Formulations comprising u0126

Abstract

The invention provides pharmaceutical preparations comprising U0126 (1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene), or a pharmaceutically acceptable salt thereof, and sorbitol. The pharmaceutical preparations of the invention possess characteristics including stability, solubility, and a long shelf-life. The invention also provides solutions comprising the pharmaceutical preparation, a method of treating ischemic damage comprising administration of the pharmaceutical preparation, and a kit of parts comprising the pharmaceutical preparation and a physiologically acceptable solution that is a solvent for the pharmaceutical preparation. The invention further provides a method for preparing the pharmaceutical preparation.

Description

[0001] FORMULATIONS COMPRISING U0126

[0002] Field of the invention

[0003] The present invention is directed to pharmaceutical preparations comprising compound (I) or pharmaceutically acceptable salts or solvates thereof, methods fortheir preparation, compositions comprising the lyophilised pharmaceutical preparations, and their use in the treatment of ischemic damage.

[0004] Background

[0005] Stroke is a leading cause of death, neurological damage, and a major cause of long-term disabilities. Ischemic strokes are caused by interruption of the blood supply to the brain, while haemorrhagic strokes result from the rupture of a blood vessel or an abnormal vascular structure. Ischemic stroke is the most common type of stroke (about 87% of strokes), while haemorrhagic stroke is responsible for more deaths and disability.

[0006] Subarachnoid haemorrhage (SAH) is caused by bleeding in the space that surrounds the brain. Most often, it occurs when a weak area in a blood vessel (aneurysm) on the surface of the brain bursts and leaks. The blood then builds up around the brain and inside the skull, increasing pressure on the brain. SAH is difficult to treat and can be fatal. It has been discovered that the damage is associated with early enhanced expression of contractile receptors via protein kinase induced activation (Edvinsson and Povlsen, J. Cereb. Blood Flow Metab., 2011 , 31 (7), 1554-1571). The damage can be mitigated following early blockade of this pathway (Edvinsson and Krause, Transl. Stroke Res., 2024, DOI: 10.1007 / s12975-024-01234-z).

[0007] Patients who initially survive the damage, whether it is from a large blood clot or the rupture of an aneurysm, may experience a devastating turn for the worse days later and die or suffer permanent neurologic deficits (Macdonald, Nat. Rev. Neurol., 2014, 10, 44-58). This is attributed to secondary cerebral ischaemia (also called delayed cerebral ischemia (DCI)) following aneurysmal subarachnoid haemorrhage (aSAH), caused byvasospasm- constriction of blood vessels and restriction of blood flow in the brain. Blood supply to parts of the brain can become reduced to a dangerous extent, thereby disrupting the normal functions of the brain. Secondary cerebral ischemia can be mitigated by pharmacological intervention with the calcium channel blocker nimodipine. The dosing of nimodipine recommended by the UK’s National Institute for Health and Care Excellence is 60 mg every 4 hours, to be started within 4 days of aneurysmal subarachnoid haemorrhage, and continued for 21 days. From preliminary, pre-clinical and clinical testing the compound U0126 has shown promise for the treatment of delayed cerebral ischemia typically observed in haemorrhagic stroke, particularly SAH. U0126 has the formula:

[0008]

[0009] U0126 is known to be an inhibitor of MEK1 / 2, and has been proposed for use in the treatment of ischemic disorders, such as subarachnoid haemorrhage. Duncia etal., Bioorg. & Med. Chem. Lett., 1998, 8, 2839-2844 describes the MEK inhibitor activity of U0126. EP2139512 discloses the use of U0126for administration from 1 to 6 hours after onset of ischemic damage. Ahnstedt etal., J. Cereb. Blood Flow Metab., 2015, 35, 454-460 discloses that U0126 improves long-term neurologic outcome after stroke in female rats.

[0010] U0126 is a difficult compound to formulate and administer, being highly insoluble in most common solvents, highly labile in solution due to intramolecular reactions, and sensitive to both acid and base (see for example Duncia etal., Bioorg. & Med. Chem. Lett., 1998, 8, 2839-2844). Formulations which are soluble in physiological fluids, and which are hence able to transport the active material directly to the brain for the treatment of ischemic disorders, are required for the treatment of ischemic disorders. U0126 is known to be soluble in dimethylsulfide (DMSO), but a high concentration of this solvent is not suitable for use in liquid formulations. Furthermore, the stability of liquid formulations of U0126 at room temperature is poor.

[0011] Consequently, there is a need for alternative pharmaceutical preparations of U0126 having sufficient solubility in physiologically acceptable solutions, and sufficient stability for practical use as a medicament. Summary of the invention

[0012] The invention provides a pharmaceutical preparation comprising compound (I):

[0013]

[0014] or a pharmaceutically acceptable salt or solvate thereof, and sorbitol.

[0015] The invention also provides a solution comprising a pharmaceutical preparation comprising compound (I) and sorbitol.

[0016] The invention also provides a kit of parts comprising a pharmaceutical preparation comprising compound (I) and sorbitol, and a physiologically acceptable solution that is a suitable solvent for compound (I).

[0017] The invention also provides a pharmaceutical preparation comprising compound (I) or a pharmaceutically acceptable salt or solvate thereof, and sorbitol, for use as a medicament. The invention also provides a pharmaceutical preparation comprising compound (I) or a pharmaceutically acceptable salt or solvate thereof, and sorbitol, for use in the treatment of ischemic damage in a human patient.

[0018] The invention also provides a method for the treatment of ischemic damage, comprising administering the solution comprising the pharmaceutical preparation to a human patient. The invention also provides a pharmaceutical preparation comprising compound (I) or a pharmaceutically acceptable salt or solvate thereof, and sorbitol, for the manufacture of a medicament for the treatment of ischemic damage.

[0019] The invention also provides a method for preparing a dry (for example lyophilized) pharmaceutical preparation of compound (I), comprising:

[0020] a. Providing a solution of compound (I), or a pharmaceutically acceptable salt or solvate thereof, in an organic solvent;

[0021] b. Providing a solution of sorbitol in water;

[0022] c. Combining the solution of compound (I) in organic solvent and the aqueous solution of sorbitol to form a miscible mixture; d. Optionally concentrating the mixture by removal of solvent;

[0023] e. Subjecting the solution of compound (I) (optionally concentrated in step (d)) to drying (for example by lyophilisation).

[0024] Brief description of drawings

[0025] Figure 1: HPLC results from stability study of lyophilised preparations of compound (I) prepared using different excipients (ambient temperature).

[0026] Figure 2: HPLC results from stability study of lyophilised preparations of compound (I) prepared using different excipients (temperature 40°C).

[0027] Figure 3:1H NMR results from stability study of lyophilised preparations of compound (I) prepared using sorbitol (ambient temperature, day 1).

[0028] Figure 4:1H NMR results from stability study of lyophilised preparations of compound (I) prepared using sorbitol (ambient temperature, day 85).

[0029] Figure 5: HPLC results from stability study of compound (I) and reconstituted solution of lyophilised preparation of compound (I) prepared with sorbitol (ambient temperature).

[0030] Figure 6: HPLC analysis of compound (I) atT=0.

[0031] Figure 7: HPLC analysis of compound (I) after stirring for 4 h in Kolliphor / H2O (3:1).

[0032] Detailed description

[0033] The compound of formula (I) is known as U0126. Its chemical name is 1 ,4-diamino-2,3-dicyano-1 ,4-bis(o-aminophenylmercapto)butadiene. U0126 is an MEK1 / 2 inhibitor which has been classified as an orphan drug by the US Food and Drug Administration (593217) and the European Medicines Agency (EU / 3 / 17 / 1935) for the treatment of brain ischemia associated with severe subarachnoid haemorrhage. The mechanism of action of U0126 is to counter-act the delayed vasoconstriction that occurs in cerebral arteries and arterioles in conditions such as subarachnoid haemorrhage and ischemia, allowing circulation of oxygen and other essential nutrients to brain cells, thus preventing ischemic brain damage.

[0034] It has been well documented that U0126 suffers from solution instability. The inventors have found that a dry (for example lyophilised) formulation of compound (I) also comprising sorbitol as an excipient possesses excellent characteristics including stability, solubility, and a long shelf-life. The lyophilised preparation of the invention shows enhanced stability and an improved shelf life compared with the compound alone. Furthermore, the very low dose of the active compound used for treating stroke and ischemic damage makes preparation of individual sterile doses extremely challenging. The preparation of a dry (for example lyophilised) formulation of compound (I) comprising a large amount of the benign ingredient sorbitol makes manufacturing possible at scale.

[0035] The invention provides a pharmaceutical preparation comprising compound (I) or a pharmaceutically acceptable salt or solvate thereof, and sorbitol, that is dry. As used herein, “dry” means essentially free from water, for example comprising less than 5% w / w water. In a preferred embodiment, the pharmaceutical preparation comprises less than 1% w / wwater, for example less than 0.5% w / wwater, or less than 0.1% w / wwater.

[0036] The invention further provides a lyophilised pharmaceutical preparation comprising compound (I) or a pharmaceutically acceptable salt or solvate thereof, and sorbitol. The inventors have found that lyophilised preparations of compound (I) with sorbitol exhibit enhanced stability when compared with lyophilised preparations of compound (I) without an excipient, and compared with lyophilised preparations of compound (I) with other common physiologically acceptable excipients.

