COMPOSITIONS COMPRISING SHORT-ACTING BENZODIAZEPINES

Abstract

The present invention provides novel compositions comprising benzodiazepine derivatives according to formula (I). Also provided are compositions comprising at least one hygroscopic excipient, in particular lactose and/or dextran.

##STR00001##

Claims

1. A storage-stable pharmaceutical composition of methyl 3-[(4S)-8-bromo-1-methyl-6-(pyridine-2-yl)-4H-imidazo[1,2-a][1,4]benzodiazepin-4-yl]propanoate besylate, comprising (a) methyl 3-[(4S)-8-bromo-1-methyl-6-(pyridine-2-yl)-4H-imidazo[1,2-a][1,4]benzodiazepin-4-yl]propanoate besylate, (b) dextran having a molecular weight of less than 80 kD, and (c) lactose, wherein the combined weight % ratio of the dextran and lactose to the methyl 3-[(4S)-8-bromo-1-methyl-6-(pyridine-2-yl)-4H-imidazo[1,2-a][1,4]benzodiazepin-4-yl]propanoate is at least 4:1, and wherein the weight % ratio of the lactose to the dextran is from 1:1 to 1:10.

2. The storage-stable pharmaceutical composition of claim 1, wherein the combined weight % ratio of the dextran and lactose to the methyl 3-[(4S)-8-bromo-1-methyl-6-(pyridine-2-yl)-4H-imidazo[1,2-a][1,4]benzodiazepin-4-yl]propanoate is between 5:1 and 25:1.

3. The storage-stable pharmaceutical composition of claim 1, wherein the combined weight % ratio of the dextran and lactose to the methyl 3-[(4S)-8-bromo-1-methyl-6-(pyridine-2-yl)-4H-imidazo[1,2-a][1,4]benzodiazepin-4-yl]propanoate is between 5:1 and 7:1.

4. The storage-stable pharmaceutical composition of claim 1, wherein the combined weight % ratio of the dextran and lactose to the methyl 3-[(4S)-8-bromo-1-methyl-6-(pyridine-2-yl)-4H-imidazo[1,2-a][1,4]benzodiazepin-4-yl]propanoate is 5:1, 6:1, or 7:1.

5. The storage-stable pharmaceutical composition of claim 1, wherein the weight % ratio of the lactose to the dextran is from 1:1 to 1:5.

6. The storage-stable pharmaceutical composition of claim 1, wherein the weight % ratio of the lactose to the dextran is from 1:1 to 1:4.5.

7. The storage-stable pharmaceutical composition of claim 1, wherein the weight % ratio of the lactose to the dextran is from 1:1.5 to 1:4.

8. The storage-stable pharmaceutical composition of claim 1, wherein the weight % ratio of the lactose to the dextran is 1:1.5.

9. The storage-stable pharmaceutical composition of claim 1, wherein the dextran is dextran 40.

10. The storage-stable pharmaceutical composition of claim 1, wherein the dextran is dextran 70.

11. The storage-stable pharmaceutical composition of claim 1, wherein the combined weight % ratio of the dextran and lactose to the methyl 3-[(4S)-8-bromo-1-methyl-6-(pyridine-2-yl)-4H-imidazo[1,2-a][1,4]benzodiazepin-4-yl]propanoate is between 5:1 and 7:1, and the weight % ratio of the lactose to the dextran is between about 1:1 and about 1:5.

12. The storage-stable pharmaceutical composition of claim 11, wherein the weight % ratio of the lactose to the dextran is from 1:1 to 1:4.5.

13. The storage-stable pharmaceutical composition of claim 11, wherein the weight % ratio of the lactose to the dextran is from 1:1.5 to 1:4.

14. The storage-stable pharmaceutical composition of claim 11, wherein the weight % ratio of the lactose to the dextran is 1:1.5.

15. The storage-stable pharmaceutical composition of claim 11, wherein the dextran is dextran 40.

16. The storage-stable pharmaceutical composition of claim 11, wherein the dextran is dextran 70.

17. A storage-stable pharmaceutical composition of methyl 3-[(4S)-8-bromo-1-methyl-6-(pyridine-2-yl)-4H-imidazo[1,2-a][1,4]benzodiazepin-4-yl]propanoate besylate, comprising (a) methyl 3-[(4S)-8-bromo-1-methyl-6-(pyridine-2-yl)-4H-imidazo[1,2-a][1,4]benzodiazepin-4-yl]propanoate besylate, (b) dextran 40, and (c) lactose, wherein the combined weight % ratio of the dextran 40 and lactose to the methyl 3-[(4S)-8-bromo-1-methyl-6-(pyridine-2-yl)-4H-imidazo[1,2-a][1,4]benzodiazepin-4-yl]propanoate is between 5:1 and 7:1, and wherein the weight % ratio of the lactose to the dextran is from 1:1.5.

18. The storage-stable pharmaceutical composition of claim 1, wherein less than 1% of the carboxylic ester moiety of the methyl 3-[(4S)-8-bromo-1-methyl-6-(pyridine-2-yl)-4H-imidazo[1,2-a][1,4]benzodiazepin-4-yl]propanoate is hydrolyzed after storage for at least 13 weeks at 25° C.

19. The storage-stable pharmaceutical composition of claim 1, wherein less than 1% of the carboxylic ester moiety of the methyl 3-[(4S)-8-bromo-1-methyl-6-(pyridine-2-yl)-4H-imidazo[1,2-a][1,4]benzodiazepin-4-yl]propanoate is hydrolyzed after storage for at least 13 weeks at 40° C.

20. The storage-stable pharmaceutical composition of claim 1, wherein less than 1% of the carboxylic ester moiety of the methyl 3-[(4S)-8-bromo-1-methyl-6-(pyridine-2-yl)-4H-imidazo[1,2-a][1,4]benzodiazepin-4-yl]propanoate is hydrolyzed after storage for at least 6 months at 25° C.

Description

[0133] Particularly, the invention relates to the following embodiments: In the embodiment 1, the invention relates to a composition comprising at least one benzodiazepine comprising at least one carboxylic acid ester moiety or a pharmaceutically acceptable salt thereof, wherein the composition

a) comprises at least one pharmaceutically acceptable hygroscopic excipient, and/or
b) the composition is at least in part amorphous.

[0134] Embodiment 2 relates to a composition according to embodiment 1, wherein the benzodiazepine is a compound according to formula (I)

##STR00005##

wherein
W is H, a C.sub.1-C.sub.4 branched or straight chain alkyl;
X is CH.sub.2, NH, or NCH.sub.3; n is 1 or 2;

Y is O or CH.SUB.2.; m is 0 or 1;

Z is O; p is 0 or 1;

[0135] R.sup.1 is a C.sub.1-C.sub.7 straight chain alkyl, a C.sub.3-C.sub.7 branched chain alkyl, a C.sub.1-C.sub.4 haloalkyl, a C.sub.3-C.sub.7 cycloalkyl, an aryl, a heteroaryl, an aralkyl, or a heteroaralkyl;
R.sup.2 is phenyl, 2-halophenyl or 2-pyridyl,
R.sup.3 is H, Cl, Br, F, I, CF.sub.3, or NO.sub.2;
(1) R.sup.4 is H, a C.sub.1-C.sub.4 alkyl, or a dialkylaminoalkyl and R.sup.5 and R.sup.6 together represent a single oxygen or S atom which is linked to the diazepine ring by a double bond and p is zero or 1; or (2) R.sup.4 and R.sup.5 together is a double bond in the diazepine ring and R.sup.6 represents the group NHR.sup.7 wherein R.sup.7 is H, C.sub.1-4 alkyl, C.sub.1-4 hydroxyalkyl, benzyl or benzyl mono or disubstituted independently with halogen substituents, C.sub.1-4 alkylpyridyl or C.sub.1-4 alkylmidazolyl and p is zero; or (3) R.sup.4, R.sup.5 and R.sup.6 form the group-CR.sup.8═U—V=wherein R.sup.8 is hydrogen, C.sub.1-4 alkyl or C.sub.1-3 hydroxyalkyl, U is N or CR.sup.9 wherein R.sup.9 is H, C.sub.1-4 alkyl, C.sub.1-3 hydroxyalkyl or C.sub.1-4 alkoxy, V is N or CH and p is zero.

[0136] Embodiment 3 relates to a composition according to embodiment 2, wherein p is zero and R.sup.4, R.sup.5 and R.sup.6 form the group —CR.sup.8═U—V=wherein R.sup.8 is hydrogen, C.sub.1-4 alkyl or C.sub.1-3 hydroxyalkyl, U is N or CR.sup.9 wherein R.sup.9 is H, C.sub.1-4 alkyl, C.sub.1-3 hydroxyalkyl or C.sub.1-4 alkoxy, V is N or CH.

[0137] Embodiment 4 relates to a composition according to embodiment 2 or 3, wherein W is H; X is CH.sub.2, n is 1; Y is CH.sub.2, m is 1;

R.sup.1 is CH.sub.3, CH.sub.2CH.sub.3, CH.sub.2CH.sub.2CH.sub.3, CH(CH.sub.3).sub.2 or CH.sub.2CH(CH.sub.3).sub.2:
R.sup.2 is 2-fluorophenyl, 2-chlorophenyl or 2-pyridyl;

R.SUP.3 .is Cl or Br.

[0138] Embodiment 5 relates to a composition according to any of embodiments 2 to 4, wherein p is zero and R.sup.4, R.sup.5 and R.sup.6 form the group —CR.sup.8═U—V=wherein R.sup.8 is methyl, U is CH.sub.2, V is N;

W is H; X is CH.sub.2, n is 1; Y is CH.sub.2, m is 1;
R1 is CH.sub.3; R2 is 2-pyridyl; R3 is Br.

[0139] Embodiment 6 relates to a composition according to any of the embodiments 1 to 5, wherein the benzodiazepine is in the form of a pharmaceutically acceptable salt.

[0140] Embodiment 7 relates to a composition according to any of the embodiments 1 to 6, wherein in the pharmaceutically acceptable salt the benzodiazepine is formulated in cationic form and the counter ion is selected from halogenides, in particular fluoride, chloride or bromide, sulfate, organic sulfates, sulfonate, organic sulfonates, nitrate, phosphate, salicylate, tartrate, citrate, maleate, formiate, malonate, succinate, isethionate, lactobionate and sulfamate.

[0141] Embodiment 8 relates to a composition according to embodiment 7, wherein the counter ion is selected from organic sulfates and sulfonates, in particular aromatic sulfates and sulfonates.

[0142] Embodiment 9 relates to a composition according to embodiment 8, wherein the counter ion is benzene sulfonate (besylate).

[0143] Embodiment 10 relates to a composition according to embodiment 9, wherein the benzodiazepine salt is crystalline remimazolam besylate.

[0144] Embodiment 11 relates to a composition according to any of the embodiments 1 to 10, wherein the hygroscopic excipient is a compound which is able to form stable hydrates.

[0145] Embodiment 12 relates to a composition according to any of the embodiments 1 to 11, wherein the hygroscopic excipient is an organic substance, preferably selected from carbohydrates and/or organic polymers.

[0146] Embodiment 13 relates to a composition according to embodiment 12, wherein the hygroscopic excipient possesses a molecular weight of less than 150 kD.

[0147] Embodiment 14 relates to a composition according to embodiment 12 or 13, wherein the carbohydrate is a dextran molecule.

[0148] Embodiment 15 relates to a composition according to embodiment 12 or 13, wherein the carbohydrate is selected from monosaccharides and C.sub.2-6-oligosaccharides.

[0149] Embodiment 16 relates to a composition according to embodiment 15, wherein the carbohydrate is a disaccharide, preferably selected from the group consisting of lactose, maltose, sucrose and trehalose.

[0150] Embodiment 17 relates to a composition according to embodiment 12, wherein the organic polymer is a polyvinylpyrrolidone and preferably possesses a molecular weight of between 5 and 40 kD.

[0151] Embodiment 18 relates to a composition according to any of the embodiments 1 to 17, wherein the wt. % ratio between the total amount of hygroscopic excipients and the total amount of benzodiazepines or salts thereof in the composition is at least 1:1, preferably at least 2:1, most preferably at least 5:1.

