Cyclodextrin-panobinostat adduct

Abstract

The present invention provides a method for producing a cyclodextrin-panobinostat adduct, comprising: a) providing a first aqueous solution comprising a buffering agent, the solution having a pH in the range 2.0 to 4.0; b) dissolving panobinostat in said first aqueous solution to provide a second aqueous solution; and c) mixing said second aqueous solution comprising panobinostat with a third aqueous solution comprising a cyclodextrin to form a fourth aqueous solution comprising a cyclodextrin-panobinostat adduct. Also provided is an artificial cerebrospinal fluid (CSF) solution comprising the cyclodextrin-panobinostat adduct and medical uses of the cyclodextrin-panobinostat adduct, including in the treatment of brain tumours.

Claims

1. A method of treatment of a brain tumour in a mammalian subject, comprising administering a therapeutically effective amount of a cyclodextrin-panobinostat adduct to a mammalian subject in need thereof, wherein the cyclodextrin-panobinostat adduct is administered at a concentration of between 1 μM and 100 μM.

2. The method according to claim 1, wherein the brain tumour comprises a glioma.

3. The method according to claim 2, wherein the glioma comprises Diffuse Intrinsic Pontine Glioma (DIPG).

4. The method according to claim 1, wherein the method comprises administration via convection-enhanced delivery (CED).

5. The method according to claim 1, wherein the cyclodextrin-panobinostat adduct is dissolved in artificial cerebrospinal fluid (CSF).

6. The method according to claim 1, wherein the cyclodextrin-panobinostat adduct is 2-hydroxypropyl-β-cyclodextrin-panobinostat.

7. The method according to claim 1, wherein the cyclodextrin-panobinostat adduct has a total concentration of dimethyl sulphoxide (DMSO) of less than 1000 parts per million (ppm).

8. The method according to claim 1, wherein the subject is a human child under 10 years of age.

9. A method of treatment of a brain tumour in a mammalian subject, comprising administering a therapeutically effective amount of a cyclodextrin-panobinostat adduct to a mammalian subject in need thereof, wherein the cyclodextrin-panobinostat adduct is administered at a concentration of between 1 μM and 100 μM, and wherein the method comprises administration via convection-enhanced delivery (CED).

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIGS. 1A-1D show MTT-assessed cell viability (%) plotted against drug concentration (μM) for: (FIG. 1A) HP-β-CD panobinostat (squares), cyclodextrin (triangles) and artificial CSF (circles) against U87MG cells after 72 hours treatment; (FIG. 1B) HP-β-CD panobinostat (squares), cyclodextrin (triangles) and artificial CSF (circles) against U87MG cells after 48 hours treatment; (FIG. 1C) HP-β-CD panobinostat (squares), cyclodextrin (triangles) and artificial CSF (circles) against HEPG2 cells after 72 hours treatment; and (FIG. 1D) HP-β-CD panobinostat against U87MG (circles), HEPG2 (diamonds) and h19 (triangles) cells after 72 hours treatment. Error bars indicate plus or minus standard deviation of triplicate samples. N=1 experimental replicate.

(2) FIGS. 2A and 2B show: (FIG. 2A) cell viability (%) of SF8628 DIPG cells plotted against HP-β-CD panobinostat concentration (μM) following 72 hours treatment; (FIG. 2B) cell viability (% live cells) plotted against logio dose of HP-β-CD panobinostat concentration following 72 hours treatment (circles), 6 hours treatment (squares) and 30 minutes treatment (diamonds). Error bars indicate plus or minus standard deviation of triplicate samples.

(3) FIG. 3 shows panobinostat concentration (μM) over time for a 24-week storage test at 15° C.-25° C. The measured panobinostat concentration of the stored HP-β-CD panobinostat adduct was remarkably stable even out to the full 24-week period of the study.

(4) FIG. 4 shows total impurities (diamonds) as a percentage plotted against time for a 24-week storage test at 15° C.-25° C. Relative retention time (RRT) 0.86 (squares) and RRT 1.12 (triangles) refer to chromatogram peaks either side of the main panobinostat drug peak. The plot shows that the RRT peaks and total % impurities remained stable and low throughout the study period implying that formation of panobinostat degradation products is negligible.