[0037] Pharmaceutical preparations

[0038] In an embodiment of the invention, the dry (for example lyophilised) preparation comprises compound (I) and sorbitol at a molar ratio (mol / mol) of between 1 :2 and 1 :500.

[0039] In a preferred embodiment, the molar ratio (mol / mol) between compound (I) and sorbitol is from 1:2 to 1 :250, for example from 1:2 to 1 :100, from 1:5 to 1 :75, from 1:10 to 1 :50, or from 1:20 to 1:40.

[0040] In a preferred embodiment, the molar ratio (mol / mol) between compound (I) and the sorbitol is selected from 1:2, 1:5, 1:10, 1:20, 1:25, 1:30, 1:40, 1:50, 1:75, 1:100, 1:250 or 1:500.

[0041] In an especially preferred embodiment, the molar ratio (mol / mol) between compound (I) and the sorbitol is 1 :30. In another embodiment of the invention, the dry (for example lyophilised) preparation comprises compound (I) and sorbitol at a molar ratio (mol / mol) of between 1 :1 and 1 :500. In a preferred embodiment, the molar ratio (mol / mol) between compound (I) and sorbitol is from 1:1 to 1:30, for example from 1:1 to 1:20, from 1:1 to 1:10, orfrom 1:10to 1:20.

[0042] In another preferred embodiment, the molar ratio (mol / mol) between compound (I) and the sorbitol is selected from 1 :1 , 1 :2, 1 :5, 1 :10, 1 :20, 1 :25 or 1 :30. More preferably, the molar ratio (mol / mol) between compound (I) and the sorbitol is selected from 1 :2, 1 :5 or 1 :10.

[0043] In an especially preferred embodiment, the molar ratio (mol / mol) between compound (I) and the sorbitol is 1 :5.

[0044] In an embodiment of the invention, the dry (for example lyophilised) preparation comprises compound (I) and sorbitol at a weight ratio (w / w) of between 1 :0.96 and 1 :239.39.

[0045] In a preferred embodiment, the weight ratio (w / w) between compound (I) and sorbitol is from 1 :0.96 to 1 :119.69, for example from 1 :0.96 to 1 :47.88, from 1 :2.39 to 1 :35.91 , from 1 :4.79 to 1:23.94, orfrom 1:9.58 to 1:19.15.

[0046] In a preferred embodiment, the weight ratio (w / w) between compound (I) and the sorbitol is selected from 1:0.96, 1:2.39, 1:4.79, 1:9.58, 1:11.97, 1:14.36, 1:19.15, 1:23.94, 1:35.91, 1:47.88, 1:119.69 or 1:239.39.

[0047] In an especially preferred embodiment, the weight ratio (w / w) between compound (I) and the sorbitol is 1 :14.36.

[0048] In another embodiment of the invention, the dry (for example lyophilised) preparation comprises compound (I) and sorbitol at a weight ratio (w / w) of between 1 :0.48 and 1 :239.39. In another preferred embodiment, the weight ratio (w / w) between compound (I) and the sorbitol is from 1 :0.48 to 1 :14.36, for example from 1 :0.48 to 1 :9.58, from 1 :0.48 to 1 :4.79, or from 1 :4.79 to 1:9.58.

[0049] In another preferred embodiment, the weight ratio (w / w) between compound (I) and the sorbitol is selected from 1 :0.48, 1 :0.96, 1 :2.39, 1 :4.79, 1 :9.58, 1:11.97, or 1 :14.36. More preferably, the weight ratio (w / w) between compound (I) and the sorbitol is selected from 1 :0.96, 1 :2.39, or 1:4.79. In an especially preferred embodiment, the weight ratio (w / w) between compound (I) and the sorbitol is 1 :2.39.

[0050] In an embodiment of the invention, the dry (for example lyophilised) preparation comprises compound (I) ethanolate and sorbitol at a weight ratio (w / w) of between 1 :0.85 and 1 :213.53. In a preferred embodiment, the weight ratio (w / w) between compound (I) ethanolate and sorbitol is from 1 :0.85 to 1:106.77, for example from 1:0.85 to 1 :42.71, from 1 :2.14to 1:32.03, from 1:4.27 to 1:21.35, orfrom 1:8.54 to 1:17.08.

[0051] In a preferred embodiment, the weight ratio (w / w) between compound (I) ethanolate and the sorbitol is selected from 1:0.85, 1:2.14, 1:4.27, 1:8.54, 1:10.68, 1:12.81, 1:17.08, 1:21.35, 1:32.03, 1:42.71, 1:106.77 or 1:213.53.

[0052] In an especially preferred embodiment, the weight ratio (w / w) between compound (I) ethanolate and the sorbitol is 1 : 12.81.

[0053] In another embodiment of the invention, the dry (for example lyophilised) preparation comprises compound (I) ethanolate and sorbitol at a weight ratio (w / w) of between 1 :0.43 and 1:213.53.

[0054] In a preferred embodiment, the weight ratio (w / w) between compound (I) ethanolate and sorbitol is from 1 :0.43 to 1:12.81 , for example from 1 :0.43 to 1 :8.54, from 1 :0.43 to 1 :4.27, or from 1:4.27 to 1:8.54.

[0055] In another preferred embodiment, the weight ratio (w / w) between compound (I) ethanolate and the sorbitol is selected from 1:0.43, 1:0.85, 1:2.14, 1:4.27, 1:8.54, 1 :10.68 or 1 :12.81. More preferably, the weight ratio (w / w) between compound (I) ethanolate and the sorbitol is selected from 1:0.85, 1:2.14 or 1:4.27.

[0056] In an especially preferred embodiment, the weight ratio (w / w) between compound (I) ethanolate and the sorbitol is 1 :2.14. In an embodiment of the invention, the dry (for example lyophilised) pharmaceutical preparation is a bulk preparation. In such an embodiment, the dry (for example lyophilised) pharmaceutical preparation comprises 250-1000 mg of compound (I). In a preferred embodiment, the pharmaceutical preparation comprises 300-700 mg of compound (I), for example 400-600 mg of compound (I). In an especially preferred embodiment, the pharmaceutical preparation of the invention is lyophilised and the lyophilised pharmaceutical preparation comprises 500 mg of compound (I).

[0057] In such an embodiment of the invention, the dry (for example lyophilised) pharmaceutical preparation comprises 500 mg of compound (I) and 7182 mg of sorbitol. Alternatively, the dry (for example lyophilised) pharmaceutical preparation comprises 500 mg of compound (I) and 1197 mg of sorbitol.

[0058] In another embodiment of the invention, dry (for example lyophilised) pharmaceutical preparation comprises 500 mg of compound (I) ethanolate and 6406 mg of sorbitol.

[0059] Alternatively, the dry (for example lyophilised) pharmaceutical preparation comprises 500 mg of compound (I) ethanolate and 1067 mg of sorbitol.

[0060] In an alternative embodiment, the dry (for example lyophilised) pharmaceutical preparation comprises a single dose of compound (I). In such an embodiment of the invention, the pharmaceutical preparation comprises 10-100 pg of compound (I), In a preferred embodiment, the pharmaceutical preparation comprises 10-50 pg of compound (I), for example 20-40 pg of compound (I). In an especially preferred embodiment, the pharmaceutical preparation comprises 28.5 pg of compound (I). In another preferred embodiment, the pharmaceutical preparation comprises 30-100 pg of compound (I), for example 50-75 pg of compound (I). In an especially preferred embodiment, the pharmaceutical preparation comprises 57 pg of compound (I) and 818.71 pg sorbitol. Alternatively, the pharmaceutical preparation comprises 57 pg of compound (I) ethanolate and 730.29 pg sorbitol.

[0061] In another embodiment of the invention, the dry (for example lyophilised) pharmaceutical preparation comprises a single dose of compound (I), comprising 1-100 mg of compound (I). In a preferred embodiment, the pharmaceutical preparation comprises 2-40 mg of compound (I), for example 2-20 mg of compound (I). In an especially preferred embodiment, the pharmaceutical preparation comprises 5-15 mg of compound (I). In an especially preferred embodiment, the pharmaceutical preparation comprises 10 mg of compound (I) and 23.94 mg sorbitol. Alternatively, the pharmaceutical preparation comprises 10 mg of compound (I) ethanolate and 21.35 mg sorbitol.

[0062] Salts and solvates

[0063] Pharmaceutical preparations of salt forms of compound (I) also form part of the invention. In some embodiments the salt is a salt of compound (I).

[0064] Salts of compounds of the invention include those which are pharmaceutically acceptable, i.e. which are suitable for use in medicine. Suitable salts according to the invention include those formed with organic or inorganic acids or bases. Pharmaceutically acceptable acid addition salts include those formed with hydrochloric, hydrobromic, sulphuric, nitric, citric, tartaric, acetic, phosphoric, lactic, pyruvic, acetic, trifluoroacetic, succinic, perchloric, fumaric, maleic, glycolic, salicylic, oxaloacetic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic, and isethionic acids. Other acids such as oxalic acid may be useful as intermediates in obtaining the compounds of the invention in final form.

[0065] Pharmaceutically acceptable salts with bases include ammonium salts, alkali metal salts, for example potassium and sodium salts, alkaline earth metal salts, for example calcium and magnesium salts, and salts with organic bases, for example dicyclohexylamine and N-methyl-D-glucomine.