[0152] Embodiment 19 relates to a composition according to any of the embodiments 1 to 18, wherein the composition is in the solid state and is preferably a lyophilized solid.

[0153] I. Stability of CNS 7056; Formulations with Selected Excipients

[0154] 1. Formulations

[0155] A total of 11 formulations of the besylate salt of remimazolam with a selection of suitable excipients as detailed in FIGS. 1 and 2 were lyophilized. In addition a formulation containing the besylate salt of remimazolam alone and matching placebos for each formulation were also prepared (see FIG. 3). In the following the abbreviation “REM” is used for the besylate salt of remimazolam.

[0156] Each formulation was prepared as follows and filled into vials prior to freeze drying: Excipient was dissolved in approximately 50 ml water. REM was added and stirred to dissolve. Once dissolved the pH of the solutions was adjusted to 3.10±0.05 with 0.5 M hydrochloric acid/2 M sodium hydroxide. Placebo solutions and the solution containing REM alone were prepared in the same manner. Each solution was made up to 100 ml and 1.2 ml of each solution was aliquoted into 2 ml vials. The formulations were lyophilized using a Virtis Genesis 25 EL freeze dryer according to the following cycle:

TABLE-US-00003 Freezing steps Temperature Time Pressure Hold/ Step (° C.) (minutes) (mTorr) Ramp 1 4 10 — H 2 −45 490 — R 3 −45 170 — H

TABLE-US-00004 Drying steps Temperature Time Pressure Hold/ Step (° C.) (minutes) (mTorr) Ramp 1 −45 10 100 H 2 −25 200 100 R 3 −25 3640 100 H 4 30 275 70 R 5 30 1300 70 H

[0157] After freeze drying the samples were stored in storage cabinets at 25° C./60% RH and at 40° C./75% RH for 13 weeks, respectively. (“RH” means relative humidity.)

[0158] Analysis

[0159] a) Reconstitution Time

[0160] After 13 weeks of storage the vials were reconstituted in duplicate with 1.2 ml of water for irrigation/injection and swirled gently to mix. The time taken for complete dissolution was recorded.

[0161] b) HPLC

[0162] For HPLC each vial was reconstituted with sample solvent (50/50% v/v acetonitrile/water) and the contents transferred to a 25 ml volumetric flask (with exception of REM only vial, which was transferred to a 50 ml volumetric flask) with several rinsings. The dextran formulation was insoluble in sample solvent and was diluted in 100% water. For each formulation a placebo was also analysed in the same way. Analyses were performed in duplicate, unless otherwise stated.

[0163] Results

[0164] a) Reconstitution Time

[0165] Reconstitution time was acceptable for all samples.

[0166] b) HPLC

[0167] The investigation of the emergence of the hydrolysis degradant of REM (results are summarized in FIG. 4), which is formed by hydrolysis of the ester bond, revealed that the sample with REM only as well as the sample containing mannitol, which is a commonly used excipient for lyophilization of pharmaceuticals, exhibited only poor stability of REM, showing a degradation after 13 weeks at storage conditions of 40° C./75% RH of more than 8%.

[0168] Samples containing glycine showed moderate degradation, whereas all samples containing hygroscopic excipients (carbohydrates or polyvinylpyrrolidone) showed good or excellent stability. In particular the samples containing carbohydrates (disaccharides or dextran) showed excellent stability, i.e., a degradation after 13 weeks at storage conditions of 40° C./75% RH of less than 1%.

[0169] The samples with differing amounts of lactose revealed that the greater the amount of carbohydrate relative to the amount of REM, the better the stability of the REM. Further by incorporating a carbohydrate (e.g. lactose) as a component of a formulation of CNS 7056 that is inherently unstable e.g. mannitol it is possible to improve the stability of this formulation.

[0170] 2. Stability Data of CNS 7056: Lactose Based Formulation Batches after Storage for Up to 36 Months

[0171] 2.1 Introduction

[0172] CNS 7056 is presented for clinical use as a sterile lyophilized powder for reconstitution in 20 mL vials with a Bromobutyl stopper, suitable for intravenous injection. Each vial contains 26 mg of CNS 7056. During development further batches with 25, 23 and 26 mg of CNS 7056 have been prepared. On reconstitution with a defined volume of Water for Injection, the concentration of the dose solution is 5 mg/mL CNS7056. All these products contain the same CNS 7056 to lactose ratio in the lyophilized product (i.e. 1:13 CNS 7056: lactose monohydrate). Stability data were collected for all intervals through the month shown in bold in the following Table 1:

TABLE-US-00005 TABLE 1 Summary of Stability studies for CNS 7056 batches Summary of CNS 7056 for Injection on Stability Storage Test Interval Batch Conditions Type of Batch (Months) A01P310 25° C./60% RH GMP clinical batch, stability 0, 1, 3, 6, 9, 12, 18, 24, 36.sup.c, 48.sup.c 30° C./65% RH 0, 1, 3, 6, 9, 12 40° C./75% RH 0, 1, 3, 6 P310-01(B) 25° C./60% RH Lab development batch, stability 0, 1, 3, 6, 9, 12, 18 30° C./65% RH 0, 6, 9, 12 40° C./75% RH 0, 1, 3, 6, 9, 12 025CNS27 25° C./60% RH Lab development batch, stability 0, 1, 2, 3, 6, 9, 12, 18.sup.ab 40° C./75% RH 0, 1, 2, 3, 6, 12.sup.b 026CNS27 25° C./60% RH Lab development batch, stability 0, 1, 2, 3, 6, 9, 12, 18.sup.ab 40° C./75% RH 0, 1, 2, 3, 6.sup.b G384 25° C./60% RH Lab development batch, stability 0, 1, 2, 3, 6, 9, 12, 18.sup.ab 40° C./75% RH 0, 1, 2, 3, 6.sup.b P02308 25° C./60% RH GMP clinical batch, stability 0, 1, 2, 3, 6, 9, 12, 18, 24, 36.sup.c, 48.sup.c 40° C./75% RH 0, 1, 2, 3, 6, 9.sup.a, 12 .sup.a= Stability test interval added. Test vials taken from remaining spare vials at specified temperature. .sup.b= Stability studies on 025CNS27, 026C0NS27 and G384 are now complete. .sup.c= Optional time points

[0173] 2.1.1 CNS 7056 Batch Composition

TABLE-US-00006 TABLE 2 Composition of the different CNS7056 batches Batch# Substance 025CNS27 026CNS27 G384 P02308 P310-01(B) A01P310 CNS7056  25 mg  25 mg  25 mg  23 mg   26 mg   26 mg base Lactose 433 mg 433 mg 433 mg 398 mg 450.3 mg 450.3 mg monohydrate 0.12M qs to pH 3.1 qs to pH 3.1 qs to pH 3.1 qs to pH 3.1 qs to pH 3.1 qs to pH 3.1 NaOH/ 0.12M HCl [0174] qs=to add sufficiently quantity to

[0175] 2.1.2 Freeze Drying Conditions

[0176] The freeze drying conditions for the batches are given in the following tables 3 to 7:

TABLE-US-00007 TABLE 3 Freeze drying cycle for batch 025CNS27: Shelf temperature Ramp rate Hold time Pressure Step Process (° C.) (° C./min) (min) (mTorr) 1 Load 4 0 30 n/a 2 Freezing −45 0.1 180 n/a 3 Primary −25 0.1 1700 100 drying 4 Secondary 30 0.2 1300 75 drying 5 Finish Vials stoppered under 95% pure nitrogen

TABLE-US-00008 TABLE 4 Freeze drying cycle for batch 026CNS27: Shelf temperature Ramp rate Hold time Pressure Step Process (° C.) (° C./min) (min) (mTorr) 1 Load 4 0 120 n/a 2 Freezing −45 0.1 300 n/a 3 Primary drying −30 0.1 2885 100 4 Primary drying −25 0.2 4100 100 4 Secondary 30 0.2 1580 75 drying 5 Finish Vials stoppered under 95% pure nitrogen

TABLE-US-00009 TABLE 5 Freeze drying cycle for batch G384: Shelf temperature Ramp rate Hold time Pressure Step Process (° C.) (° C./min) (min) (mTorr) 1 Load 4 0 10 n/a 2 Freezing −45 0.1 300 n/a 3 Primary drying −25 0.1 3640 100 4 Secondary 30 0.2 1125 70 drying 5 Finish Vials stoppered under 95% pure nitrogen

TABLE-US-00010 TABLE 6 Freeze drying cycle for batch P02308: Shelf temperature Ramp rate Hold time Pressure Step Process (° C.) (° C./min) (min) (mTorr) 1 Load 4 0 60 n/a 2 Freezing −45 0.1 180 n/a 3 Primary drying −25 0.1 3640 100 4 Secondary 30 0.2 1300 75 drying 5 Finish Vials stoppered under 95% pure nitrogen

TABLE-US-00011 TABLE 7 Freeze drying cycle for batch A01P310: Shelf temperature Ramp rate Hold time Pressure Step Process (° C.) (° C./min) (min) (mTorr) 1 Load 4 0  60 n/a 2 Freezing −45 0.1   210.sup.a n/a 3 Primary drying −25 0.1 3640 100 4 Secondary 30 0.2 1300  75 drying 5 Finish Vials stoppered under 95% pure nitrogen .sup.a= Includes 30 min for condenser preparation

[0177] 2.2 Methods of Analysis

[0178] 2.2.1 Appearance of Lyophilized Product

[0179] The same CNS 7056 vials (6 at each storage condition) were inspected visually, the appearance recorded and the vials placed back on storage. A comparison was also made to a set of vials that had been stored in the same secondary packaging at 2 to 8° C. to assess whether there were any differences (in particular in colour) between these controls and those stored at elevated temperatures.

[0180] 2.2.2 CNS7056 Vial Content, Concentration on Reconstitution and Related Substances

[0181] The CNS7056 assay and related substances determination was determined by HPLC. For this purpose, the appropriate volume of WFI was added to each vial and swirled until complete dissolution was achieved. The seal and the stopper were carefully removed and the stopper was rinsed thoroughly into a 100 ml volumetric flask. The contents of the vial with washings of diluent were transferred to a volumetric flask. The diluent was added to reach a volume of 100 mL (equals a concentration of 0.23, 0.25 or 0.26 mg/mL, respectively). The samples were analysed by HPLC by using the following conditions:

Column: YMC ODS-AQ, 250×4.6 mm, 3 μm particle size
Mobile phase: Res A: 0.01% trifluoroacetic acid in water [0182] Res B: 0.01% trifluoroacetic acid in acetonitrile

Gradient:

[0183]

TABLE-US-00012 Time (mins) % A % B 0 75 25 20.0 60 40 30.0 20 80 32.0 20 80 32.5 75 25 40.0 75 25
Flow rate: 1.0 ml/min
Column temperature: 40° C.
Autosampler: ambient

Detection: UV at 230 nm

[0184] Injection volume: 10 μl
Run time: 40 min

[0185] The retention time for CNS7056 is about 15 minutes. The CNS 7056 content was assayed by comparison with similarly chromatographed reference solutions. Related substances were determined by normalised area %.