DETAILED DESCRIPTION OF THE INVENTION

(5) In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

(6) “Panobinostat” (Farydak®, LBH-589, 2-(E)-N-hydroxy-3-[4[[[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]-2-propenamide) is a non-selective HDAC inhibitor having the chemical structure depicted below:

(7) ##STR00001##

(8) As used herein, unless context specifies otherwise (such as when described as “free base panobinostat”), “panobinostat” includes salt forms (e.g. panobinostat citrate, panobinostat lactate, and the like). Methods for producing hydroxamate derivatives useful as deacetylase inhibitors, including panobinostat, are detailed in WO2002/022577, the entire content of which is expressly incorporated herein by reference.

(9) “Cyclodextrin” is a cyclic oligosaccharide and specifically includes α-cyclodextrins, β-cyclodextrins, and γ-cyclodextrins. The cyclodextrin is typically a pharmaceutically acceptable cyclodextrin. In particular cases, the cyclodextrin is 2-hydroxypropyl-β-cyclodextrin.

(10) “Convection-enhanced delivery (CED)” is a method of delivering a drug directly to the brain through one or more very small catheters which are surgically placed into or around the brain tumour. The placement of the catheters may be stereotactically directed, e.g. to minimize off-target effects. WO2013/135727, the entire content of which is expressly incorporated herein by reference, describes a method for treatment of glioma by convection enhanced delivery (CED) using a composition of carboplatin in artificial cerebrospinal fluid (CSF). CED typically employs in-line sterilising filters. A number of sterilising filters are not compatible with DMSO (for example, cellulose acetate; cellulose nitrate; polycarbonate; polyether sulfone; Sartobran P; some PVDFs; PVC; Metricet; Nylon; and PES). It is therefore desirable that the panobinostat artificial CSF formulation for CED delivery should be substantially free from certain organic solvents such as DMSO. The use of cyclodextrin-panobinostat of the present invention in a formulation for CED delivery is therefore advantageous in the context of commonly used in-line sterilising filters.

(11) “Artificial Cerebrospinal Fluid (CSF)”

(12) Artifical cerebrospinal fluid (CSF) is intended to match the electrolyte concentrations of CSF. Preferably, artificial CSF is prepared from high purity water and analytical grade reagents or can be obtained from medical and commercial suppliers (e.g. South Devon Healthcare NHS Foundation Trust, UK or Tocris Bioscience, Bristol, UK). The final ion concentrations in artificial CSF may be as follows (in mM):

(13) Na 150;

(14) K 3.0;

(15) Ca 1.4;

(16) Mg 0.8;

(17) P 1.0;

(18) Cl 155.

(19) Optionally, each ionic constituent may be at a concentration plus or minus (±) 10%, ±5%, ±2%, ±1% or ±0.5% from the above-listed concentration values. Further optionally, the artificial CSF may further comprise glucose and/or one or more proteins at concentrations typically found in human CSF. In preferred cases, the artificial CSF does not comprise glucose or protein.

(20) The following is presented by way of example and is not to be construed as a limitation to the scope of the claims.

EXAMPLES

Example 1

Procedure for Production of Panobinostat-2-Hydroxypropyl-β-Cyclodextrin Adduct

(21) Panobinostat free base is poorly soluble in water. It is similarly insoluble in concentrated aqueous solutions of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) (450 mg/ml). However, Panobinostat is soluble in citrate buffer at pH 3.0.

(22) Solution A: 0.1M Citric Acid in Water

(23) Citric acid (Sigma Aldrich C-0759; Lot #21K0042) (2.101 g) was weighed out and dissolved in 100 mL ultrapure water.

(24) Solution B: 0.1M Sodium Citrate in Water

(25) Sodium citrate (tribasic) (Sigma Aldrich C3434; Lot #1304640/41807230 (2.944 g) was weighed out and dissolved in 100 mL ultrapure water.