[0066] Compound (I) does not readily form salts. In a preferred embodiment of the invention, the pharmaceutical preparation comprises compound (I) in a non-salt form.

[0067] Those skilled in the art of organic and / or medicinal chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. Such complexes are known as "solvates". For example, a complex with water is known as a "hydrate". The invention encompasses solvates of the compound (I), and solvates of salts of compound (I). The invention also encompasses hemisolvates of compound (I) and salts of compound (I).

[0068] Those skilled in the art of organic and / or medicinal chemistry will also appreciate than many organic compounds can exist in different forms, including as amorphous material and / or in one or more crystalline forms. Different physical forms of organic compounds are known as polymorphs. The invention also encompasses pharmaceutical preparations comprising all such different physical forms of compound (I), as well as different physical forms of its salts. In a preferred embodiment, the pharmaceutical preparation comprises compound (I) ethanolate. In another preferred embodiment, the pharmaceutical preparation comprises the ethanolate hemisolvate of compound (I).

[0069] Solutions comprising the pharmaceutical preparations

[0070] Before use, the dry (for example lyophilised) pharmaceutical preparation comprising compound (I), or a pharmaceutically acceptable salt or solvate thereof, and sorbitol, is diluted in a solvent, for example a physiologically acceptable solution, in order to obtain a solution that can be administered to a patient.

[0071] The dry (for example lyophilised) preparation of compound (I), for example in the form of a powder, may be dissolved first in a solvent, surfactant, or emulsifier, for example a polyoxyethylene castor oil such as Kolliphor, followed by the addition of a physiologically acceptable solution, for example artificial cerebrospinal fluid (aCSF). Alternatively, the dry (for example lyophilised) preparation of compound (I) may be first dissolved in the physiologically acceptable solution such as aCSF, followed by the addition of a solvent, surfactant, or emulsifier, such as polyoxyethylene castor oil. The preparation of compound (I) may also be dissolved in a premixed mixture of a physiologically acceptable solution such as aCSF and a solvent such as polyoxyethylene castor oil.

[0072] In an alternative embodiment, the dry (for example lyophilised) preparation of compound (I), for example in the form of a powder, may be dissolved in a physiologically acceptable solution, for example artificial cerebrospinal fluid (aCSF).

[0073] In an embodiment of the invention, there is provided a solution prepared from a preparation of compound (I) according to the invention.

[0074] In an embodiment, the solution comprises a preparation of compound (I), or a pharmaceutically acceptable salt or solvate thereof, and the solution is isotonic.

[0075] In a preferred embodiment, the solution comprises sorbitol at a concentration of 2-10 wt%, preferably a concentration of 3-8 wt% most preferably a concentration of 5 wt%. In another embodiment of the invention, the solution comprises a preparation of compound (I) of the invention and a physiologically acceptable solution. Examples of physiologically acceptable solutions include a NaCI solution, such as 0.9 wt% NaCI (normal saline), or Ringer’s solution.

[0076] In certain embodiments, the physiologically acceptable solution is artificial cerebrospinal fluid (aCSF).

[0077] “Artificial cerebrospinal fluid” is a term of art, and aCSF is widely used in research and in medicine, often as an improvement over the use of sterile saline. The composition of aCSF is designed to mirror as closely as possible the composition of human cerebrospinal fluid. Such fluids are sterile aqueous solutions of electrolytes including sodium, potassium, calcium and magnesium cations, togetherwith anions selected from phosphate, sulfate, chloride and bicarbonate. Sugars such as glucose or dextrose may also be included. Atypical aCSF comprises, for example, Na+: between 130 mM and 170 mM, K+: between 2 mM and 5 mM, Ca2+: between 1 mM and 2.5 mM, Mg2+: between 0.5 mM and 2.5 mM. Suitably, the aCSF comprises Na+: between 140 mM and 160 mM, K+: between 2.5 mM and 4.5 mM, Ca2+: between 1 mM and 2 mM, Mg2+: between 0.5 mM and 1.5 mM. More suitably, the aCSF comprises Na+: between 145 mM and 155 mM, K+: between 2.5 mM and 4 mM, Ca2+: between 1 mM and 1.5 mM, Mg2+: between 0.5 mM and 1.3 mM. Most suitably, the aCSF comprises Na+: about 150 mM, K+: between 3 mM and 4 mM, Ca2+: between 1.2 mM and 1.5 mM, Mg2+: between 0.7 mM and 1.3 mM.

[0078] Counterions included in the aCSF may be, for example, phosphate, between 0.3 mM and 1.5 mM, chloride, between 130 mM and 165 mM, and / or bicarbonate, between 20 mM and 30 mM. In certain embodiments, the aCSF comprises Na+: between 140 mM and 160 mM, K+: between 2.5 mM and 4.5 mM, Ca2+: between 1 mM and 2 mM, Mg2+: between 0.5 mM and 1.5 mM, P: between 0.3 mM and 1.5 mM, and Cl’: between 130 mM and 165 mM. Suitably, the aCSF comprises Na+: between 145 mM and 155 mM, K+: between 2.5 mM and 4 mM, Ca2+: between 1 mM and 1.5 mM, Mg2+: between 0.5 mM and 1.5 mM, P: between 0.4 mM and 1.2 mM, and Cl’: between 130 mM and 155 mM. More suitably, the aCSF comprises Na+: between 145 mM and 155 mM, K+: between 3 mM and 4 mM, Ca2+: between 1 mM and 1.5 mM, Mg2+: between 0.7 mM and 1.3 mM, P: between 0.4 mM and 1.1 mM, and Cl’: between 145 mM and 155 mM. One suitable aCSF comprises: Na+: 150 mM, K+: 3 mM, Ca2+: 1.4 mM, Mg2+: 0.8 mM, P: 1.0 mM, and Cl": 155 mM.

[0079] In other embodiments, the aCSF comprises, per 500 ml of artificial cerebrospinal fluid:

[0080] • between 3500 and 3800 mg sodium chloride;

[0081] • between 900 and 1000 mg sodium bicarbonate;

[0082] • between 350 mg and 450 mg dextrose;

[0083] • between 120 mg and 180 mg magnesium sulfate.7H2O;

[0084] • between 120 mg and 180 mg potassium chloride;

[0085] • between 80 mg and 120 mg calcium chloride.2H2O;

[0086] • between 45 mg and 60 mg sodium phosphate, dibasic, anhydrous; and

[0087] • balance distilled water.

[0088] In some embodiments, the aCSF comprises, per 500 ml of artificial cerebrospinal fluid:

[0089] • between 3600 and 3700 mg sodium chloride;

[0090] • between 930 and 990 mg sodium bicarbonate;

[0091] • between 375 mg and 425 mg dextrose;

[0092] • between 140 mg and 160 mg magnesium sulfate.7H2O;

[0093] • between 140 mg and 160 mg potassium chloride;

[0094] • between 90 mg and 110 mg calcium chloride.2H2O;

[0095] • between 50 mg and 55 mg sodium phosphate, dibasic, anhydrous; and

[0096] • balance distilled water.

[0097] In some embodiments, the aCSF comprises, per 500 ml of artificial cerebrospinal fluid:

[0098] • between 3650 and 3690 mg sodium chloride;

[0099] • between 950 and 970 mg sodium bicarbonate;

[0100] • between 390 mg and 410 mg dextrose;

[0101] • between 145 mg and 155 mg magnesium sulfate.7H2O;

[0102] • between 145 mg and 155 mg potassium chloride;

[0103] • between 95 mg and 105 mg calcium chloride.2H2O;

[0104] • between 50 mg and 55 mg sodium phosphate, dibasic, anhydrous; and

[0105] • balance distilled water. A particularly preferred aCSF consists of, per 500 ml of artificial cerebrospinal fluid:

[0106] • 3670 mg sodium chloride;

[0107] • 960 mg sodium bicarbonate;

[0108] • 400 mg dextrose;

[0109] • 150 mg magnesium sulfate.7H2O;

[0110] • 150 mg potassium chloride;

[0111] • 100 mg calcium chloride.2H2O;

[0112] • 53 mg sodium phosphate, dibasic, anhydrous; and

[0113] • balance distilled water.

[0114] In some embodiments, the aCSF consists of, per ml of artificial cerebrospinal fluid:

[0115] • 6.0 to 8.0 mg sodium chloride;

[0116] • 0.2 to 0.4 mg potassium chloride;

[0117] • 0.15 to 0.25 mg calcium chloride.2H2O;

[0118] • 0.05 to 0.15 mg magnesium chloride hexahydrate;

[0119] • 1.2 to 2.3 mg sodium hydrogen carbonate;

[0120] • 0.05 to 0.15 mg disodium phosphate dihydrate;

[0121] • Ca 4.5 mg Hydrochloric acid as 1 M solution; and

[0122] • balance distilled water.

[0123] A more preferred aCSF consists of, per ml of artificial cerebrospinal fluid:

[0124] • 6.5 to 7.5 mg sodium chloride;

[0125] • 0.25 to 0.35 mg potassium chloride;

[0126] • 0.15 to 0.25 mg calcium chloride.2H2O;

[0127] • 0.05 to 0.15 mg magnesium chloride hexahydrate;

[0128] • 1.5 to 2.0 mg sodium hydrogen carbonate;

[0129] • 0.05 to 0.15 mg disodium phosphate dihydrate;

[0130] • Ca 4.5 mg Hydrochloric acid as 1 M solution; and

[0131] • balance distilled water. For example, the aCSF consists of, per ml of artificial cerebrospinal fluid:

[0132] • 7.2 mg sodium chloride;

[0133] • 0.3 mg potassium chloride;

[0134] • 0.2 mg calcium chloride.2H2O;

[0135] • 0.1 mg magnesium chloride hexahydrate;

[0136] • 1.8 mg sodium hydrogen carbonate;

[0137] • 0.1 mg disodium phosphate dihydrate

[0138] • Ca 4.5 mg Hydrochloric acid as 1 M solution; and

[0139] • balance distilled water.