[0186] The concentration of the reconstituted solution is calculated by the following equation:

[00001]$\mathrm{CNS}7056\mathrm{base}\left(\mathrm{mg}\right)\text{?}\left(\frac{\mathrm{sample}\mathrm{peak}\mathrm{area}}{\mathrm{mean}\mathrm{peak}\mathrm{area}\text{?}}\right)\times \frac{\mathrm{Wt}\text{?}}{50}\times \frac{M\mathrm{Wt}\mathrm{CNS}7056\mathrm{base}}{M\mathrm{Wt}\mathrm{CNS}7056B}\times \frac{P}{100}\times \mathrm{DF}$$\mathrm{where}$$\mathrm{Wt}\mathrm{std}\text{?}\mathrm{is}\mathrm{the}\mathrm{weight}\mathrm{of}\mathrm{CNS}7056B\mathrm{reference}\mathrm{material}\mathrm{used}\mathrm{to}\text{?}$$M\mathrm{Wt}\mathrm{CNS}7056\mathrm{base}\mathrm{is}\mathrm{the}\mathrm{molecular}\mathrm{weight}\mathrm{of}\mathrm{the}\mathrm{free}\text{?}\mathrm{of}\mathrm{CNS}7056\text{?}439.3$$M\mathrm{Wt}\mathrm{CNS}7056B\mathrm{is}\mathrm{the}\mathrm{molecular}\mathrm{weight}\mathrm{of}\mathrm{CNS}7056\text{?}$$P\mathrm{is}\mathrm{the}\text{?}\mathrm{as}\mathrm{per}\mathrm{the}C\mathrm{of}A\mathrm{for}\mathrm{the}\mathrm{reference}\mathrm{standard}$$\mathrm{DF}\mathrm{is}\mathrm{the}\mathrm{disulution}\mathrm{factor}$$\text{?}\text{indicates text missing or illegible when filed}$

[0187] The vial content is calculated according to the following formula:

[00002]$\mathrm{CNS}7056\mathrm{base}\left(\mathrm{mg}\right)\text{?}\left(\frac{\mathrm{sample}\mathrm{peak}\mathrm{area}}{\mathrm{mean}\mathrm{peak}\mathrm{area}\text{?}}\right)\times \frac{\mathrm{Wt}\text{?}}{50}\times \frac{M\mathrm{Wt}\mathrm{CNS}7056\mathrm{base}}{M\mathrm{Wt}\mathrm{CNS}7056B}\times \frac{P}{100}\times \mathrm{DF}$$\mathrm{where}$$\mathrm{Wt}\mathrm{std}\text{?}\mathrm{is}\mathrm{the}\mathrm{weight}\mathrm{of}\mathrm{CNS}7056B\mathrm{reference}\mathrm{material}\mathrm{used}\mathrm{to}\text{?}$$M\mathrm{Wt}\mathrm{CNS}7056\mathrm{base}\mathrm{is}\mathrm{the}\mathrm{molecular}\mathrm{weight}\mathrm{of}\mathrm{the}\mathrm{free}\mathrm{based}\mathrm{of}\mathrm{CNS}7056\text{?}439.3$$M\mathrm{Wt}\mathrm{CNS}7056B\mathrm{is}\mathrm{the}\mathrm{molecular}\mathrm{weight}\mathrm{of}\mathrm{CNS}7056\text{?}$$P\mathrm{is}\mathrm{the}\text{?}\mathrm{as}\mathrm{per}\mathrm{the}C\mathrm{of}A\mathrm{for}\mathrm{the}\mathrm{reference}\mathrm{standard}$$\mathrm{DF}\mathrm{is}\mathrm{the}\mathrm{disulution}\mathrm{factor}$$\text{?}\text{indicates text missing or illegible when filed}$

[0188] For determination of related substances CNS7056 is identified by comparison of the retention time to that of CNS7056 in the reference standard chromatograms. The amount of each individual detected related substance is calculated as area percent for each sample injection according to the following formula:

[00003]$\mathrm{Area}\%=\left(\frac{A}{T}\right)\times 100\mathrm{Where},A=\mathrm{area}\mathrm{of}\mathrm{the}\mathrm{related}\mathrm{substance}\mathrm{peak}T=\mathrm{total}\mathrm{area}\mathrm{of}\mathrm{all}\mathrm{peaks}\mathrm{in}\mathrm{the}\mathrm{chromatogram}$

[0189] 2.2.3 Chiral Purity

[0190] The chiral purity of CNS 7056 was determined by HPLC by using the following conditions:

Column: Chiralpak IC, 250×4.6 mm, 5 μm particle size
Mobile phase: phosphate buffer pH 7.0/water/acetonitrile 10/40/50, v/v/v
Sample solvent: water/acetonitrile 50/50, v/v
Flow rate: 0.7 ml/min
Column temperature: 40° C.
Autosampler: ambient

Detection: UV at 250 nm

[0191] Injection volume: 10 μl
Run time: 35 minutes

[0192] The retention time for the CNS 7056 S-enantiomer is about 21.3 min and for CNS 7056 R-enantiomer is about 17.8 min (RRT=0.84). The chiral purity is calculated according to the following formula:

[00004]$\mathrm{Area}\%=\left(\frac{A}{T}\right)\times 100\mathrm{where}$$A=\mathrm{area}\mathrm{of}R-\mathrm{CNS}7056\mathrm{peak}$$T=\mathrm{total}\mathrm{area}\mathrm{of}\mathrm{the}\mathrm{CNS}7056\mathrm{and}\mathrm{the}R-\mathrm{CNS}7056\mathrm{peaks}$

[0193] 2.2.4 Volume of Solution in Vial Following Reconstitution (Ph.Eur 2.9.17)

[0194] A single vial was reconstituted with 5.0 ml of Water for Injection (WFI), Ph. Eur. using a 5 ml BD syringe fitted with a suitable needle. When fully reconstituted, the contents were removed using a syringe and 21 gauge needle and transferred to a calibrated 10 ml measuring cylinder.

[0195] 2.2.5 Appearance of Reconstitution

[0196] The appearance of the solution following reconstitution was recorded.

[0197] 2.2.6 Reconstitution Time

[0198] Two vials were reconstituted with 5.0 mL of Water for Injection (WFI), Ph. Eur., using a 5 ml BD syringe and appropriate needle, and the time taken to fully dissolve recorded.

[0199] 2.2.7 pH Value

[0200] The pH was determined on two reconstituted solutions following addition of 5.0 mL Water for Injection (WFI), Ph. Eur., using a 5 ml BD syringe fitted with a suitable needle. The pH was measured on one aliquot from each of the two vials.

[0201] 2.2.8 Osmolality

[0202] The osmolality was determined on the two reconstituted solutions following addition of 5.0 mL Water for Injection (WFI), Ph. Eur., using a 5 ml BD syringe fitted with a suitable needle. Osmolality was measured on one aliquot from each of the two vials by freezing point depression with reference to a solution of known Osmolality. For this purpose 100 μl of the reconstituted CNS 7056 solution is measured in a freezing point depression osmometer.

[0203] 2.2.9 Water Content

[0204] The water content was determined by coloumetric Karl Fischer titration. The moisture content of vials of CNS 7056 drug product is determined by dissolving the entire contents of a vial of CNS 7056 lyophilised powder in anhydrous dimethylformamide (DMF) and injecting a known volume of the solution into the anolyte of a coloumetric Karl Fischer apparatus. In the Karl Fischer reaction, water reacts in a 1:1 ratio with iodine. The amount of water is determined by measuring the number of coulombs of electricity required to oxidise iodide ions to the iodine required for the Karl Fischer reaction. The number of Coulombs is used to calculate the amount of water titrated in μg, which is displayed by the apparatus.

[0205] The following equipment and reagents were used:

Karl-Fischer titratus apparatus: Mitsubishi CA-100

Anolyte: Hydranal Coulomat AG

Catholyte: Hydranal Coulomat CG

[0206] The water content of CNS7056 lyophilised powder is calculated according to the following formula:

[00005]$\mathrm{Moisture}\mathrm{per}\mathrm{vial}\left(\mathrm{mg}\right)=\frac{\left(\begin{array}{c}\mathrm{Msample}-\\ \mathrm{msolvent}\end{array}\right)}{1000}\times \frac{\left(\mathrm{Wsolvent}\text{/}\mathrm{DSolvent}\right)}{\mathrm{Vtitration}}$$\mathrm{Moisture},\%w\text{/}w=\frac{\mathrm{Moisture}\mathrm{per}\mathrm{vial}\times 100}{\left(\mathrm{Svial}\right)}$$\mathrm{Where}\text{:}$$\mathrm{Msample}=\mathrm{amount}\mathrm{of}\mathrm{water}\mathrm{in}\mathrm{the}\mathrm{sample}\mathrm{solution}\mathrm{added}\mathrm{to}\mathrm{the}\mathrm{titration}\mathrm{vessel}\left(\mathrm{.Math.g}\right)$$\mathrm{Msolvent}=\mathrm{mean}\mathrm{amount}\mathrm{of}\mathrm{water}\mathrm{in}\mathrm{the}\mathrm{solvent}\mathrm{blanks}\mathrm{added}\mathrm{to}\mathrm{the}\mathrm{titration}\mathrm{vessel}\left(\mathrm{.Math.g}\right)$$\mathrm{Wsolvent}=\mathrm{weight}\mathrm{of}\mathrm{DMF}\mathrm{added}\mathrm{to}\mathrm{the}\mathrm{vial}\left(g\right)$$\mathrm{Dsolvent}=\mathrm{density}\mathrm{of}\mathrm{the}\mathrm{solvent}\left(g\text{/}\mathrm{ml}\right\}\mathrm{For}\mathrm{DMF}d=0.944g\text{/}\mathrm{ml},\mathrm{source}\mathrm{CRC}\mathrm{handbook}{81}^{\mathrm{st}}\mathrm{edition}$$\mathrm{Vtitration}=\mathrm{volume}\mathrm{of}\mathrm{solution}\mathrm{added}\mathrm{to}\mathrm{the}\mathrm{titration}\mathrm{vessel}\left(\mathrm{ml}\right)$$\mathrm{Svial}=\mathrm{Calculated}\mathrm{total}\mathrm{weight}\mathrm{solid}\mathrm{per}\mathrm{vial},\mathrm{including}\mathrm{water}\left(\mathrm{mg}\right)$

[0207] 2.2.10 ID by UV

[0208] Analysis was performed in duplicate on a single vial. The identification by UV was confirmed by comparison of the drug product spectra to reference spectra.

[0209] 2.2.11 Sub Visible Particles (EP 2.9.19)

[0210] Ten vials were reconstituted with 5 mL WFI using an appropriate sterile syringe and needle. The vials were pooled together under aseptic conditions and analysed according to European Pharmacopeia 2.9.19.

[0211] 2.2.12 Sub Visible Particles (EP 2.9.19)

[0212] Ten vials were reconstituted with 5 mL WFI using an appropriate sterile syringe and needle. The vials were pooled together under aseptic conditions and analysed according to European Pharmacopeia 2.9.19.

[0213] 2.2.13 Bacterial Endotoxin

[0214] Bacterial Endotoxin was determined by the Limulus amebocyte lysate (LAL) gel-clot method as a limit test with a limit of <0.5 EU/mg. For this purpose a LAL with declared sensitivity equal to 0.03 EU/ml is used. The Endotoxins are quantified using the following formula:

[00006]$\mathrm{Endotoxin}\mathrm{concentration}\mathrm{in}\mathrm{the}\mathrm{sample}\left(\mathrm{EU}\text{/}\mathrm{mg}\right)=\frac{\begin{array}{c}\begin{array}{c}\\ \mathrm{lysate}\mathrm{sensitivity}\left(\lambda \right)\times \end{array}\\ \mathrm{test}\mathrm{dilution}\mathrm{factor}\end{array}}{\mathrm{sample}\mathrm{concentration}}$$\mathrm{where}\text{:}$$\mathrm{lysate}\mathrm{sensitivity}=0.03\mathrm{EU}\text{/}\mathrm{ml}$$\mathrm{sample}\mathrm{concentration}=5\mathrm{mg}\text{/}\mathrm{ml}$

[0215] 2.2.14 Sterility

[0216] Sterility was determined by reconstituting the lyophilised CNS 7056 with 5 ml of sterile peptonate water (0.1%) each and incubating the samples in 100 ml of thioglycollate medium (THG) at 30 to 35° C. and 100 ml of tryptic soy broth (TSB) at 20 to 25° C. The incubations were performed for not less than 14 days. The media are visually inspected every 2 to 3 days for the presence of microbiological proliferation. If there is no microbial growth, the examined sample meets the test requirements (sterile).

[0217] 2.3 Results

[0218] The results of the stability analyses for the above described batches after storage at 25° C./60% relative humidity (RH) or at 40° C./75% RH (so called “accelerated stability” analysis) are summarized in the FIGS. 11 to 36.