(26) Solution C: 0.1M Citrate Buffer

(27) Solution A (82 mL) and solution B (18 mL) were added to a 250 mL Duran flask and mixed briefly to form [C]. The pH of the citrate buffer was measured and found to be pH3.0.

(28) Solution D: 10 mM Panobinostat Solution in Citrate Buffer (25 mL)

(29) Concentration=10 mM=0.01M

(30) Volume=25 mL=0.025 L

(31) Therefore number of moles required=2.5×10.sup.−4

(32) Molecular weight of Panobinostat=349.43 g/mol

(33) Therefore mass of Panobinostat required=82.36 mg

(34) Thus, Panobinostat free base (81.0 mg) was weighed out and dissolved in solution C (24.59 mL).

(35) The mixture was shaken on an orbital shaker for 10 minutes to give a clear, colourless solution [D].

(36) Solution E: 100 mg/mL 2-Hydroxypropyl-β-Cyclodextrin in Water (20 mL)

(37) 2-Hydroxypropyl-β-cyclodextrin has an average molecular weight of 1460 g/mol. For simplicity it was prepared as follows.

(38) Concentration=100 mg/mL.

(39) 2-Hydroxypropyl-β-cyclodextrin (Sigma Aldrich H107; Lot #048K0672) (2.004 g) was weighed out and dissolved in 20.04 mL ultrapure water. The mixture was shaken on an orbital shaker for 10 minutes to give a clear, colourless solution [E].

(40) Solution F: Panobinostat-2-Hydroxypropyl-β-Cyclodextrin Adduct (5 mM)

(41) Equal volumes of solutions D (2 mL) and E (2 mL) were mixed in a Falcon tube. The clear, colourless solution was vortexed briefly to homogenise.

(42) Solution G: Panobinostat-2-Hydroxypropyl-β-Cyclodextrin Adduct (3.125 mM)

(43) To produce a neutral solution (˜pH7) for injection, a sample of [F] (1 mL) was mixed with NaOH.sub.(aq) (0.6 mL, 0.2M) to give a clear, colourless solution (1.6 mL in volume).

(44) Solution H: Sterile Solution of Panobinostat-2-Hydroxypropyl-β-Cyclodextrin Adduct in aCSF (Artificial Cerebrospinal Fluid) (10 μM)

(45) aCSF Part 1 solution (South Devon Healthcare; Lot #1407292) (155.5 mL) was added to a 250 mL Duran flask. Solution [G] (0.5 mL) was then added via pipette to give a final solution of Panobinostat in aCSF at concentration 10 μM. The solution was briefly mixed and then filter sterilised in a laminar flow hood using a SteriFlip sterile filter cartridge. The clear, colourless solution was then stored at 4° C.

Example 2

HDAC Enzyme Assays

(46) The inhibitory activity of panobinostat dissolved in DMSO and that of the HP-β-CD panobinostat adduct (prepared as described in Example 1) were compared against HDAC types 1-11. Both were tested in singlicate 10-dose IC.sub.50 mode with 3-fold serial dilution starting at 10 μM against 11 HDACs. HDAC reference compounds, Trichostatin A ((R,2E,4E)-6-(4-(dimethylamino)benzoyl)-N-hydroxy-4-methylhepta-2,4-dienamide) and TMP 269 (N-((4-(4-phenylthiazol-2-yl)tetrahydro-2H-pyran-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide), were tested in a 10-dose IC.sub.50 with 3-fold serial dilution started at 10 μM.

(47) The substrates were as follows:

(48) Substrate for HDAC 1, 2, 3, 6, 10: Fluorogenic peptide from p53 residues 379-382 (RHKK(Ac)AMC)

(49) Substrate for HDAC 4, 5, 7, 9 and 11: Fluorogenic HDAC Class2a Substrate (Trifluoroacetyl Lysine)

(50) Substrate for HDAC 8: Fluorogenic peptide from p53 residues 379-382 (RHK(Ac)K(Ac)AMC)

(51) IC.sub.50 values were calculated using the GraphPad Prism 4 program based on a sigmoidal dose-response equation. The blank (DMSO) value was entered as 1.00E-012 of concentration for curve fitting.