[0140] In an embodiment of the invention, the solution comprising a pharmaceutical preparation of compound (I) according to the invention and a physiologically acceptable solution, further comprises polyoxyethylene castor oil. Polyoxyethylates of castor oil are condensates of ethylene oxide with castor oil, and some are commercially available. Such polyoxyethylene castor oil derivatives may for example be prepared by reacting castor oil with ethylene oxide, for example in a molar ratio of 1 :30 to 1 :40, for example 1 :35. Such products are referred to as Macrogolylycerol ricinoleate, PEG-35 castor oil, Polyoxyl 35 hydrogenated castor oil, and Polyoxyl-35 castor oil. For example, polyoxyl 35 castor oil, for example as available under the trade name Kolliphor® EL, previously known as “Cremophor EL” (Trade Mark), or Kolliphor® HS 15, also called Solutol® HS 15.

[0141] The solution of the invention may comprise the polyoxyethylene castor oil at a concentration of 0.2 to 5.0% (v / v), for example about 0.2 to 1.0% (v / v). For example, the solution may comprise the polyoxyethylene castor oil at a concentration of about 0.3 to about 0.7% (v / v), for example about 0.5% (v / v). For example, it may comprise polyoxyl 35 castor oil at a concentration of about 0.3 to about 0.7% (v / v), for example about 0.5% (v / v).

[0142] In a preferred embodiment, the solution comprises polyoxyethylene castor oil and aCSF. In a more preferred embodiment, the composition comprises polyoxyethylene castor oil and aCSF in a volume ratio of 3:1 polyoxyethylene castor oil / aCSF.

[0143] The invention therefore provides a solution that comprises per ml: 9.5 to 76.1 pgcompound (I);

[0144] 90.25 to 1522 g sorbitol; for example 137.75 to 1103.45 pg sorbitol; 3.0 to 7.0 mg polyoxyethylene castor oil

[0145] 6.0 to 8.0 mg sodium chloride;

[0146] 0.2 to 0.4 mg potassium chloride;

[0147] 0.15 to 0.25 mg calcium chloride.2H2O;

[0148] 0.05 to 0.15 mg magnesium chloride hexahydrate;

[0149] 1.2 to 2.3 mg sodium hydrogen carbonate;

[0150] 0.05 to 0.15 mg disodium phosphate dihydrate;

[0151] ca. 4.5mg hydrochloric acid as 1 M solution; and

[0152] balance distilled water.

[0153] Preferably, the solution comprises per ml:

[0154] 12.5 to 50.0 pgcompound (I);

[0155] 118.75 to 1000 pg sorbitol; for example 181.25 to 725 pg sorbitol; 3.5 to 6.5 mg polyoxyethylene castor oil;

[0156] 6.5 to 7. 5 mg sodium chloride;

[0157] 0.25 to 0.35 mg potassium chloride;

[0158] 0.15 to 0.25 mg calcium chloride.2H2O;

[0159] 0.05 to 0.15 mg magnesium chloride hexahydrate;

[0160] 1.5 to 2.0 mg sodium hydrogen carbonate;

[0161] 0.05 to 0.15 mg disodium phosphate dihydrate;

[0162] ca. 4.5 mg hydrochloric acid as 1 M solution; and

[0163] balance distilled water. For example, the solution of the invention consists essentially of the components listed above.

[0164] More preferably, the solution comprises per mL:

[0165] 19.0 to 38.0 pg compound (I);

[0166] 180.5 to 760 pg sorbitol; for example 275.5 to 551 pg sorbitol;

[0167] 4.0 to 6.0 mg polyoxyethylene castor oil;

[0168] 6.5 to 7.5 mg sodium chloride;

[0169] 0.25 to 0.35 mg potassium chloride;

[0170] 0.175 to 0.225 mgcalcium chloride.2H2O;

[0171] 0.075 to 0.125 mg magnesium chloride hexahydrate;

[0172] 1.5 to 2.1 mg sodium hydrogen carbonate;

[0173] 0.075 to 0.125 mg disodium phosphate dihydrate;

[0174] ca. 4.5mg hydrochloric acid as 1 M solution; and

[0175] balance distilled water.

[0176] For example, the solution of the invention consists essentially of the components listed above. For example, the solution comprises per ml:

[0177] 28.5 pgcompound (I);

[0178] 270.75 to 570 pg sorbitol; for example 413.25 pg sorbitol;

[0179] 5.0 mg polyoxyethylene castor oil;

[0180] 6.0 mg sodium chloride;

[0181] 0.30 mg potassium chloride;

[0182] 0.20 mgcalcium chloride.2H2O;

[0183] 0.10 mg magnesium chloride hexahydrate;

[0184] 1.8 mg sodium hydrogen carbonate;

[0185] 0.10 mg disodium phosphate dihydrate; ca. 4.5 mg hydrochloric acid as 1 M solution; and

[0186] balance distilled water.

[0187] For example, the solution of the invention consists essentially of the components listed above. It is preferred for the pharmaceutical composition or solution of the invention to be essentially free of dimethyl sulfoxide (DMSO). However, in some embodiments, the pharmaceutical composition or solution may further comprise dimethyl sulfoxide (DMSO) in a quantity that is low enough to be tolerated in the human body. In an embodiment, the solution comprises 0-250 pL DMSO, preferably 50 pL DMSO. In a preferred embodiment, the solution comprises 0-0.5 % (v / v) DMSO.

[0188] In certain embodiments, the physiologically acceptable solution is a solution that is suitable for intravenous administration, for example by injection or infusion. In such embodiments, the lyophilised pharmaceutical preparation comprising compound (I) and sorbitol may be dissolved in a 0.9% w / v saline solution, 5% w / vdextrose solution, orwaterfor injection (WFI).

[0189] In such embodiments, the invention provides a solution that comprises:

[0190] 1-20 mg compound (I) ethanolate;

[0191] 0.427-8.54 mg sorbitol;

[0192] 10.0 mL hypotonic sorbitol solution; and

[0193] 1-75% w / v polyoxyethylene castor oil (e.g. Kolliphor® HS 15 or Cremophor® EL).

[0194] The solution of the invention may comprise other additives such as solubilizers, stabilizers, buffers, tonicity modifiers, bulking agents, viscosity modifiers, surfactants, chelating agents, and adjuvants. Examples of buffers that may be used include citric acid, sodium bicarbonate, maleic acid, tartaric acid, lactic acid, acetic acid, and sodium phosphate.

[0195] In certain embodiments, the solution of the invention may comprise one or more additives selected from a surfactant, for example, sorbitan trioleate (Span 85), sorbitan oleate (Span 80), sorbitan monostearate (Span 60), sorbitan monolaurate (Span 20), polyoxyethylene 10 stearyl ether (Brij 98), polysorbate 60 (polyoxyethylene 20 sorbitan monostearate, Tween 60), polysorbate 80 (polyoxyethylene 20 sorbitan oleate, Tween 80), macrogol cetosteayl ether (Cetomacrogol 1000), polysorbate 20 (polyoxyethylene 20 sorbitan monolaurate, Tween 20), potassium oleate, sodium dodecyl (lauryl) sulphate, macrogol 15 hydroxystearate (Kolliphor HS 15 (Solutol HS 15)), macrogolglycerol ricinoleate (Cremophor EL (Polyoxyl 35 castor oil)), or macrogolglycerol hydroxystearate (Kolliphor RH 40); a solubilizer, for example dimethyl sulfoxide (DMSO), ethanol, benzyl alcohol, polyethylene glycol 400 (PEG400), HP-0-CD (hydroxypropyl-p-cyclodextrin), orSBE-0-CD (sulfobutylether-p-cyclodextrin); ora tonicity agent or pH modifier, for example glycerol (glycerin), sorbitol, sodium chloride (NaCl), dextrose (glucose), mannitol, propylene glycol or hydrochloric acid (HCl).

[0196] In some embodiments, the pharmaceutical composition or solution may further comprise dimethyl sulfoxide (DMSO) in a quantity that is low enough to be tolerated in the human body. In an embodiment, the solution comprises 0-250 pL DMSO, for example 0-100 pL DMSO. More preferably, the solution comprises 50 pL DMSO. In a preferred embodiment, the solution comprises 0-0.5 % (v / v) DMSO.

[0197] In such embodiments, the invention provides a solution that comprises:

[0198] 1-20 mg compound (I) ethanolate;

[0199] 0.427-8.54 mg sorbitol;

[0200] 1-300 pL DMSO;

[0201] 25-100 pL sterile water for injection;

[0202] 9.7-10.0 mL hypotonic sorbitol solution; and

[0203] 1-10%w / v Kolliphor® HS 15.