[0219] 2.4 Summary

[0220] The tested formulations for CNS 7056 exhibit an excellent long term stability which already supports a shelf life of 36 month for the drug product.

[0221] 3. Stability Data after Storage for 36 Month

[0222] The CNS 7056 batch P02308 was subjected to a stability study whereby the vials were stored for 36 months at 25° C./60% RH.

[0223] For batch composition and freeze drying conditions see chapter 2.1. For description of the analytical methods see chapter 2.2.

[0224] 3.1 Results

[0225] The results of the stability analysis for the batch P02308 after storage at 25° C./60% relative humidity (RH) up to and including 36 months are summarized in FIGS. 27-30.

[0226] 3.2 Summary

[0227] All tests performed on batch P02308 after storage at 25° C./60% RH (T=36 months) were within the specified acceptance criteria. Appearance of Lyophilised Product, Completeness of Solution, Time to Reconstitute, pH, and Osmolality of all samples at T=36 months were well within specification.

[0228] CNS7056B Vial Content at 25° C./60% RH is 23.4 mg/vial, which is in keeping with all previous results. These results are well within specification. The main CNS 7056 hydrolysis product CNS7054X (RRT 0.59) has increased to 0.29% at 25° C./60% RH from 0.07% at initial months.

[0229] A total impurities result of 0.80% was observed at the T=36 month time point, compared to 0.65% at initial. These results, together with the supporting data from storage over 12 months at the accelerated stability storage condition of 40° C./75% RH, reflect only a slight increase in degradation over this significant period of time and demonstrate the stabilising effect of CNS 7056 in combination with lactose.

[0230] Moisture content at T=36 months 25° C./60% RH is 0.68% which shows an increase from 0.27% at initial. This increase is thought to be due to water desorption from the stopper, which will occur over time. These results are well within specification.

[0231] 3.3 Conclusion

[0232] All parameters are within specification and the only noticeable trends are the expected increase in the hydrolysis product CNS 7054X and moisture content. The rate of increase of CNS 7054X is similar to previous laboratory non GMP development batches of CNS 7056 for injection produced/tested.

[0233] 4. Evaluation of Crystalline Material in a Lyophilised Lactose Formulation of CNS 7056 by Raman Mapping

[0234] 4.1 Introduction

[0235] XRPD studies showed that the lyophilised formulation of CNS 7056 is amorphous, however when material is examined under polarised light microscopy there is evidence of crystalline material present in the amorphous matrix. In order to reveal if this crystalline material is due to CNS 7056 or some other component e.g. lactose monohydrate a Raman mapping analysis was performed. This study makes use of a confocal Raman dispersive microscope to study the physical form of CNS 7056 within the lyophilized lactose formulation using Raman mapping. In Raman mapping experiments, once the first Raman spectrum collection is completed from the in focus sample surface, the sample stage is moved at a predefined step (normally a few to few tens micron) and another spectrum is taken. This is continued until the chosen analytical area has been covered and a hyperspectral data set has been constructed. The sample is prepared to ensure its surface is flat as this avoids the need to refocus the microscope during the data collection from one point to another. The hyperspectral data cube is then processed to generate chemical images based on the distinguishable specific Raman peak (fingerprint) of each component of the sample under study. The chemical images thus generated can then establish each component variation over the chosen area of the studied sample. Crystalline (Form I polymorph), amorphous (lyophilized) CNS 7056B and the lyophilized (amorphous) lactose were characterized by Raman, and the characteristic Raman peak of crystalline CNS7056B were used to generate the chemical images of crystalline CNS7056B. The chemical images of the lactose were also generated based on its own characteristic peak. One area of a lyophilized formulation of CNS 7056B was mapped to determine, if present, the content (based on area ratio without calibration) and distribution of crystalline (Form 1) CNS 7056B within the mapping area of the lyophilized lactose formulation. The aim of this study was to establish whether crystalline material within the lactose formulation is due to CNS 7056B or some other component eg. lactose monohydrate.

[0236] 4.2. Material and Methods

[0237] The following samples were tested in the Raman mapping study: CNS 7056B (Form 1) [0238] Item/Lot Number: SOL 12621/5 [0239] Appearance: white powder [0240] Pharmaterials Ref no: PMO553/08

[0241] CNS 7056 for Injection (Received from Paion) [0242] Item/Lot Number: P02308 [0243] Appearance: white lyophilised powder [0244] Pharmaterials Ref no: PM0554/08

[0245] Lyophilized CNS 7056 (Amorphous) [0246] Item/Lot Number: 05/CNS/06 [0247] Appearance: white lyophilised powder [0248] Pharmaterials Ref no: PM0555/08

[0249] Lyophilized Lactose (Received from Paion) [0250] Item/Lot Number: 028/CNS/27 [0251] Appearance: white lyophilized powder [0252] Pharmaterials Ref no: PM0548/08

[0253] Raman Spectra of Supplied Materials

[0254] Raman spectroscopy on crystalline (Form I) and amorphous (lyophilized) CNS7056B and amorphous lactose (lyophilised) as supplied was performed using a confocal Nicolet Almega XR dispersive Raman microscope. A distinguishable Raman peak of crystalline (Form I) CNS7056B and amorphous lactose (lyophilised) was respectively selected for generating chemical images and establishing each variation in the examined area of a lyophilized formulation as shown later.

[0255] Raman Mapping Using a Confocal Dispersive Raman Microscope

[0256] Raman mapping was performed on one area of a lyophilised formulation. For each measurement, Raman mapping was performed on one area (e.g. 300×300 μm.sup.2). The chemical images were then produced, respectively based on the distinguishable Raman peak of crystalline CNS7056B and amorphous lactose (lyophilised). These operations allowed the identification of crystalline CNS7056B (potentially recrystallized from the lyophilized lactose formulation) and amorphous lactose (lyophilized) in the selected area of the sample. Subsequently, thus produced chemical images were used to indicate the distribution of crystalline CNS7056B and lactose (lyophilised) in the mapping area respectively.

[0257] Raman Technique

[0258] Raman Spectra

[0259] Samples were analysed by a confocal Nicolet Almega XR Dispersive Raman Microscope for its Raman spectrum using the following conditions: [0260] Exposure Time: 1.0 s [0261] Exposure Times of each spectrum: 10 [0262] Pinhole Size: 100 um [0263] Spectral range: whole (single grating) [0264] Laser: He Ne 633 nm at 100% power [0265] Objective: 50×/0.75 (magnifier/numerical aperture number)

[0266] Afterwards, the measured Raman spectra were corrected by baseline subtraction (BS) using the software OMNIC™ v7.3.

[0267] Raman Mapping

[0268] Each sample was gently pressed by hand so that the mapping area has an approximately flat surface. Raman spectra data for mapping were collected using following conditions: [0269] Exposure Time: 5.0 s [0270] Exposure Times: 10 [0271] Pinhole Size: 100 μm [0272] Wavelength range: 1700-300 cm.sup.−1 (multiple gratings) [0273] Laser: He Ne 633 nm (100% power) [0274] Objective: 50×/0.75 [0275] Area: circa 300×300 μm [0276] Scanning step: 10 μm

[0277] Then the measured Raman spectra data from mapping were modified by baseline correction and normalization using the software OMNIC™ v7.3.

[0278] 4.3 Results and Discussion

[0279] 4.3.1 Raman Spectra of Each Component in a Lyophilized Formulation

[0280] The Raman spectra of crystalline (Form I polymorph) and amorphous (lyophilized) CNS7056B and amorphous lactose (lyophilised) as supplied were collected using the procedure described under Material and Methods. As seen in FIG. 39, the Raman peak was then selected respectively: circa 1620 cm.sup.−1 for crystalline CNS7056B and circa 365 cm.sup.−1 for lyophilized lactose. These peaks are unique to both materials so that the chemical images of crystalline CNS7056B and lactose (lyophilized) can respectively be generated. The whole range of the Raman spectrum for each component contained in a lyophilized formulation is given in FIGS. 40 and 41.

[0281] 4.4 Summary

[0282] The obtained data demonstrate the presence of crystalline CNS7056B in this lyophilized formulation comprising mostly of amorphous CNS7056B. Furthermore a uniform distribution of CNS7056B and excipient in this lyophilized formulation could be shown.

[0283] In the lyophilised product tested approximately 9% of the data points contained a signal corresponding to crystalline CNS 7056B. The actual w/w (%) presence of the crystalline CNS 7056B in the amorphous lactose matrix, however, cannot be concluded from these results as calibration was not performed.

[0284] II. Preparation and Stability Assessments Analysis of Lyophilized and Spray-Dried Formulations

[0285] A lyophilised and a spray-dried formulation having the same formulation were prepared and tested for stability.

[0286] 1. Manufacture of Spray-Dried CNS7056B Formulation (with Lactose)

[0287] CNS7056B (Form 2 bn 10201126, 5.1 g) and Emprove Lactose Monohydrate, (139.2 g) were dissolved in 750 ml deionized (DI) water with heating to ˜50° C., and then filtered and cooled to room temperature. The pH was checked and not adjusted as it was at 3.1. This solution was spray-dried using the following parameters: Inlet temperature 150° C. Pump rate=10% (20 ml in 7 mins), Fan setting=50%. Yield 59.5 g. The water content was measured via Karl-Fischer and used to calculate the fill weight per vial (997 mg). 58 vials were filled with 997 mg of spray-dried formulation. 30 vials were placed in the vacuum oven with lids slightly open. These vials were dried under vacuum (˜250 psi) with a nitrogen bleed at 50° C. for 24 hours. The chamber was then flushed with nitrogen, and the vials were then closed quickly under nitrogen. These samples were called 12PM529-8-2. 28 vials were closed without drying. These samples were called 12PM529-8-1. All vials were crimped with aluminium seals.

[0288] 2. Scale Up of Spray Dried API (CNS7056B)

[0289] CNS7056B (Form 2 bn 10201126, 20 g) was dissolved in 2900 ml DI water. This solution was filtered and then spray-dried using the following parameters: Inlet temperature 130° C., outlet temp. 82-79° C. Pump rate=10% (20 ml in 7 minutes), Fan setting=30%. Yield not noted. The process was repeated with CNS7056B (Form 2 bn 10201126, 5.6 g) was dissolved in 812 ml DI water, to give 2.2 g overall yield (from both runs) of a white powder. The samples were called 12PM529-9-1.

[0290] 3. Manufacture of Freeze Dried (Lyophilized) API (CNS7056B)

[0291] A solution of CNS7056B in water was prepared (2.2 g of bn 10201126, Form 2, PM0232/12 in 230 ml water). This was placed in a round bottomed flask (rbf) and ‘shell-frozen’ in liquid nitrogen and then lyophilised over 5 days. The resulting fluffy white solid was scraped out and broken up (˜2 g). The samples were designated as 12PM529-10-1.

[0292] 4. Accelerated Stability Study on Lyophilized and Spray Dried Formulation and Spray Dried API

[0293] The spray-dried CNS7056B formulation, both dried (12PM529-8-2) and non-dried (12PM529-8-1), stored in crimped vials was placed on an accelerated stability study, along with the lyophilized CNS7056B formulation (CNS2501A) as reference, and with the spray-dried amorphous API (12PM529-9-1). Samples were stored at 40° C./75% RH for 4 and 13 weeks, and at 55° C. for 4 weeks, and analysed for appearance, assay, related substances, moisture, XRPD, reconstitution time, and appearance following reconstitution.

[0294] 4.1. Results

[0295] The results from the stability study are presented in the FIGS. 42 to 44 and can be summarized as follows:

[0296] The spray-dried formulation (sealed prior to additional drying) had a slightly higher initial total impurity level at t=0 than the lyophilised formulation CNS 2501A i.e. batch (˜0.73/0.67% vs 0.48%). This is potentially due to the manufacturing process involving higher temperature, and could be optimised on scale up.

[0297] The vacuum dried spray-dried formulation sample (12PM529-8-2) had similar water content to the lyophilised sample supplied (0.24% vs 0.34%). The non-dried spray-dried formulation sample (12PM529-8-1) had significantly higher water content (2.87%), as did the amorphous spray-dried API (CNS7056B, 12PM529-9-1).

