed daily (6 days/week) from day 3 to day 60. Graphs depicting the evolution of the Parkins score and the body weight over time are presented in FIG. 3. In FIG. 3, the curve (.diamond-solid.) corresponds to the control, the curve (*) corresponds to the placebo gel, the curve () corresponds to the gel n9, the curve (.square-solid.) corresponds to the amifostine IV.

[0189] As already observed after a single fraction irradiation, a peak of mucosal reaction was induced 15 days after the first fraction of irradiation in the control group. The healing period extended until 60 days after irradiation. A noticeable body weight loss was observed at days 12-16 and correlated with the peak of mucositis. As expected, the placebo gel did not show any effect regarding the development, the intensity and the duration of the mucositis. For the Amifostine IV group, the acute peak of mucosal reaction was also observed around day 15, but it was of lower intensity (score of 2.6 for the Amifostine IV group versus 6.4 for the control group) and mice recovered from the mucositis by the 30.sup.th day. Interestingly, the same result as for the Amifostine IV group was observed for the Amifostine thiol gel group: the score at the peak was of 2.6 and mice did not show any signs of mucositis after day 30. So, the local application of the Amifostine thiol gel protected mice from radio-induced mucositis. With this gel, the mucositis was of lower intensity and duration.

G-Preclinical Evaluation of a Thermogel According to the Invention Containing Aminothiol for the Prevention of Radiation-Related Cutaneous Erythema

Example G-1: Preparation of Gel Formulations by Reconstitution of Freeze-Dried Powder with Aqueous Solution for Efficacy Study

[0190] The following formulations were used for an efficacy study with fractionated irradiation as explained hereafter. The freeze-dried thermogel were reconstituted the day before the efficacy study, stored at 5 C. and analyzed on dosing day.

[0191] Purity of amifostine batch used was taken into account and concentration in mg/ml is expressed as equivalent amifostine concentration.

TABLE-US-00017 Formu- Final composition lation Preparation method % w/w Gel n10 Prepare the Placebo gel Kolliphor Placebo Freeze-drying the placebo gel P407: 21% Gel Reconstitute freeze-dried powder Kolliphor P188: 4% using water to volume Water 75% Gel n11 Prepare the gel by mixing 8% v/v of Amifostine 40 mg/ml amino- a 500 mg/ml concentrated solution converted in thiol thiol of amifostine previously converted (25 mg/ml) at 25 into thiol (heating 1 h at 60 C. in HCl 4M: ~8% mg/ml HCl 4M) with 92% v/v of a Kolliphor P407: placebo gel (P407/P188: 20/4) 18.4% Freeze-drying the gel Kolliphor Reconstitution freeze-dried powder P188: 3.7% using water to volume Water: ~69.9%

[0192] After reconstitution, final formulation Gels n10 and 11 have the same composition as that before lyophilization, according to the invention.

Example G-2: Efficacy Study of the Gel Formulations of Example G-1

[0193] The aim of this example was to evaluate the efficacy of the thermogel compositions of example G-1 containing amifostine against radiation-induced cutaneous erythema in vivo.

[0194] Female C57BL/6 mice (12 weeks old) purchased from Janvier CERT (Le Genest St. Isle, France) were used after an acclimation time of 7 days. They had free access to food (SAFE reference R0340, Augy, France) and water. They were housed on a 12 hours light/dark cycle at a room temperature of 22 C.2 C. and a relative humidity of 55%15%.

[0195] Irradiation was carried out locally with a X-ray XRAD320 tube (320 kV, 12.5 mA) at a dose rate of 1.08 Gy/min, the rest of the body being protected by a lead shield. Only the skin on the back of mice was irradiated. Mice were euthanized when body weight loss exceeded 20% of the initial weight for more than 24 hours, in case of severe clinical signs or at the end of the experiment (ethical endpoints).

[0196] At high doses of irradiation, epidermal reactions (erythema, desquamation, ulceration, . . . ) are observed, beginning approximately 15 days after irradiation and reaching a peak about 20 days after irradiation. An arbitrary score system was used to evaluate the development of the radiation-induced cutaneous erythema.

Scoring Scale

[0197]

TABLE-US-00018 Score normal 0 dry skin 0.5 cracking 1 one wound-slight desquamation 2 2-3 non extended wounds (desquamation of 3 <25% of irradiated area) desquamation of <50% of irradiated area 4 desquamation of <75% of irradiated area 5

Experiment

[0198] The purpose of this efficacy experiment was to evaluate the impact of Amifostine thiol on radiation-induced cutaneous erythema in a setting of a fractionated irradiation regimen and using reconstituted freeze-dried thermogels.

[0199] The experiment was conducted with a placebo gel and a gel containing 40 mg/ml Amifostine (corresponding to 25 mg/ml amifostine thiol). Four fractions of 12 Gy were delivered for four consecutive days (everyday from day 1 to day 4; day 1 corresponding to the first day of treatment) to the skin on the back of mice. The same amount of compound (corresponding to 4 mg amifostine per mouse) was administered to mice before each fraction of irradiation. Thus, the compound was applied 4 times for four consecutive days.

[0200] For each day, a similar experimental procedure was used. Briefly, mice were anesthetized, the Amifostine thiol gel or the placebo gel was applied directly onto the depilated skin on the back of mice 10 minutes later or not depending on the group (final volume of gel=100 l). The gel was removed 1 hour later with water soaked compresses. Mice were then irradiated at a 12 Gy dose and awakened 10 minutes later. The same experimental procedure was repeated for 4 consecutive days.

[0201] Ten mice were allocated to 3 groups: [0202] Irradiation without gel (n=4 mice); [0203] Irradiation with Amifostine thiol gel (Gel n11) (25 mg/ml, corresponding to 4 mg amifostine or 2.5 mg amifostine thiol/mouse/day) (n=3 mice); [0204] Irradiation with Placebo gel (Gel n10) (n=3 mice);

[0205] For each group, the development of cutaneous erythema and body weight were analyzed daily (6 days/week) from day 3 to day 50. The epidermal reaction was monitored through macroscopic observation and scoring. Graphs depicting the evolution of the erythema score are presented in FIG. 4. Individual curves for the control group, the placebo gel (Gel n10) group and the gel n11 group are depicted in FIGS. 4A, 4B and 4C respectively.

[0206] In our experiment, mouse skin irradiation induced a severe desquamation with a peak of epidermal reaction around day 20. The desquamation reached a plateau and was still present at the end of the observation period. No body weight loss was observed during the course of the experiment. As expected, the placebo gel did not show any effect regarding the development, the intensity and the duration of the epidermal reaction. Interestingly, in the group of mice treated with the amifostine thiol gel, the development of erythema was delayed in 2 mice out of 3, and in this group, one mouse healed from the cutaneous reaction and recovered from the erythema by the 50.sup.th day (score of 0.5 at day 50). So, the local application of the Amifostine thiol gel seems to protect mice from radio-induced erythema.