[0204] The dry (for example lyophilised) preparation or the solution according to the invention may comprise one or more further therapeutic agents. Any desired additional active ingredient may be used. For example, a further therapeutic agent may be selected from an anti-inflammatory agent (for example a steroid, e.g. dexamethasone), a calcium channel blocker (for example nimodipine), or an inhibitor of a component of an inflammation response (for example a TNFa or interleukin blocker, typically an antibody).

[0205] The solution may be prepared by a process which comprises admixing the ingredients together, followed if necessary by filtration, e.g. microfiltration. The use of vigorous solubilisation techniques may be desirable, for example vigorous stirring or shaking, e.g. using a magnetic stirrer, or the application of ultrasound. The admixing may take place at any suitable temperature and / or pressure. Suitably, the ingredients are mixed at a temperature of between 20 °C and 45 °C, suitably between 25 °C to 40 °C and most suitably between 25 °C and 30 °C. Furthermore, the ingredients are preferably mixed at atmospheric pressure (e.g. 1 atm). As the composition of the invention should be sterile, the process of the invention should be carried out under controlled conditions, for example the process may include sterile filtration with cooling.

[0206] In an embodiment, the solution of the invention comprises compound (I) at a concentration of 1 nM to 100 mM, for example 100 nM to 75 mM, for example 500 nM to 50 mM. In a preferred embodiment, the solution of the invention comprises compound (I) at a concentration of 10 mM to 50 mM. In a more preferred embodiment, the solution of the invention comprises compound (I) at a concentration of 20 mM to 30 mM, for example 22 mM to 25 mM. In an especially preferred embodiment, the solution of the invention comprises compound (I) at a concentration of 23.45 mM.

[0207] In a preferred embodiment, the solution of the invention comprises compound (I) at a concentration of 10 mM, 15 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 30 mM, 40 mM or 50 mM.

[0208] In an embodiment of the invention, the solution comprises a preparation of compound (I), or a pharmaceutically acceptable salt or solvate thereof, and sorbitol, sterile water for injection, DMSO, sorbitol solution and a polyoxyethylene castor oil, for example Kolliphor® HS 15.

[0209] In another embodiment of the invention, the solution comprises a preparation of compound (I), or a pharmaceutically acceptable salt or solvate thereof, and sorbitol, sterile water for injection, sorbitol solution and a polyoxyethylene castor oil, for example Kolliphor® HS 15 or Cremophor® EL.

[0210] Lipid emulsions

[0211] In an embodiment of the invention, there is provided a lipid emulsion prepared from a preparation of compound (I) according to the invention. The lipid emulsion comprises a preparation of compound (I) according to the invention, a triglyceride-containingoil and an emulsifier.

[0212] The dry (for example lyophilised) preparation of compound (I), for example in the form of a powder, may be dissolved first in a triglyceride-containing oil, for example a long-chain fatty acid such as soybean oil, safflower oil, olive oil, fish oil or coconut oil, or a medium-chain fatty acid such as Miglyol® 812, Myritol® 318, Captex® 355 or Neobee® M-5, or a mixture of triglyceride-containing oils, followed by addition of the oil phase to an aqueous solution of a phospholipid or mixture of phospholipids, for example egg lecithin soy lecithin, DSPC, DPPC or DSPE-PEG2000, while stirring. A nanoemulsion is then created using a mixing device on the mixture, for example a probe sonicator or high-shear homogenizer.

[0213] In a preferred embodiment, the lipid emulsion comprises a preparation of compound (I) according to the invention, soybean oil and egg lecithin.

[0214] In a preferred embodiment, the lipid emulsion comprises a preparation of compound (I) comprising 10 mg compound (I) and 128.1 mg sorbitol, dissolved in 1 g (10% w / v) soybean oil, and 120 mg egg lecithin dissolved in 9-10 mL sterile water for injection. The oil phase is slowly added to the aqueous phase while stirring, and a high-shear homogenizer or probe sonicator is used to create nanoemulsion. pH is adjusted to 7-8 if needed, and the emulsion is filtered to reduce the droplet size.

[0215] Kits

[0216] The invention also provides a kit of parts comprising a dry (for example lyophilised) pharmaceutical preparation comprising compound (I) of the invention, and a physiologically acceptable solution.

[0217] In an embodiment, the kit of parts comprises two or more separate components, wherein the components are provided in containers, for example vials.

[0218] In an embodiment, the kit of parts comprises two or more separate components, where a component (a) comprising the preparation of compound (I) is supplied together with a component (b) comprising the polyoxyethylene castor oil, and optionally a component (c) the aCSF. Preferably, component (a) is provided as a powder. The powder may then be formed into a composition of the invention by combiningwith component (b) and component (c). The invention therefore further provides a kit suitable for the preparation of a solution of the invention, which comprises as separate components (a) a powder comprising compound (I), and (b) a polyoxyethylated castor oil. The kit may optionally also contain (c) a pharmaceutically acceptable isotonic solution (e.g. aCSF).

[0219] In another embodiment, the invention provides a kit suitable for the preparation of a solution of the invention, which comprises as separate components (a) a powder comprising compound (I), and (b) a pharmaceutically acceptable isotonic solution (e.g. aCSF). The kit may optionally also contain (c) a polyoxyethylated castor oil.

[0220] In a preferred embodiment, the kit suitable for the preparation of a solution of the invention comprises as separate components (a) a powder comprising compound (I), (b) a polyoxyethylated castor oil, and (c) aCSF.

[0221] In another embodiment, the kit suitable for the preparation of a solution of the invention comprises as separate components (a) a powder comprising compound (I), and (b) sterile water for injection made isotonic with, for example, sodium chloride.

[0222] The kit may comprise a dry component (such as a powder) comprising compound (I), and a liquid component comprising a mixture of polyoxyethylated castor oil and pharmaceutically acceptable isotonic solution (e.g. aCSF). The kit may alternatively comprise a dry component (such as a powder) comprising compound (I) and the dry mass ingredients that make up the pharmaceutically acceptable isotonic solution (e.g. aCSF), and a liquid component comprising a polyoxyethylated castor oil.

[0223] The mass of the solid components and the volume of the liquid components in a kit of the invention are selected so as provide a pharmaceutical composition or solution of the invention when they are reconstituted. The kit is preferably provided with instructions for the preparation of a pharmaceutical composition or solution of the invention as defined herein.

[0224] For example, immediately before use, the dry compound (I) is reconstituted using pharmaceutically acceptable isotonic solution (e.g. aCSF) in the presence of the polyoxyethylated castor oil, the aCSF being either supplied with the kit or supplied by the pharmacist or medical professional carrying out the reconstitution. In embodiments in which the kit comprises a powder comprising compound (I) and the dry mass ingredients that make up the pharmaceutically acceptable isotonic solution (e.g. aCSF), immediately before use, the powder is reconstituted using (sterile) distilled water, thereby forming the pharmaceutically acceptable isotonic solution in situ (e.g. aCSF). The polyoxyethylated castor oil component of the kit may be added either together with the distilled water or separately therefrom (e.g. before or after the addition of the distilled water).

[0225] In an embodiment of the invention, the kit comprises a device for mixing the contents of the two containers with each other, and / or for transferring the resulting mixture to a device for the administration to a patient. In a preferred embodiment, the kit comprises a dual chamber syringe. In an embodiment of the invention, compound (I) in the kit is in admixture with a pharmaceutically acceptable carrier and sorbitol. One example of a pharmaceutically acceptable carrier is polyoxyethylene castor oil with a small amount (approximately 33% of the volume of polyoxyethylene castor oil) H2O or aCSF. The amount of physiologically acceptable solution may either be a small amount in order to prepare a concentrated solution of the pharmaceutical preparation comprising compound (I), ora larger amount in order to enable the preparation of a solution having the desired concentration for administration to a patient. In an alternative embodiment, the kit comprises both a container comprising a physiologically acceptable solution for preparing a concentrated solution of the pharmaceutical preparation and a second container, such as a bagfor infusion, comprising a larger amount of a physiologically acceptable solution for preparation of the more diluted solution for administration to a subject.

[0226] In an embodiment of the invention, the kit comprises a dry (for example lyophilised) component comprising compound (I) and sorbitol, and one or more liquid components comprising DMSO, a solution of sorbitol, sterile water for injection, and optionally a solution of polyoxyethylene castor oil, for example Kolliphor® HS 15.

[0227] In such an embodiment, the dry component of the kit may comprise 1 to 20 mg of compound (I), and 1 to 150 mg of sorbitol, and the one or more liquid components comprises 0 to 250 pL DMSO, 300 to 600 mg sorbitol in solution, 0 to 500 mg polyoxyethylene castor oil (for example Kolliphor® HS 15), and 5 to 15 mL sterile water for injection.

[0228] In a preferred embodiment, the dry component of the kit may comprise 10 mg of compound (I), and 1 to 150 mg of sorbitol, and the one or more liquid components comprises 50 to 250 pL DMSO, 300 to 600 mg sorbitol in solution, 0 to 500 mg polyoxyethylene castor oil (for example Kolliphor® HS 15), and 9 to 10 mL sterile water for injection.

[0229] In such an embodiment, the kit comprises 3 containers, comprising:

[0230] Vial 1 : Dry powder

[0231] Small cylindrical polypropylene vial with conical base with flip or snap cap, ora small clear cylindrical type 1 glass bottle with rubber stopper and aluminium cap, containing:

[0232] Dry (for example lyophilised) preparation of compound (I) in ethanolate form and sorbitol (1-20 mg compound (I) and 1-30 molar equivalents sorbitol). Vial 2: Primary co-solvent

[0233] Small cylindrical polypropylene vial with conical base with flip or snap cap, ora small clear cylindrical type 1 glass bottle with rubber stopper and aluminium cap, containing:

[0234] Mixture of 1-300 pL methyl sulfoxide (DMSO) and 25-100 pL sterile water for injection.