[0298] The ‘dry’ spray-dried formulation (12PM529-8-2) showed similar stability to the lyophilized formulation. The total impurities increased ˜0.2% for both samples after 4 weeks (slightly more at 55° C. than at 40/75), and actually only increased ˜0.05% for both samples after 13 weeks at 40/75.

[0299] The ‘wet’ spray-dried formulation (12PM529-8-1) had slightly inferior stability than the other formulation samples, but was still within specification for impurities after 13 weeks at 40/75 (total impurities increased from 0.67% at t=0, to 1.33% at t=13 weeks).

[0300] The spray-dried API (CNS7056B, 12PM529-9-1) showed significant instability, with increase of total impurities to 1.94% (4 weeks at 55° C.), 2.56% (4 weeks at 40/75) and 3.35% (13 weeks at 40/75). This confirms that the lactose formulation is stabilising the API significantly during the stability trial, even when there are similar levels of water present in the formulation to the API sample.

[0301] As expected the major impurity that was observed was the hydrolysis product CNS7054X.

[0302] 5. Investigation of API Distribution and Form in the Spray-Dried and Lyophilized Formulations Using Raman Mapping

[0303] A vial of lyophilized formulated product (CNS7056B in lactose, batch number CNS2501A) was opened and sampled randomly four times. Each sampled portion was then presented on a microscope slide and Raman mapping was carried out over a small area of the surface of the formulation sample (˜300×300 μm). The data was processed in comparison with reference samples of lyophilized (amorphous) and crystalline API (CNS7056B, forms 1 and 2) and lyophilized (amorphous) and crystalline (monohydrate) lactose. The mapping was analysed to determine the distribution of the API within the formulation, and then if any phase separation (regions of API) was detected, these would be analysed to assess the physical form of the API. A second experiment was carried out where a new vial of lyophilized formulated product (CNS7056B in lactose, batch number CNS2501A) was opened and sampled from top, middle and bottom of the cake. These three samples were again analysed by Raman mapping as above. Also, two regions from the top and bottom samples were mapped over a smaller region (˜20×20 μm) in more detail. Three more batches of lyophilized formulated product (CNS7056B in lactose) were also analysed by Raman mapping: batches P02308, A01P301 and P301-02N. The size of region mapped in these experiments was ˜120×100 μm.

[0304] 5.1 Results for Batch CNS2501 A

[0305] The Raman mapping data was processed to give a ‘chemical image’ which shows the similarity of the Raman spectra detected at each point on the map with:

a) the excipient main peak at 355 cm-1 (i.e. lactose)
b) the API (CNS7056B) main peak at 1580 cm-1
c) correlation with the whole excipient spectra (lactose).

[0306] The data showed that no phase separation and re-crystallization of API was found in batch CNS2501A as supplied after analysis of 7 different grab-samples from 2 different vials. The distribution of API and lactose was uniform and no separate regions or particles of API were found. This suggests that a true molecular dispersion of the API in lactose has been formed in the lyophilised formulation batch CNS2501A.

[0307] 5.2 Results for Batches P02308, A01P301 and P301-02N

[0308] The Raman mapping data was processed as described in chapter 5.1.

[0309] The data revealed no phase separation and re-crystallization of API in batches P02308, A01P301 and P301-02N as supplied based on one set of mapping data for each batch. The distribution of API and lactose was uniform and no separate regions or particles of API were found. This suggests that a molecular dispersion of the API in lactose has been formed in the lyophilised formulation in batches P02308, A01P301 and P301-02N. (Note: some phase separation was found in previous mapping performed on batch P02308. This suggests that the distribution of separated (crystalline) API in this batch is not uniform.)

[0310] 6. Summary

[0311] An equivalent spray dried formulation to the current lyophilised (freeze-dried) product could be successfully developed and tested.

[0312] Both the spray dried and lyophilised formulations were shown to be fully amorphous and single phase by XRPD and Raman analysis (i.e. no detectable separated crystalline API). The spray dried formulation had a slightly higher impurity level (˜0.7% total impurities vs. ˜0.5% for the lyo product). This is presumed to be formed during spray-drying manufacture and could be reduced with process optimisation.

[0313] The fully dried spray-dried formulation showed equivalent stability to the lyo product over 13 weeks at 40° C./75% RH and 4 weeks at 55° C. The non-dried spray-dried formulation (3% water) showed slightly worse stability, but stayed within the specification over 13 weeks at 40° C./75% RH.

[0314] The spray-dried formulation showed similar colour change to the lyo product in the light stability trial, both turning grey/blue. Physical analysis of the light stressed samples of API and formulation showed some recrystallisation and absorption of water, but no evidence of changes in physical form contributing to the colour changes.

[0315] Raman mapping analysis of the current lyo batch (CNS2501A) and previous lyo batches (P02308, A01P301 and P301-02N) of formulated product showed uniform distribution of API and excipients, with no evidence of separation of API and subsequent crystallisation.

[0316] III. Preparation and Stability Analysis of Disaccharide Binary Excipient Containing Formulations

[0317] 1. Purpose and Study Outline

[0318] The purpose of the present study was to evaluate the stability of selected formulations.

[0319] Several lyophilized formulations of CNS 7056 were prepared containing lactose monohydrate and the pH adjusted to 3.1, the API is present as besylate salt. Two fill concentrations of CNS 7056 were investigated: 5 mg/ml and 10 mg/ml. The formulations were filled in ISO 10R and ISO 6R clear glass vials. Fill volume was reduced to 4 mL/vial (current fill volume is 5.2 mL). The existing formulation was filled in ISO 10R vials that were stoppered with both West 4023/50 art. 1346 stoppers and West S87 J 4416/50 stoppers. The stability of the new formulations manufactured in ISO 10R vials were evaluated together with the existing formulation. In addition to this, the existing formulation lyophilized in the frame of the last clinical batch manufacturing (batch number A01 P310, fill volume 5.2 mL, ISO 20R clear glass vial) was tested to generate comparative stability data.

[0320] 2. Methods

[0321] The following tests were performed on the stability samples: [0322] Appearance of the lyophyilisate. [0323] Reconstitution time. [0324] Appearance of the reconstituted solution. [0325] Moisture content by Karl Fischer titration. [0326] HPLC Assay/Related Substances. [0327] Osmolality (only at time 0)

[0328] 3. Batch Description

[0329] The product composition of the batches submitted to stability is summarised here below.

TABLE-US-00013 Formulation CNS 7056 (Excipient concen- Fill Product weight ratio) tration Vials volume Stoppers Reference Lactose  5 mg/mL  6R 4 mL West 4023/50 L6R5 Reference art. 1346 (Current) 10R 4 mL West 4023/50 L10R5 Formulation art. 1346 West S87 J L10R5S87 4416/50 20R 5.2 West 4023/50 L20R5 mL art. 1346 10 mg/mL  6R 4 mL West 4023/50 L6R10 art. 1346 10R 4 mL West 4023/50 L10R10 art. 1346 Lactose:  5 mg/mL  6R 4 mL West 4023/50 L4M16R5 Mannitol art. 1346 (4:1) 10R 4 mL West 4023/50 L4M110R5 art. 1346 10 mg/mL  6R 4 mL West 4023/50 L4M16R10 art. 1346 10R 4 mL West 4023/50 L4M110R10 art. 1346 Lactose:  5 mg/mL  6R 4 mL West 4023/50 L2M16R5 Mannitol art. 1346 (2:1) 10R 4 mL West 4023/50 L2M110R5 art. 1346 10 mg/mL  6R 4 mL West 4023/50 L2M16R10 art. 1346 10R 4 mL West 4023/50 L2M110R10 art. 1346

[0330] 4. Stability Program

[0331] The stability program is summarized in the following table:

TABLE-US-00014 Formulation (Excipient weight ratio) Product Reference Stability Lactose L6R5 1 month Reference (Current) Formulation L10R5  3 months L10R5S87  3 months L20R5  3 months L6R10 1 month L10R10 1 month Lactose 4: Mannitol 1 L4M16R5 1 month L4M110R5 1 month L4M16R10 1 month L4M110R10 1 month Lactose 2: Mannitol 1 L2M16R5 1 month L2M110R5 1 month L2M16R10 1 month L2M110R10 1 month

[0332] 5. Stability Schedules

[0333] The stability schedules are summarize in the following tables:

TABLE-US-00015 Storage conditions 40° C. ± 2° C./75% ± 5% RH and 55° C. ± 5° C. Tests for 1 month study Time 0 1M Lyo appearance (to be noted on all 5 vials) ✓ ✓ Reconstitution time ✓ ✓ Appearance of reconstituted solution ✓ ✓ Moisture content (KF titration) ✓ ✓ HPLC (Assay/Related Substances) ✓ ✓ Osmolality ✓ —

TABLE-US-00016 Storage conditions 55° C. ± 5° C. Tests for 3 months study Time 0 1M Lyo appearance (to be noted on all 5 vials) ✓ ✓ Reconstitution time ✓ ✓ Appearance of reconstituted solution ✓ ✓ Moisture content (KF titration) ✓ ✓ HPLC (Assay/Related Substances) ✓ ✓ Osmolality ✓ —

TABLE-US-00017 Storage conditions 25° C. ± 2° C./60% ± 5% RH Tests for 3 months study Time 0 1M 3M Lyo appearance (to be noted on all 5 vials) ✓ — ✓ Reconstitution time ✓ — ✓ Appearance of reconstituted solution ✓ — ✓ Moisture content (KF titration) ✓ — ✓ HPLC (Assay/Related Substances) ✓ — ✓ Osmolality ✓ — ✓

TABLE-US-00018 Storage conditions 40° C. ± 2° C./75% ± 5% RH Tests for 3 months study Time 0 1M 3M Lyo appearance (to be noted on all 5 vials) ✓ ✓ ✓ Reconstitution time ✓ ✓ ✓ Appearance of reconstituted solution ✓ ✓ ✓ Moisture content (KF titration) ✓ ✓ ✓ HPLC (Assay/Related Substances) ✓ ✓ ✓ Osmolality ✓ — —

[0334] 6. Stability Results

[0335] The results collected in the frame of the present study are presented in FIGS. 45 to 51 and can be summarised as follows:

[0336] Samples Stored at 40° C. 75% RH (1 Month). [0337] Some changes in the appearance of the lyo cake in the formulations L2M110R5 and L2M110R10. Some vials of L20R5 (clinical batch vials rejected after visual inspection) showed a different lyo cake appearance [0338] Expected increase of moisture content (not observed in L20R5; L2M110R10) [0339] Small increase in total impurities (not observed in L10R10; L10R5S87; L20R5). The HPLC assay kept practically constant. [0340] Increase of known impurity CNS7054X.

[0341] Samples after 3 Months Storage at 40° C. 75% RH (Only L10R5: L10R5S87 and L20R5 Formulations).

[0342] The appearance of both the cake and the reconstituted solutions didn't undergo any variation (some of the cakes of the L20R5 samples were found to be shrunk).

[0343] The assay remained unvaried.

[0344] L10R5 [0345] Further increase of moisture content (anyway the % H.sub.2O<1.0%). [0346] Further slight increase of the impurities due to the CNS7054X.

[0347] L10R5S87 [0348] Further increase of moisture content (anyway the % H.sub.2O<1.0%). [0349] Further slight increase of the impurities content due to CNS7054X.

[0350] L20R5 (Visual Inspection) [0351] Further increase of moisture content (anyway the % H2O<1.0%). [0352] Increase of the impurities content mainly due to CNS7054X.

[0353] Samples Stored at 55° C. (1 Month). [0354] The lyo cake of the formulations L2M110R5, L2M110R10, L4M110R5 and L4M110R10 was found to be shrunk and yellowish colored. Some vials of L20R5 (clinical batch vials rejected after visual inspection) showed a charred (insoluble) lyo cake. [0355] Increase of moisture content (not observed in L20R5) [0356] Increase in total impurities (total impurities below 1.00% in L10R5; L10R10). Concurrently negligible reduction of the HPLC assay. [0357] Increase of known impurity CNS7054X. [0358] Additional impurities exceeding the LOQ in L20R5, L4M110R5; L4M110R10; L2M110R5; L2M110R10 [0359] Slight presence of foam (not persistent) upon reconstitution of. [0360] L10R5; L10R5S87 and L20R5 formulations after 1 month storage at 25° C./60% RH. [0361] The appearance of both the cake and the reconstituted solutions didn't undergo any variation. [0362] The assay remained unvaried.

[0363] L10R5

[0364] Slight increase of moisture content (anyway the % H.sub.2O<1.0%).

[0365] Slight increase of the impurities due to the CNS7054X.

[0366] L10R5S87

[0367] The moisture content didn't increase.

[0368] The impurities content remained practically unvaried.

[0369] L20R5 (Visual Inspection)

[0370] Increase of moisture content (anyway the % H.sub.2O<1.0%).

[0371] Increase of the impurities content mainly due to CNS7054X.

[0372] IV. Preparation and Stability Analysis of Disaccharide/Dextran Containing Formulations, as a Means to Reduce Lyophilization Time

[0373] 1. Purpose

[0374] Within this study several CNS7056 lyophylisate formulations containing lactose and dextran were studied. The ratio of the disaccharide to dextran was changed in order to manipulate the glass transition temperature (Tg′) and collapse temperature Tc and therefore reduce the lyophylisation time. Compared to the disaccharide lactose the dextran possesses a higher Tg′ and therefore can act as a collapse temperature modifier.

[0375] Altogether 10 formulations were prepared and tested in different lyophilization protocols.

[0376] 2. Formulations

[0377] 2.1. Formulation Compositions

[0378] Two CNS7056 formulations were prepared containing dextran only (001/PAN/13) or a mixture of lactose and dextran (002/PAN/13) as summarized in the following table:

TABLE-US-00019 Name Formulation 001/PAN/13 50: 440, 7056: Dextran 002/PAN/13 50:220:220, 7056:Lactose:Dextran

[0379] 2.2. Formulation Preparation

[0380] 2.2.1. Preparation of the Solution

[0381] The solutions containing 12 mg/mL CNS7056 were prepared according to the following protocol: [0382] API (CNS 7056 besylate salt) added with magnetic stirring to 85% final volume [0383] Stirred for 3 hours at ambient, light protected [0384] Checked pH, nominal pH 3.2 for all formulations, and adjusted to pH 3.0 [0385] Stirred for further 20 minutes: no significant change in appearance [0386] Made to 90% final volume [0387] Stirred for further 20 minutes [0388] Formulations 1-2 appeared light yellow, slightly turbid. [0389] Checked pH, all nominal pH 3 [0390] Made to final volume and stirred for further 20 minutes [0391] No change in formulation 1-2 appearance, less undissolved material in the concentrated formulation [0392] Filtered (0.22 μm PVDF) formulations 1-2 [0393] Further 25 minutes stirring of concentrated formulation Filtered (0.22 μm PVDF) concentrated formulation [0394] All filtrates clear, light yellow, free from visible particles [0395] Filtrates filled in 4.2 mL volumes and lyophilised with protocol as described in 2.1.2

[0396] 2.2.2. Lyophilisation Protocol

[0397] The samples were lyophilized according to the following protocol:

TABLE-US-00020 Temperature Pressure Time Step Cycle stage (° C.) (mTorr) (min) 1 Load 25 n/a 0 2 Ramp 0 n/a 25 3 Ramp −45 n/a 225 4 Freezing −45 n/a 180 5 Hold −45 93 0 6 Ramp −25 93 30 7 Primary drying −25 93 4890 8 Ramp 30 20 120 9 Secondary drying 30 20 480 10  Finish 30 Vials stoppered to 722000 mTorr with (pure) nitrogen. Total cycle duration ~9 hours (~4.1 days)

[0398] 2.3 Analysis of the Lyophilized Samples

[0399] The lyophylisate showed a good appearance and a rapid reconstitution time for the carbohydrate:dextran-containing lyophilisates. Both formulations exhibit a purity above 99.72%.

TABLE-US-00021 Recon time 7054X Name Formulation Appearance in saline pH (%) Purity (%) 001/PAN/13 50:440, 7056:Dextran Off white plug 1 m 50 s 3.241 0.11 99.72 002/PAN/13 50:220:220, 7056 Off white plug 35 s 3.229 0.09 99.74 Lactose:Dextran

[0400] 2.3.1 Appearance

[0401] The appearance of the lyophilized samples was determined. The results are listed in the following table:

TABLE-US-00022 Sample details Appearance 001/PAN/13 Initial (T = 0) Off white plug 40° C./75% RH T = 1 m Off white plug with signs of shrinkage 002/PAN/13 Initial (T = 0) Off white plug 40° C./75% RH T = 1 m Off white plug with signs of shrinkage

[0402] 2.3.2 Moisture

[0403] The moisture content of the lyophilized samples was determined. The results are listed in the following table:

TABLE-US-00023 Mean moisture Sample details Vial 1 Vial 2 Vial 3 (% w/w) 001/PAN/13 Initial (T = 0) 0.04 0.12 0.19 0.12 40° C./ T = 1 m 0.19 0.19 — 0.19 75% RH 002/PAN/13 Initial (T = 0) 0.16 0.16 0.38 0.23 40° C./ T = 1 m 0.36 0.38 — 0.37 75% RH

[0404] 2.3.3 Reconstitution Time and pH of Reconstituted Solution

[0405] Each vial was reconstituted with 10 mL 0.9% saline. The results regarding reconstitution time and pH are listed in the following table.

TABLE-US-00024 Recon. time Sample details (seconds) pH 001/PAN/13 Initial (T = 0) 110 3.24 40° C./75% RH T = 1 m 153 3.21 002/PAN/13 Initial (T = 0)  35 3.23 40° C./75% RH T = 1 m  85 3.26

[0406] 2.3.4 Vial Content

[0407] The vial content for the samples at T=1 m 40° C./75% RH was determined after each vial was reconstituted with 10 mL 0.9% saline. The results are given in the following table:

TABLE-US-00025 [7056] (mg/vial) Details Vial 1 Vial 2 Mean 001/PAN/13 49.1136 49.6891 49.401 002/PAN/13 49.0496 49.2652 49.157

[0408] 2.3.4 Impurities

[0409] The impurities for the different formulations at T=1 m 40° C./75% RH were determined. The results are given in the following tables: 001/PAN/13:

TABLE-US-00026 Initial.sup.*2 40° C./75% RH RRT Name (T = 0) T =.Math. 1 m Impurity profile 0.27 n.a. N.D. 0.03 (area %) 0.42 n.a. N.D. <LOQ 0.47 n.a. <LOQ <LOQ 0.51 n.a. 0.07 0.07 0.57 n.a. <LOQ <LOQ 0.59 7054X 0.11 0.17 0.64 n.a. N.D. <LOQ 0.68 n.a. <LOQ <LOQ 0.71 n.a. N.D. <LOQ 0.89 n.a. 0.10 N.D. 0.93 n.a. N.D. 0.10 1.00 7056B 99.59 99.45 1.13 n.a. N.D. 0.03 1.31 n.a. <LOQ <LOQ 1.46 n.a. N.D. <LOQ 1.73 n.a. N.D. <LOQ 1.78 n.a. <LOQ <LOQ 1.84 n.a. <LOQ <LOQ 1.91 n.a. <LOQ N.D. Total imps.sup.*1 (area %) 0.3 0.4 .sup.*1Sum of all impurities ≥0.03% by area .sup.*2Impurities from post lyo samples Impurities are mean of 2 determinations n.d. = not detected

[0410] 002/PAN/13:

TABLE-US-00027 Initial 40° C./75% RH RRT Name (T = 0) T = 1 m Impurity profile 0.27 n.a. N.D. <LOQ (area %) 0.47 n.a. <LOQ N.D. 0.51 n.a. 0.07 0.07 0.56 n.a. N.D. <LOQ 0.59 7054X 0.09 0.14 0.64 n.a. N.D. <LOQ 0.68 n.a. <LOQ <LOQ 0.71 n.a. N.D. <LOQ 0.83 n.a. N.D. <LOQ 0.89 n.a. <LOQ N.D. 0.93 n.a. 0.10 0.10 1.00 7056B 99.62 99.49 1.10 n.a. N.D. 0.03 1.31 n.a. <LOQ <LOQ 1.73 n.a. N.D. <LOQ 1.78 n.a. <LOQ <LOQ 1.84 n.a. <LOQ <LOQ Total imps* (area%) 0.3 0.3 *Sum of all impurities ≥0.03% by area Impurities are mean of 2 determinations n.d. = not detected

[0411] 3. Formulations—Potential Process Improvement with Dextran 40 Based Formulations

[0412] 3.1. Formulation Compositions

[0413] Two CNS7056 formulations were prepared containing dextran (007/PAN/13), or a mixture of lactose and dextran 40(009/PAN/13) as summarized in the following table:

TABLE-US-00028 7056:excipient(s) Batch Formulation (mg) 007/PAN/13 7056:dextran 40 50:440 009/PAN/13 7056:lactose monohydrate:dextran 40 50:88:352

[0414] 3.2. Formulation Preparation

[0415] 3.2.1. Preparation of the Solution

[0416] Dissolution of CNS7056B was facilitated by overhead stirring under ambient laboratory conditions, protected from light. A summary of the salient points for the preparation of each formulation are presented below.

[0417] 007/PAN/13 [0418] API addition (<5 minutes) to ˜85% final volume [0419] Stirring started at 500 rpm and increased to 700 rpm by 120 minutes [0420] Adjusted to pH 3.0 after 70 minutes [0421] Increased to ˜90% after 120 minutes [0422] Adjusted to pH 2.8 after 150 minutes [0423] After 180 minutes, checked pH (2.9), adjusted to pH 3.0, and made to final volume

[0424] 009/PAN/13 [0425] API addition (<5 minutes) to ˜95% final volume [0426] Following API (500 rpm) stirring immediately increased to 700 rpm and then [0427] increased to 800 rpm by 30 minutes [0428] Adjusted to pH 3.0 after 10 minutes [0429] After 75 minutes, checked pH (pH 3.1), adjusted to pH 3.0, and made to final volume

[0430] Following preparation all formulations were filtered through a 0.22 μm PVDF membrane filter

[0431] 3.2.2. Lyophilisation Protocol

[0432] Filtrates were filled in 4.2 mL volumes into 20 mL clear Type glass vials and lyophilised, directly from the shelf, with the cycle shown in the following table:

TABLE-US-00029 Temperature Pressure Time Step Cycle stage (° C.) (mTorr) (min) 1 Load 25 n/a 0 2 Ramp 0 n/a 25 3 Ramp −45 n/a 225 4 Freezing −45 n/a 180 5 Hold −45 350 30 6 Ramp −15 350 60 7 Primary drying −15 350 2861 8 Ramp 30 20 112 9 Secondary drying 30 20 459 10 Finish 30 Vials stoppered to 722000 mTorr with (pure) nitrogen. Total cycle duration ~66 hours (~2.8 days)

[0433] 3.3 Sample Analysis

[0434] There was no significant difference in the appearance of the lyophilised plugs among batches 007 and 009/PAN/13. Lyophilised plugs appeared white/off-white, homogeneous and well formed.

[0435] Duplicate vials of each product were used for initial T=0 testing, a summary of the analytical results is presented below.