[0235] Vial 3: Co-solvent

[0236] 15 mL clear type 1 glass bottle for injection with rubber stopper and aluminium cap, containing: Mixture of 9.7-10.0 mL hypotonic sorbitol solution and 1-10% w / v polyoxyethylene castor oil (for example Kolliphor® HS 15).

[0237] In an alternative embodiment of the invention, the kit comprises a dry component comprising compound (I) and sorbitol, and one or more liquid components comprising a solution of sorbitol, sterile water for injection, and optionally a solution of a polyoxyethylene castor oil, for example Kolliphor® HS 15 orCremophor® EL.

[0238] In such an embodiment, the dry component of the kit may comprise 1 to 20 mg of compound (I), and 1 to 150 mg of sorbitol, and the one or more liquid components comprises 300 to 600 mg sorbitol in solution, 50 to 7500 mg polyoxyethylene castor oil (for example Kolliphor® HS 15 or Cremophor® EL), and sterile water for injection in an amount sufficient to give a total volume of 10 to 20 mL.

[0239] In a preferred embodiment, the dry component of the kit may comprise 10 mg of compound (I), and 1 to 150 mg of sorbitol.

[0240] In such an embodiment, the kit comprises 2 containers, comprising:

[0241] Vial 1 : Dry powder

[0242] Small cylindrical polypropylene vial with conical base with flip or snap cap, ora small clear cylindrical type 1 glass bottle with rubber stopper and aluminium cap, containing:

[0243] Dry (for example lyophilised) preparation of compound (I) in ethanolate form and sorbitol (1-20 mg compound (I) and 1-30 molar equivalents sorbitol).

[0244] Vial 2: Solvent

[0245] 15 mL clear type 1 glass bottle for injection with rubber stopper and aluminium cap, containing: Mixture of 10.0 mL hypotonic sorbitol solution, and 1-75% w / v polyoxyethylene castor oil (for example Kolliphor® HS 15 or Cremophor® EL (polyoxyl 35 castor oil)).

[0246] In an embodiment of the invention, the kit comprises equipment for administration in an acute setting, for example in an ambulance or emergency room. In a preferred embodiment, the kit comprises equipment for administration in an acute setting, and further comprises as separate components (a) a powder comprising compound (I), and (b) a pharmaceutically acceptable solution that is suitable for intravenous administration, for example by injection or infusion. For example, the kit according to the 3-vial embodiment described above can be used to prepare a solution suitable for intravenous administration in an acute setting such as in an ambulance. The formulation allows the rapid and safe preparation of a pharmaceutical composition of compound (I), which would otherwise be time consuming.

[0247] The contents of vial 2 are added to vial 1 with a syringe or pipette and mixed for 1-2 minutes to dissolve the contents of vial 1, forming a yellow / orange clear solution. This solution is transferred to vial 3 using a syringe or pipette, and mixed for a few minutes until the solution is clearwith no visible particles. The resulting solution is isotonic, and ready for administration by injection using a syringe with a 0.22 pm filter to remove any invisible, undissolved particles.

[0248] Conditions

[0249] The invention provides a composition comprising a lyophilised pharmaceutical preparation of compound (I) for use as a medicament.

[0250] In an embodiment of the invention, the composition comprising a lyophilised pharmaceutical preparation of compound (I) is for use in the treatment of ischemic damage in a human patient. In an embodiment of the invention, the ischemic damage results from ischemic stroke (for example, global ischemic stroke or focal ischemic stroke), subarachnoid haemorrhage (SAH) (for example aneurysmal subarachnoid haemorrhage (aSAH)), intracerebral haemorrhage, or traumatic brain injury (TBI). The ischemic damage may result from other types of stroke, such as haemorrhagic stroke, transient ischemic attack (TIA), or it can be the result of a heart stop or dramatic lowering of systemic blood pressure by other means, e.g. heart fibrillation. In an alternative embodiment, the ischemic damage occurs in conjunction with the removal of a blood clot in a major brain artery, either physically or with thrombolysis, or other intervascular procedures in brain vessels.

[0251] In an embodiment of the invention, the composition comprising compound (I) for use in the treatment of ischemic damage is administered to a human patient suffering from ischemic damage up to 8 hours from the onset of stroke.

[0252] For example, the composition comprising compound (I) is administered to a patient from 0 to 8 hours, from 0 to 6 hours, from 0 to 4 hours or from 0 to 2 hours from the onset of stroke.

[0253] In another embodiment, the composition comprising compound (I) is administered to a patient from 2 to 8 hours, from 2 to 6 hours, or from 2 to 4 hours from the onset of stroke.

[0254] In another embodiment, the composition comprising compound (I) is administered to a patient from 4 to 8 hours or from 4 to 6 hours from the onset of stroke.

[0255] In an embodiment, the composition comprising compound (I) is administered to a patient up to 8 hours, up to 7 hours, up to 6 hours, up to 5 hours, up to 4 hours, up to 3 hours, up to 2 hours or up to 1 hour from the onset of stroke.

[0256] In an embodiment, the composition comprising compound (I) is administered to a patient 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, or 30 minutes from the onset of stroke.

[0257] In an embodiment of the invention, the composition comprising compound (I) for use in the treatment of ischemic damage is administered to a human patient suffering from ischemic damage as a single dose.

[0258] Alternatively, or additionally, the composition comprising compound (I) for use in the treatment of ischemic damage may be administered up to a maximum of 2 additional doses (a total of 3 doses), up to 48 hours after onset of stroke.

[0259] The invention also provides a method of treating ischemic damage in a human patient comprising administration of a therapeutically effective amount of a solution of the invention. Preferably, the solution is administered by injection. In an especially preferred embodiment, the solution is administered by intracerebroventricular (ICV) injection. In another embodiment of the invention, the solution is administered intravenously, for example by injection or infusion.

[0260] Methods of preparation

[0261] Another aspect of the invention provides a method for preparing a dry (for example lyophilized) pharmaceutical preparation, whereby:

[0262] a. Providing a solution of compound (I), or a pharmaceutically acceptable salt or solvate thereof, in an organic solvent;

[0263] b. Providing a solution of sorbitol in water;

[0264] c. Combiningthe solution of compound (I) in organic solvent and the aqueous solution of sorbitol to form a mixture;

[0265] d. Optionally concentrating the mixture by removal of the aqueous and organic solvent; e. Subjecting the solution of compound (I) (optionally concentrated in step (d) to drying (for example by lyophilisation).

[0266] In an embodiment, the weight ratio (w / w) between the amount of compound (I) in step (a) and the amount of sorbitol in step (b) is between 1 :0.96 and 1 :239.39, for example from 1 :0.96 to 1:119.69, for example from 1:0.96 to 1:47.88, from 1 :2.39to 1 :35.91, from 1:4.79 to 1:23.94, or from 1:9.58 to 1 :19.15, for example 1 :0.96, 1 :2.39, 1 :4.79, 1 :9.58, 1 :11.97, 1 :14.36, 1 :19.15, 1 :23.94, 1 :35.91 , 1 :47.88, 1:119.69 or 1 :239.39. In a preferred embodiment, the weight ratio (w / w) between the amount of compound (I) in step (a) and the amount of sorbitol in step (b) is 1:14.36.

[0267] In another embodiment, the weight ratio (w / w) between the amount of compound (I) in step (a) and the amount of sorbitol in step (b) is between 1 :0.48 and 1 :239.39, for example from from 1 :0.48 to 1 :14.36, for example from 1 :0.48 to 1 :9.58, from 1 :0.48 to 1 :4.79, or from 1 :4.79 to 1 :9.58, for example 1 :0.48, 1 :0.96, 1 :2.39, 1 :4.79, 1 :9.58, 1:11.97, or 1 :14.36. In a preferred embodiment, the weight ratio (w / w) between the amount of compound (I) in step (a) and the amount of sorbitol in step (b) is 1 :0.96, 1 :2.39, or 1 :4.79. In an especially preferred embodiment, the weight ratio (w / w) between the amount of compound (I) in step (a) and the amount of sorbitol in step (b) is 1 :2.39. In an alternative embodiment, the weight ratio (w / w) between the amount of compound (I) ethanolate in step (a) and the amount of sorbitol in step (b) is between 1 :0.85 and 1 :213.53, for example from 1:0.85 to 1:106.77, for example from 1:0.85 to 1:42.71, from 1:2.14to 1:32.03, from 1:4.27 to 1 :21.35, orfrom 1 :8.54 to 1 :17.08, for example 1 :0.85, 1:2.14, 1:4.27, 1:8.54, 1:10.68, 1:12.81, 1:17.08, 1:21.35, 1:32.03, 1:42.71, 1 :106.77 or 1 :213.53. In a preferred embodiment, the weight ratio (w / w) between the amount of compound (I) ethanolate in step (a) and the amount of sorbitol in step (b) is 1 : 12.81.