[0436] 3.3.1 Analysis after Reconstitution

[0437] 10 mL normal saline was added to a lyophilised sample, the vial was swirled and observed. The reconstitution time, reconstitution solution appearance, pH and purity (HPLC) were determined. The results are shown in the following table:

TABLE-US-00030 Recon- Reconstituted solution stitution 7056 7054X Batch time (s) Appearance pH (% area) (% area) 007/PAN/ 91 Clear, colourless, free from 3.3 99.58 0.10 13 visible particulates 009/PAN/ 81 Clear, colourless, free from 3.2 99.60 0.08 13 visible particulates

[0438] 3.3.2 Appearance

[0439] The appearance of the lyophilized samples was determined. The results are listed in the following table:

TABLE-US-00031 Sample details Appearance 007/ Initial (T = 0) Off white plug PAN/13 40° C./75% RH T = 1 m Off white plug with signs of shrinkage 55° C. T = 1 m Off white plug with signs of shrinkage 009/ Initial (T = 0) Off white plug PAN/13 40° C./75% RH T = 1 m Off white plug with signs of shrinkage 55° C. T = 1 m Off white plug with signs of shrinkage

[0440] 3.3.3 Moisture

[0441] The moisture content of the lyophilized samples was determined. The results are listed in the following table:

TABLE-US-00032 Mean moisture Sample details Vial 1 Vial 2 Vial 3 (% w/w) 007/PAN/13 Initial (T = 0) 0.00 0.00 0.00 40° C./75% RH T = 1 m 0.14 0.12 — 0.13 55° C. T = 1 m 0.24 0.32 — 0.28 009/PAN/13 Initial (T = 0) 0.00 0.00 0.00 40° C./75% RH T = 1 m 0.20 0.21 — 0.21 55° C. T = 1 m 0.35 0.38 — 0.37

[0442] 3.3.4 Reconstitution Time and pH of Reconstituted Solution

[0443] Each vial was reconstituted with 10 mL 0.9% saline. The results regarding reconstitution time and pH are listed in the following table.

TABLE-US-00033 Recon. time Sample details (seconds) pH 007/PAN/13 Initial (T = 0) 165* 3.25 40°C./75% RH T = 1 m  79  3.21 55° C. T = 1 m  62  3.28 009/PAN/13 Initial (T = 0)  95* 3.22 40°C./75% RH T = 1 m  59  3.21 55° C. T = 1 m  50  3.21

[0444] 3.3.5 Vial Content

[0445] The vial content for the samples at T=1 m 40° C./75% RH was determined after each vial was reconstituted with 10 mL 0.9% saline. The results are given in the following table:

TABLE-US-00034 Mean Recovery.sup.1 Sample details Vial 1 Vial 2 [7056] (mg/vial) (%) 007/PAN/13 Initial (T = 0) 48.6048 48.5855 48.595 — 40° C./75% RH T= 1 m 48.0353 47.9755 48.005  98.8 55° C. T = 1 m 47.9019 48.6901 48.296  99.4 009/PAN/13 Initial (T = 0) 48.9599 48.9696 48.965 — 40° C./75% RH T = 1 m 48.4752 49.4228 48.949 100.0 55° C. T = 1 m 49.0987 48.3462 48.722  99.5 .sup.1Recovery is calculated as a percentage of T = 0 result.

[0446] 3.3.6 Impurities

[0447] The impurities for the different formulations at T=1 m 40° C./75% RH were determined. The results are given in the following tables:

[0448] 007/PAN/13:

TABLE-US-00035 Initial (T = 0) 40° C./75% RH 55° C. RRT Name T = 1 m T = 1 m T = 1 m Impurity profile 0.36 n.a. N.D. N.D. <LOQ (area %) 0.41 n.a. <LOQ N.D. N.D. 0.46 n.a. <LOQ <LOQ <LOQ 0.51 n.a. 0.07 0.07 0.07 0.57 n.a. N.D. <LOQ <LOQ 0.59 7054X 0.10 0.16 0.43 0.63 n.a. <LOQ <LOQ <LOQ 0.67 n.a. <LOQ <LOQ <LOQ 0.70 n.a. <LOQ <LOQ <LOQ 0.88 n.a. <LOQ <LOQ <LOQ 0.92 n.a. 0.11 0.11 0.11 1.00 7056B 99.58 99.54 99.23 1.31 n.a. <LOQ <LOQ <LOQ 1.46 ONO N.D. N.D. <LOQ 1.73 n.a. <LOQ <LOQ <LOQ 1.77 n.a. <LOQ N.D. N.D. 1.79 n.a. <LOQ <LOQ <LOQ 1.84 n.a. <LOQ N.D. N.D. 1.86 n.a. N.D. <LOQ <LOQ Total imps* (area %) 0.3 0.3 0.6 *Sum of all impurities ≥0.03% by area Impurities are mean of 2 determinations n.d. = not detected

[0449] 009/PAN/13:

TABLE-US-00036 Initial 40° C./75% RH 55° C. RRT Name (T = 0) T = 1 m T = 1 m Impurity profile 0.27 n.a. N.D. N.D. <LOQ (area %) 0.36 n.a. N.D. N.D. <LOQ 0.41 n.a. <LOQ N.D. N.D. 0.46 n.a. <LOQ <LOQ <LOQ 0.51 n.a. 0.07 0.07 0.07 0.57 n.a. N.D. <LOQ <LOQ 0.59 7054X 0.08 0.12 0.38 0.63 n.a. <LOQ <LOQ <LOQ 0.67 n.a. <LOQ <LOQ <LOQ 0.70 n.a. <LOQ <LOQ <LOQ 0.88 n.a. <LOQ <LOQ <LOQ 0.92 n.a. 0.11 0.11 0.10 1.00 7056B 99.60 99.57 99.27 1.31 n.a. <LOQ <LOQ <LOQ 1.47 ONO N.D. N.D. <LOQ 1.73 n.a. <LOQ N.D. N.D. 1.75 n.a. N.D. <LOQ <LOQ 1.77 n.a. <LOQ N.D. N.D. 1.79 n.a. <LOQ <LOQ <LOQ 1.84 n.a. <LOQ N.D. N.D. 1.86 n.a. N.D. <LOQ <LOQ Total imps* (area %) 0.3 0.3 0.6 *Sum of all impurities ≥0.03% by area Impurities are mean of 2 determinations n.d. = not detected

[0450] 4. Further Formulations

[0451] 4.1 Parameters: [0452] Fill concentration of CNS 7056 base=12 mg/mL [0453] Vial size=20R (30 mm diameter) [0454] Fill solution volume 4.2 mL [0455] Approx 50 vials of each product to be prepared [0456] Also prepare placebos

[0457] 4.2 Formulation Compositions (Vial Equivalent)

TABLE-US-00037 Approx Ratio CNS 7056 Lactose CNS7056: (base Total Dextran Mono- Lactose equivalent) Excipient 40 hydrate Monohydrate 80 20 012/PAN/13 50 mg 440 mg 352 mg 88 mg 1:9   011/PAN/13 50 mg 330 mg 264 mg 66 mg 1:6   010/PAN/13 50 mg 220 mg 176 mg 44 mg 1:4.5

[0458] Predicted Tc of formulations from Thermal Assessments=−15° C.

[0459] 4.3 Parameters [0460] Freezing to −30° C. @ 0.2° C./min. Held [0461] Primary Drying −7° C. @ 700-750 mTorr [0462] Secondary Drying 30° C.

[0463] 4.4 Test Criteria for Analysis of Lyo Samples [0464] Appearance [0465] Recon time—addition of 10 mL saline [0466] Moisture [0467] Related substances [0468] Stability—1 m 55C+1, 3 m 40C/75% RH

[0469] 4.4.1 Appearance

[0470] The appearance of the lyophilized samples was determined. The results are listed in the following table:

TABLE-US-00038 Sample details Appearance 010/PAN/13 Initial (T = 0) Off white plug 011/PAN/13 Initial (T = 0) Off white plug with material on vial wall 012/PAN/13 Initial (T = 0) Off white plug

[0471] 4.4.2 Moisture

[0472] The moisture content of the lyophilized samples was determined. The results are listed in the following table:

TABLE-US-00039 Mean moisture Sample details Vial 1 Vial 2 Vial 3 (% w/w) 010/PAN/13 Initial (T = 0) 0.08 0.02 — 0.05 011/PAN/13 Initial (T = 0) 0.05 0.05 — 0.05 012/PAN/13 Initial (T = 0) 0.11 0.06 — 0.09

[0473] 4.4.3 Reconstitution Time and pH of Reconstituted Solution

[0474] Each vial was reconstituted with 10 mL 0.9% saline. The results regarding reconstitution time and pH are listed in the following table.

TABLE-US-00040 Recon. time Sample details (seconds) pH 010/PAN/13 Initial (T = 0) 48 3.23 40° C./75% RH T = 1 m — — 55° C. T = 1 m — — 011/PAN/13 Initial (T = 0) 63 3.20 40° C./75% RH T = 1 m — — 55° C. T = 1 m — — 012/PAN/13 Initial (T = 0) 64 3.23 40° C./75% RH T = 1 m — — 55° C. T = 1 m — —

[0475] 4.4.4 Vial Content

[0476] The vial content for the samples at T=1 m 40° C./75% RH was determined after each vial was reconstituted with 10 mL 0.9% saline. The results are given in the following table:

TABLE-US-00041 Mean Sample details Vial 1 Vial 2 [7056] (mg/vial) 010/PAN/13 Initial (T = 0) 49.8316 49.4836 49.658 011/PAN/13 Initial (T = 0) 49.9339 49.1202 49.527 012/PAN/13 Initial (T = 0) 49.0690 47.6608 48.365

[0477] 4.4.5 Impurities

[0478] The impurities for the different formulations at T=1 m 40° C./75% RH were determined. The results are given in the following tables:

[0479] 010/PAN/13:

TABLE-US-00042 Initial RRT Name (T = 0) Impurity profile 0.31 n.a. <LOQ (area %) 0.47 n.a. <LOQ 0.52 n.a. 0.07 0.57 n.a. <LOQ 0.59 7054X 0.10 0.65 n.a. <LOQ 0.68 n.a. <LOQ 0.72 n.a. <LOQ 0.89 n.a. <LOQ 0.93 n.a. 0.11 1.00 7056B 99.57 1.14 n.a. 0.03 1.31 n.a. <LOQ 1.74 n.a. <LOQ 1.80 n.a. <LOQ 1.86 n.a. <LOQ 2.00 n.a. <LOQ Total imps* (area %) 0.3 *Sum of all impurities ≥ 0.03% by area Impurities are mean of 2 determinations n.d. = not detected

[0480] 011/PAN/13:

TABLE-US-00043 Initial RRT Name (T = 0) Impurity profile 0.37 n.a. <LOQ (area %) 0.52 n.a. 0.07 0.59 7054X 0.10 0.68 n.a. <LOQ 0.89 n.a. <LOQ 0.93 n.a. 0.11 1.00 7056B 99.56 1.10 n.a. 0.03 1.74 n.a. <LOQ 1.80 n.a. <LOQ 1.86 n.a. <LOQ 2.00 n.a. <LOQ Total imps* (area %) 0.3 *Sum of all impurities ≥ 0.03% by area Impurities are mean of 2 determinations n.d. = not detected

[0481] 012/PAN/13:

TABLE-US-00044 Initial RRT Name (T = 0) Impurity profile 0.31 n.a. <LOQ (area %) 0.47 n.a. <LOQ 0.52 n.a. 0.07 0.59 7054X 0.08 0.64 n.a. <LOQ 0.68 n.a. <LOQ 0.71 n.a. <LOQ 0.89 n.a. <LOQ 0.93 n.a. 0.11 1.00 7056B 99.58 1.14 n.a. 0.03 1.31 n.a. <LOQ 1.74 n.a. <LOQ 1.80 n.a. <LOQ 1.86 n.a. <LOQ Total imps* (area %) 0.3 *Sum of all impurities ≥ 0.03% by area Impurities are mean of 2 determinations n.d. = not detected

[0482] 5. Further Formulations

[0483] 5.1 Parameters: [0484] Fill concentration of CNS 7056 base=12 mg/mL [0485] Vial size=20R (30 mm diameter) [0486] Fill solution volume 4.2 mL [0487] Approx 50 vials of each product to be prepared [0488] Also prepare placebos

[0489] 5.2 Formulation Compositions (Vial Equivalent)

TABLE-US-00045 Approx Ratio CNS CNS7056: 7056 Lactose Lactose (base Total Dextran Mono- Mono- equivalent) Excipient 40 hydrate hydrate 60 40 015/PAN/13 50 mg 440 mg 264 mg 176 mg 1:9 014/PAN/13 50 mg 330 mg 198 mg 132 mg 1:6 013/PAN/13 50 mg 220 mg 132 mg 88 mg 1:4.5

[0490] Predicted Tc of formulations from Thermal Assessments=−19° C.