[0268] In another embodiment, the weight ratio (w / w) between the amount of compound (I) ethanolate in step (a) and the amount of sorbitol in step (b) is between 1 :0.43 and 1 :213.53, for example from 1 :0.43 to 1:12.81 , for example from 1 :0.43 to 1 :8.54, from 1 :0.43 to 1 :4.27, or from 1 :4.27 to 1 :8.54, for example 1:0.43, 1:0.85, 1 :2.14, 1 :4.27, 1 :8.54, 1:10.68, or 1 :12.81. In a preferred embodiment, the weight ratio (w / w) between the amount of compound (I) ethanolate in step (a) and the amount of sorbitol in step (b) is 1 :0.85, 1 :2.14, or 1 :4.27. In an especially preferred embodiment, the weight ratio (w / w) between the amount of compound (I) ethanolate in step (a) and the amount of sorbitol in step (b) is 1 :2.14.

[0269] In an embodiment, the organic solvent is selected from the group comprising: MeCN, DMSO, tBuOH, DMF, THF, MeOH, and acetone. In a preferred embodiment the organic solvent is MeCN. The concentration of the aqueous solution of compound (I) in step (d) can be carried out using a rotary evaporator, or other standard techniques.

[0270] In a preferred embodiment, the method for preparing a lyophilized pharmaceutical preparation comprises the steps:

[0271] a. 250 mg compound (I) ora pharmaceutically acceptable salt or solvate thereof, is dissolved 35 mL MeCN;

[0272] b. 3.36 g sorbitol is dissolved in 35 mL water;

[0273] c. The solution of compound (I) in organic solvent and the aqueous solution of sorbitol are combined to form a miscible mixture;

[0274] d. The mixture is concentrated by removal of solvent with a rotary evaporator;

[0275] e. The concentrated aqueous solution of compound (I) is subjected to lyophilisation. Lyophilisation comprises the following steps:

[0276] 1. Freezing the sample, for example using liquid N2;

[0277] 2. Primary drying, by loweringthe pressure around the sample to sublimate ice in the frozen sample; and

[0278] 3. Secondary drying, by raising the temperature of the sample above the temperature used in the primary drying phase, and optionally further loweringthe pressure below that used in the primary drying phase, to encourage desorption of any remaining water molecules.

[0279] The present invention is also directed to a lyophilised pharmaceutical preparation as defined herein, obtainable by the above disclosed method.

[0280] Examples

[0281] Example 1 : Synthesis of Compound (I) U0126

[0282] NaOH

[0283]

[0284] A 3-necked round-bottom flask fitted with a N2bubbler, and a temperature probe was charged with H2O (8 vol) and NaOH (2.63 eq.) and stirred until fully dissolved. The vessel was placed in an ice / water bath and the solution sparged with N2for~ 5 min. 2-Aminothiophenol (4.2 eq.) was added and the yellow solution stirred for ~ 30 min, maintaining the internal temperature between 15-20 °C. Tetracyanoethane (1 eq.) was then added dropwise as a solution in 2-MeTHF (9.25 vol) over~ 15 min, again maintaining the reaction temperature between 15-20 °C. Once addition was complete the reaction was stirred for 2 h, after which time the starting material had been consumed. The reaction was filtered and the solid washed with H2O (3x5 vol) then redissolved in 2-butanone (50 vol). The reaction was filtered to remove any inorganic salts and then concentrated in vacuo. The resulting solid was suspended in EtOH (10 vol) and stirred for ~ 15 min, then again concentrated in vacuo. The solid was then triturated in EtOH (8 vol) at 70 °C for ~ 30 min, then cooled to ~ 35 °C and filtered whilst still warm. The off-white solid was washed with EtOH (3x5 vol) and dried to provide compound (I) as the hemi-ethanolate (~ 0.6 eq EtOH). Example 2: Lyophilised formulations of compound (I)

[0285] Investigations of a range of excipients (Table 1 ), was proposed in the formulation of compound (I). The excipients investigated were mono- and disaccharides, and polyols. Formulation would be carried out by solubilising the compound (I) and the excipient in a H2O / organic mixture, followed by lyophilisation to provide a solid.

[0286] Table 1 : Proposed excipients for formulation of compound (I)

[0287] Monosaccharides Disaccharides Polyols

[0288] Glucose Sucrose Mannitol

[0289] Fructose Maltose Sorbitol

[0290] Trehalose

[0291]

[0292] Choice of solvent:

[0293] Solvent trials were carried out to ascertain an organic solvent to use in the formulations that could fully solubilise compound (I) and the excipients and allow successful lyophilisation. MeCN, DMSO and tBuOH were initially considered as they are known to be favourable organic solvents to use in lyophilisation (providing that the quantities are kept relatively low). However, due to the very poor solubility of compound (I) in H2O, a higher than ideal proportion of organic solvent was required to fully solubilise the material with the excipients. Lyophilisation trials of the resulting solutions were unsuccessful. DMF, THF, MeOH and acetone were used as the organic portion of the mixture, however these solvents also resulted in unsuccessful lyophilisation. Neat DMSO was used, as this solvent is often used as a lyophilisation solvent on its own, however the lyophilisation was also unsuccessful.

[0294] Based on these observations a different approach was adopted. The excipient / compound (I) mixture was dissolved in a mixture of H2O / organic solvent as before, however the mixture was then concentrated on a rotary evaporator to remove the bulk of the organic solvent and increase the H2O:organic solvent ratio. MeCN was found to be the most suitable organic solvent for this method, with removal of the solvent on the rotary evaporator typically producing a slightly cloudy solution that could be successfully lyophilised to give a free flowing solid. Quantity of excipient:

[0295] A study was undertaken to compare differences in using a variety of excipient quantities which led to the choice of 30 molar equivalents. A 500 mg quantity of compound (I) was combined with each of the seven excipients listed above in Table 1 to provide the different formulations for the subsequent stability study.

[0296] Method of preparing lyophilised formulations:

[0297] Compound (I) (1 eq.) was dissolved in the minimum quantity of MeCN required to dissolve the compound. Separately, the excipient (30 eq.) was dissolved in the minimum quantity of H2O to dissolve the excipient in a round-bottom flask. The compound (I) solution was added to the excipient solution and the mixture concentrated on a rotary evaporator with an ambient temperature water bath. The resulting cloudy solution was frozen with liquid nitrogen and lyophilised to produce an off-white free-flowing solid.

[0298] Example 3: Stability of lyophilised preparations of compound (I)

[0299] The seven formulated samples were portioned into individual vials for storage during the trial; 10 mg portions were stored from which both HPLC and 1 H NMR samples would be taken and 100 mg portions were stored for measurement of H2O content by Karl Fischer titration. The vials were stored at both ambient temperature and at 40°C for an accelerated stability study.

[0300] Additionally, samples of compound (I) were taken to serve as a control.

[0301] HPLC samples were taken initially daily, then less frequently over the 3 month study.1H NMR and Karl Fischer titrations were taken initially weekly and less frequently over the 3 month study, at the same frequency as HPLC sampling.

[0302] 3.1 : HLPC Analysis

[0303] HPLC conditions used for reaction monitoring / stability study:

[0304] Column: PhenomenexGemini-NXC18, 5 pm, 250x4.6

[0305] Mobile Phase A: 10 mM ammonium formate in H2O (pH 9) Mobile Phase B: 10 mM H2O / MeCN (1 :9)

[0306] Gradient:

[0307] Time A% B%

[0308] 0 75 25

[0309] 1 75 25

[0310] 18 80 20

[0311] 19 80 20

[0312] 20 75 25

[0313] 24.5 75 25

[0314]

[0315] Flow rate: 1.0 ml / min

[0316] Column Temp: 25°C

[0317] Detection: 246 nm

[0318] Sample preparation: 0.1 mg / ml in A / B (1:1)

[0319] HPLC samples were taken initially daily, then less frequently over the course of the 3-month study. The HPLC results for both the ambient and accelerated studies are shown below (Tables 2 and 3). In summary, the results clearly show that the formulation with sorbitol is superior to both the other excipients and compound (I) alone, with very little degradation (<1%) observed at ambient temperature over the duration of the study.

[0320] Table 2: %Area by HPLC of compound (I) and lyophilised formulations (stored at ambient temperature)

[0321] Day Compound Glucose Sucrose Sorbitol Maltose Trehalose Mannitol (I)

[0322] 1 99.12 98.97 98.50 99.15 98.76 98.96 98.63 2 98.53 98.87 98.29 99.07 98.80 98.93 98.67 3 98.51 98.85 98.53 99.13 98.72 98.81 98.48 4 98.63 98.85 97.60 99.14 98.58 98.72 98.44 5 99.14 99.04 98.51 99.29 98.61 99.01 98.36 8 99.04 98.50 98.38 99.07 98.61 98.78 98.20 10 98.57 98.67 98.57 99.13 98.31 98.76 98.06

[0323]

[0324] 12 98.83 98.58 98.46 99.30 98.33 98.62 97.92 15 98.94 98.54 X 99.08 X 98.64 97.54 22 98.91 98.15 X 98.94 X 97.67 96.74 36 98.59 97.13 X 98.69 X 96.73 96.48 57 97.87 X X 98.27 X X X 85 97.78 X X 98.21 X X X

[0325]

[0326] X- HPLC analysis was halted early due to poor stability compared to compound (I) control sample.

[0327] Table 3: %Area by HPLC of compound (I) and lyophilised formulations (stored at 40°C) Day Compound Glucose Sucrose Sorbitol Maltose Trehalose Mannitol (I)

[0328] 1 99.12 98.97 98.50 99.15 98.76 98.96 98.63 2 97.14 98.11 98.45 99.04 97.66 98.67 97.50 3 98.01 98.00 98.25 99.10 97.16 97.27 96.99 4 98.79 97.39 98.20 99.05 96.95 97.00 96.54 5 98.67 98.87 97.85 98.84 96.70 96.52 96.38 8 98.66 96.64 97.90 99.09 95.60 95.18 95.05 10 98.35 95.54 97.70 99.08 95.92 94.44 94.63 12 98.44 95.73 97.05 99.00 94.58 94.82 94.77 15 98.53 94.32 X 99.07 X 93.40 94.26 22 97.97 93.62 X 98.76 X 90.43 93.24 36 96.98 90.90 X 97.59 X 89.38 92.11 57 94.68 X X 97.46 X X X 85 94.70 X X 98.02 X X X

[0329]

[0330] X- HPLC analysis was halted early due to poor stability compared to compound (I) control sample.

[0331] 3.2:1H NMR

[0332] 1H NMRwas taken initially weekly and less frequently over the 3-month study, at the same intervals as the HPLC sampling.

[0333] Very little change in the1H NMR spectra was observed over the course of the study, particularly in the case of the sorbitol sample. This was to be expected given that the HPLC results showed very little degradation in the material. The spectra (Figures 3 and 4) show that very little observable degradation can be seen in either the compound (I) peaks (5.4-7.6 ppm) or in the sorbitol peaks (3.2-4.7 ppm) between Day 1 and Day 85 of the study.

[0334] Example 4: Solubility of lyophilised preparation of compound (I) and sorbitol

[0335] Trials were undertaken to confirm that the formulated samples were soluble in a typical Kolliphor / aCSF mixture that would be suitable for use in the clinic. The samples were prepared in accordance with the processes described in the examples above, with an increased quantity of the formulated material used so that the same quantity of compound (I) (i.e. 28.5 pg / mL) would be included in the sample. The solution was sterile filtered using a 0.22pm filter and filled into 2mL vials. The sterile filter was integrity tested after filtration. The vials were sealed under sterile conditions.

[0336] Studies were carried out to assess the solubility limit of compound (I) in Kolliphor. It was found that 100 mg of compound (I) could be solubilised in 4 mL of 3:1 Kolliphor / H2O (i.e. 25 mg / mL) however at higher concentrations of compound (I) the samples remained cloudy. Under these conditions full solubilisation of the material was only observed after stirring for approximately 30 minutes at ambient temperature. Warming the sample was found to increase the rate of dissolution, however this would also likely result in an increased rate of degradation.

[0337] The sorbitol formulation was also found to efficiently dissolve in the 3:1 Kolliphor / H2O mixture, however dissolution was observed to occur in less solvent (100 mg in 1 mL) and full solubility was observed after stirring for only 5 minutes. After further investigations it was found that the solubility limit of the formulated material could be increased by using a larger proportion of H2O in the Kolliphor / H2O mixture. Utilising a 1 :1 mixture allowed 200 mg of the formulated material to be dissolved in 1.5 mL of solvent. Based on there being 30 molar equivalents of sorbitol in the formulation this equates to ~8.67 mg / mL of compound (I).

[0338] Example 5: Stability of reconstituted solutions of lyophilised preparations of compound (I) and sorbitol

[0339] Atrial of the stability of both compound (I) and the sorbitol formulated material in solution was carried out to ascertain if the formulated material would show less degradation. Compound (I) and the sorbitol formulation were both dissolved in a 3:1 Kolliphor / H2O mixture so that they could be directly compared, and HPLC samples were taken at three time points over a 4-hour period.

[0340] The results are summarised in Table 4:

[0341] Table 4: %Area by HPLC of compound (I) and the sorbitol formulated material in solution (Kolliphor / H2O, 3:1)

[0342] Time (min) Compound (I) Sorbitol formulation

[0343] 0 99.12 99.15

[0344] 40 91.94 93.38

[0345] 120 83.08 82.75

[0346] 240 66.63 76.37

[0347]

[0348] T = 0 purities were taken as the purities of the parent solids. The first time point was taken at 40 minutes (i.e. approximately 10 minutes after full dissolution of the compound (I) sample was observed), with both samples showing similar degradation. This was also observed at the 2 h time point, with only the 4 h time point showing any major difference between the two samples. The Sorbitol formulation showed less degradation at this stage, although purity had still decreased to 76.37%. Figure 5 suggests that the compound (I) sample underwent a very linear degradation, whilst the rate of degradation in the Sorbitol formulation appears to decrease slightly overtime.

[0349] In both cases the main degradation product observed is the assumed isomerised material, a small quantity of which can been seen in theT = 0 samples as a small peak in the HPLC trace immediately before the product peak (Figures 6 and 7).

[0350] The results above demonstrate that compound (I) could be successfully lyophilised with a sorbitol excipient. The lyophilised sorbitol formulation showed superior stability over the course of 3 months to compound (I) alone, as well as to lyophilised preparations with other sugar excipients.

[0351] The lyophilised sorbitol formulations could be successfully dissolved to prepare solutions suitable for administration in a clinic, and the reconstituted solutions showed improved stability compared with corresponding solutions of compound (I) alone.

Claims

ClaimsA pharmaceutical preparation comprising compound (I)J sNH2CN NH2ora pharmaceutically acceptable salt or solvate thereof, and sorbitol.

2. A pharmaceutical preparation accordingto claim 1, wherein the pharmaceutical preparation is lyophilised.

3. A pharmaceutical preparation accordingto claim 1 or claim 2, wherein the molar ratio (mol / mol) between compound (I) and sorbitol is from 1 :2 to 1 :500.

4. A pharmaceutical preparation according to claim 3, wherein the molar ratio (mol / mol) between compound (I) and sorbitol is 1 :30, or 1 :5.

5. A pharmaceutical preparation accordingto claim 1 or claim 2, wherein the weight ratio (w / w) between compound (I) and the sorbitol is between 1 :0.5 and 1 :250.

6. A pharmaceutical preparation according to claim 5, wherein the weight ratio (w / w) between compound (I) and the sorbitol is 1 :14.5.

7. A pharmaceutical preparation according to any one of claims 1 to 6, wherein the preparation comprises 250 to 1000 mg compound (I).

8. A pharmaceutical preparation according to claim 1 , wherein the pharmaceutical preparation comprises less than 1% w / w water, for example less than 0.5% w / w water, or less than 0.1% w / w water.

9. A solution comprising a pharmaceutical preparation accordingto anyone of claims 1 to 8.

10. A solution according to claim 9, wherein the solution comprises a pharmaceutical preparation according to any one of claims 1 to 8 and a physiologically acceptable solution.

11. A solution according to claim 10, wherein the physiologically acceptable solution is artificial CSF (aCSF).

12. A solution according to claims 9 to 11 , wherein the preparation further comprises polyoxyethylene castor oil.

13. A solution comprising a pharmaceutical preparation comprising:compound (I)NH2CN NH2J s yNH2CN NH2or a pharmaceutically acceptable salt or solvate thereof,sorbitol,and a physiologically acceptable solution.

14. A solution according to claim 13, wherein the solution further comprises sterile water for injection.

15. A solution according to claim 13 or claim 14, wherein the solution further comprises polyoxyethylene castor oil.

16. A kit of parts comprising:A pharmaceutical preparation according to any one of claims 1 to 8; anda physiologically acceptable solution that is a suitable solvent for compound (I).

17. A kit of parts according to claim 16 wherein the physiologically acceptable solution is aCSF.

18. A kit of parts according to claim 16 or 17 wherein the kit additionally comprises polyoxyethylene castor oil.

19. A pharmaceutical preparation according to any one of claims 1 to 8, for use as a medicament.

20. A pharmaceutical preparation according to any one of claims 1 to 8, for use in the treatment of ischemic damage in a human patient.

21. A pharmaceutical preparation for use accordingto claim 20, wherein the ischemic damage is due to global or focal ischemic stroke, subarachnoid haemorrhage, intracerebral haemorrhage, traumatic brain injury (TBI), transient ischemic attack (TIA), dramatic lowering of systemic blood pressure, for example due to heart stop or heart fibrillation, or in conjunction with removal of a clot or other intravascular procedures in brain vessels.

22. A method for the treatment of ischemic damage comprising administering the pharmaceutical preparation as claimed in anyone of claims 1 to 8 to a human patient.

23. Use of the pharmaceutical preparation accordingto anyone of claims 1 to 8 for the manufacture of a medicament for the treatment of ischemic damage.

24. A method for preparing a lyophilized pharmaceutical preparation accordingto claim 2, comprising:a. Providing a solution of compound (I), or a pharmaceutically acceptable salt or solvate thereof, in an organic solvent;b. Providing a solution of sorbitol in water;c. Combiningthe solution of compound (I) in organic solvent and the aqueous solution of sorbitol to form a miscible mixture;d. Optionally concentrating the mixture by removal of solvent;e. Subjecting the aqueous solution of compound (I), optionally concentrated in step (d), to lyophilisation.

25. The method according to claim 24, wherein the organic solvent is step (a) is selected from MeCN, DMSO, tBuOH, DMF, THF, MeOH, and acetone.

26. The method according to claim 25, wherein the organic solvent is MeCN.

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