[0491] 5.3 Parameters [0492] Freezing to −30° C. @ 0.2° C./min [0493] Primary Drying −15° C. @ 400 mTorr [0494] Secondary Drying 30° C.

[0495] 5.4 Test Criteria for Analysis of Lyo Samples [0496] Appearance [0497] Recon time—addition of 10 mL saline [0498] Moisture [0499] Related substances [0500] Stability—1 m 55C+1, 3 m 40C/75% RH

[0501] 5.4.1 Appearance

[0502] The appearance of the lyophilized samples was determined. The results are listed in the following table:

TABLE-US-00046 Sample details Appearance 013/PAN/13 Initial (T = 0) Off white plug some material on walls of vial 014/PAN/13 Initial (T = 0) Off white plug some material on walls of vial 015/PAN/13 Initial (T = 0) Off white plug some material on walls of vial

[0503] 5.4.2 Moisture

[0504] The moisture content of the lyophilized samples was determined. The results are listed in the following table:

TABLE-US-00047 Mean moisture Details Vial 1 Vial 2 Vial 3 (% w/w) 013/PAN/13 Initial (T = 0) 0.06 0.05 — 0.06 014/PAN/13 Initial (T = 0) 0.00 0.09 0.09 015/PAN/13 Initial (T = 0) 0.09 0.00 — 0.09

[0505] 5.4.3 Reconstitution Time and pH of Reconstituted Solution

[0506] Each vial was reconstituted with 10 mL 0.9% saline. The results regarding reconstitution time and pH are listed in the following table.

TABLE-US-00048 Recon. time Sample details (seconds) pH 013/PAN/13 Initial (T = 0) 39 3.21 014/PAN/13 Initial (T = 0) 35 3.22 015/PAN/13 Initial (T = 0) 43 3.24

[0507] 5.4.4 Vial Content

[0508] The vial content for the samples at T=1 m 40° C./75% RH was determined after each vial was reconstituted with 10 mL 0.9% saline. The results are given in the following table:

TABLE-US-00049 Mean Sample details Vial 1 Vial 2 [7056] (mg/vial) 013/PAN/13 Initial (T = 0) 48.1356 48.5325 48.334 014/PAN/13 Initial (T = 0) 49.9574 49.7535 49.855 015/PAN/13 Initial (T = 0) 48.3542 47.9459 48.150

[0509] 5.4.5 Impurities

[0510] The impurities for the different formulations at T=1 m 40° C./75% RH were determined. The results are given in the following tables:

[0511] 013/PAN/13:

TABLE-US-00050 Initial RRT Name (T = 0) Impurity profile 0.31 n.a. <LOQ (area %) 0.47 n.a. <LOQ 0.52 n.a. 0.07 0.59 7054X 0.09 0.64 n.a. <LOQ 0.68 n.a. <LOQ 0.71 n.a. <LOQ 0.89 n.a. <LOQ 0.93 n.a. 0.11 1.00 7056B 99.60 1.31 n.a. <LOQ 1.74 n.a. <LOQ 1.77 n.a. <LOQ 1.79 n.a. <LOQ 1.85 n.a. <LOQ Total imps* (area %) 0.3 *Sum of all impurities ≥ 0.03% by area Impurities are mean of 2 determinations n.d. = not detected

[0512] 014/PAN/13:

TABLE-US-00051 Initial RRT Name (T = 0) Impurity profile 0.31 n.a. <LOQ (area %) 0.47 n.a. <LOQ 0.51 n.a. 0.07 0.58 n.a. <LOQ 0.59 7054X 0.09 0.64 n.a. <LOQ 0.68 n.a. <LOQ 0.71 n.a. <LOQ 0.89 n.a. <LOQ 0.92 n.a. 0.11 1.00 7056B 99.61 1.31 n.a. <LOQ 1.74 n.a. <LOQ 1.79 n.a. <LOQ 1.85 n.a. <LOQ Total imps* (area %) 0.3 *Sum of all impurities ≥ 0.03% by area Impurities are mean of 2 determinations n.d. = not detected

[0513] 015/PAN/13:

TABLE-US-00052 Initial RRT Name (T = 0) Impurity profile 0.31 n.a. <LOQ (area %) 0.47 n.a. <LOQ 0.51 n.a. 0.07 0.56 n.a. <LOQ 0.59 7054X 0.08 0.64 n.a. <LOQ 0.68 n.a. <LOQ 0.71 n.a. <LOQ 0.89 n.a. <LOQ 0.92 n.a. 0.11 1.00 7056B 99.61 1.31 n.a. <LOQ 1.74 n.a. <LOQ 1.79 n.a. <LOQ 1.85 n.a. <LOQ Total imps* (area %) 0.3 *Sum of all impurities ≥ 0.03% by area Impurities are mean of 2 determinations n.d. = not detected

[0514] V. THERMAL ANALYSIS OF REMIMAZOLAM FORMULATIONS

[0515] 1. Purpose of the Study

[0516] In order to increase the lyophilisation temperature, the relative amount of dextran of the lactose:dextran mixture was increased and the critical temperature was determined by differential scanning calorimetry (DSC) and freeze drying microscopy (FDM), for CNS7056B formulations 1-6 as shown in the following Table.

TABLE-US-00053 T.sub.g′ by DSC T.sub.c by FDM Formulation (° C.) (° C.) 001 −13 −11 002 −23 −21 003 −28 −27 004 −24 −20 005 −29 −28 006 −29 −27

[0517] Critical temperatures were plotted with respect to dextran, relative to total formulation solute content and shown in FIG. 52.

[0518] From the linear equations (FIG. 52), for a given critical temperature, the theoretical dextran content of a CNS7056B formulation can be calculated. As shown in the following table, there was a good correlation between data generated for formulations containing lactose.

TABLE-US-00054 Target Tc Theoretical (° C.) 2.sup.nd excipient Technique dextran (%) −20 Lactose DSC 48.8 FDM 56.8 DSC mean 49 FDM mean 58 −17.5 Lactose DSC 60.3 FDM 69.1 DSC mean 60 FDM mean 70 −15 Lactose DSC 71.9 FDM 81.3 DSC mean 71 FDM mean 82

[0519] An alternative presentation of the data, expressing collapse temperature relative to the dextran:lactose ratio in each formulation is shown in FIG. 53. The Phase I/II sedation formulation was used to represent a formulation containing no dextran (zero on the abscissa). The collapse temperature onset for this formulation has been reported as −31° C.

[0520] Similarly, the linear equation from FIG. 53 may be used to calculate the theoretical dextran:lactose composition of CNS7056B formulations for given collapse temperatures as shown in the following table:

TABLE-US-00055 Theoretical excipient Example formulation Target Tc composition (%) API:lactose:dextran (° C.) Lactose Dextran (mg/vial) −20 45.4 54.6 50:200:240 −17.5 32.8 67.2 50:145:295 −15 20.2 79.8 50:90:350

FIGURE LEGENDS

[0521] FIG. 1: Excipients

[0522] FIG. 2: Active formulations

[0523] FIG. 3: Placebo formulations

[0524] FIG. 4: Formulation of hydrolysis degradant of remimazolam (REM) given in % after storage for 13 weeks at 25° C./60% relative humidity (RH) or 40° C./75% RH.

[0525] FIG. 5A-D: Crystallographic co-ordinates and other relevant data tabulated in the form of a SHELX File for Compound of formula (I) besylate Form 1 of WO2008/007071 A1

[0526] FIG. 6A-C: Crystallographic co-ordinates and other relevant data tabulated in the form of a SHELX File for Compound of formula (I) besylate Form 2 of WO2008/007071 A1.

[0527] FIG. 7A-B: Bond lengths for Compound of formula (I) besylate Form 1 of WO2008/007071 A1

[0528] FIG. 8A-C: Bond angles for Compound of formula (I) besylate Form 1 of WO2008/007071 A1

[0529] FIG. 9: Bond Lengths for Compound of formula (I) besylate Form 2 of WO2008/007071 A1

[0530] FIG. 10: Bond angles for Compound of formula (I) besylate Form 2 of WO2008/007071 A1

[0531] FIG. 11: Stability data for lot A01P310

[0532] FIG. 12: Stability data for lot A01P310, continued

[0533] FIG. 13: Accelerated stability data for lot A01 P310

[0534] FIG. 14: Accelerated stability data for lot A01 P310, continued

[0535] FIG. 15: Long term stability data for lot P310-01

[0536] FIG. 16: Long term stability data for lot P310-01, continued

[0537] FIG. 17: Accelerated stability data for lot P310-01

[0538] FIG. 18: Accelerated stability data for lot P310-01, continued

[0539] FIG. 19: Long term stability data for lot 026CNS27

[0540] FIG. 20: Long term stability data for lot 026CNS27, continued

[0541] FIG. 21: Accelerated stability data for lot 026CNS27

[0542] FIG. 22: Accelerated stability data for lot 026CNS27, continued

[0543] FIG. 23: Long term stability data for lot G384

[0544] FIG. 24: Long term stability data for lot G384, continued

[0545] FIG. 25: Accelerated stability data for lot G384

[0546] FIG. 26: Accelerated stability data for lot G384, continued

[0547] FIG. 27: Long term stability data for lot P02308

[0548] FIG. 28: Long term stability data for lot P02308, continued

[0549] FIG. 29: Accelerated stability data for lot P02308

[0550] FIG. 30: Accelerated stability data for lot P02308, continued

[0551] FIG. 31: Long term stability data for lot 25CNS27

[0552] FIG. 32: Long term stability data for lot 25CNS27, continued

[0553] FIG. 33: Long term stability data for lot 25CNS27, continued

[0554] FIG. 34: Accelerated stability data for lot 25CNS27

[0555] FIG. 35: Accelerated stability data for lot 25CNS27, continued

[0556] FIG. 36: Accelerated stability data for lot 25CNS27, continued

[0557] FIG. 37: Long term stability data (T=36M) for lot P02308

[0558] FIG. 38: Long term stability data (T=36M) for lot P02308, continued

[0559] FIG. 39: Raman spectra of each component in a lyophilized formulation: each rectangle range showing distinctive peak(s) of crystalline and lyophilized CNS7056B (L) and lyophilized lactose (R).

[0560] FIG. 40: Raman spectra of each component in a lyophilized formulation of CNS7056B in lactose: crystalline CNS7056B (top), lyophilized (amorphous) CNS7056B (middle) and lyophilized lactose (amorphous) (bottom).

[0561] FIG. 41: Representative Raman spectra of crystalline CNS7056B (top 3) selected within the Raman mapping area of the lyophilized formulation and pure crystalline (Form 1) CNS7056B (bottom) as a reference.

[0562] FIG. 42: Table summarizing the results for the stability study of the Lots 12PM529-8-1, 12PM529-8-2, 12PM529-9-1 and PM0232/12 at initial time point t=0

[0563] FIG. 43: Table summarizing the results for the stability study of the 12PM529-8-2, 12PM529-9-1 and PM0232/12 at time point t=4 weeks

[0564] FIG. 44: Table summarizing the results for the stability study of the Lots 12PM529-8-1, 12PM529-8-2, 12PM529-9-1 and PM0232/12 at time point t=13 weeks

[0565] FIG. 45 Table summarizing the results for the stability study of the Lot L10R5

[0566] FIG. 46 Table summarizing the results for the stability study of the Lot L10R10

[0567] FIG. 47 Table summarizing the results for the stability study of the Lot L10R5S87

[0568] FIG. 48 Table summarizing the results for the stability study of the Lot L20R5

[0569] FIG. 49 Table summarizing the results for the stability study of the Lots L4M110R5 and L4M110R10

[0570] FIG. 50 Table summarizing the results for the stability study of the Lot L2M110R5

[0571] FIG. 51 Table summarizing the results for the stability study of the Lot L2M110R10

[0572] FIG. 52 Critical temperature as a function of dextran content for CNS7057B:lactose:dextran formulations

[0573] FIG. 53 Collapse temperature relative to the dextran: lactose ratio for CNS7056B formulations