(52) The IC.sub.50 values for each compound against each HDAC enzyme are shown below.

(53) TABLE-US-00001 HDAC1 HDAC2 HDAC3 HDAC4 Compound ID IC50 (M) IC50 (M) IC50 (M) IC50 (M) HP-β-CD 8.65E−09 1.59E−08 4.02E−09 5.67E−07 Panobinostat Panobinostat 1.61E−09 4.97E−09 7.51E−10 1.55E−06 in DMSO Trichostatin 1.05E−08 8.83E−09 9.33E−09 ND A TMP 269 ND ND ND 3.76E−07 HDAC5 HDAC6 HDAC7 HDAC8 Compound ID IC50 (M) IC50 (M) IC50 (M) IC50 (M) HP-β-CD 1.10E−07 7.49E−09 1.82E−06 5.57E−08 Panobinostat Panobinostat 1.66E−07 1.77E−09 3.68E−06 5.19E−08 in DMSO Trichostatin ND 1.41E−09 ND 3.66E−07 A TMP 269 3.54E−07 ND 1.53E−07 ND HDAC9 HDAC10 HDAC11 Compound ID IC50 (M) IC50 (M) IC50 (M) HP-β-CD 9.27E−07 2.03E−08 1.38E−06 Panobinostat Panobinostat in 1.04E−06 1.27E−08 1.67E−06 DMSO Trichostatin A ND 3.96E−08 7.89E−06 TMP 269 4.33E−08 ND ND ND indicates compound no tested against enzyme.

(54) The results show that HP-β-CD panobinostat is equipotent to panobinostat in DMSO across the range of HDAC enzymes 1-11.

Example 3

In Vitro Cell Toxicity

(55) Cytotoxicity of HP-β-CD panobinostat was assessed in Heptacellular Carcinoma and Glioblastoma.

(56) U87MG (Glioblastoma) and HEPG2 (Hepatocellular carcinoma) cell lines were exposed to increasing doses of HP-β-CD panobinostat, Cyclodextrin alone or Artificial Cerebral Spinal Fluid (Artificial-CSF) alone for either 48 or 72 hrs. Cell viability was then assessed using MTT reagent. The IC.sub.50 of HP-β-CD panobinostat was determined to be approximately 0.01 μM (see FIGS. 1A-D). Cyclodextrin alone and artificial CSF alone were essentially non-toxic under the conditions tested.

(57) Cytotoxcity of HP-β-CD panobinostat was assessed in human SF8628 DIPG cells (gift from Gupta group at UCSF, USA).

(58) A fluorescent live-dead cell viability assay (Invitrogen) was employed. Cells cultured in presence of HP-β-CD panobinostat in artificial CSF for 72 hours at doses of 10 μM, 5 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, and 0.01 μM in triplicate. The assay was repeated three times in three different passage. Cell viability compared to live and dead controls (& vehicle only control). The dose response curve was plotted using GraphPad Prism software (see FIG. 2A). The IC.sub.50 value was calculated to be 0.01 μM.

(59) These results demonstrate that panobinostat formulated as an HP-μ-CD panobinostat adduct is soluble in artificial CSF at neutral pH and displays potent cancer cell killing activity against a range of tumour cell lines, including glioma and, in particular, DIPG cells in vitro.

(60) In order to investigate the time-course of HP-β-CD panobinostat action on DIPG cells, cells were cultured in presence of HP-β-CD panobinostat for 72 hours, 6 hours and 30 minutes at doses of 5 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM in triplicate, with three separate cell passage. Short time point incubation (6 hours and 30 minutes) assayed at 72 hours post dosing. The results are shown in FIG. 2B. IC.sub.50 values are shown for each time point below:

(61) TABLE-US-00002 Incubation time IC.sub.50 (μM) 72 hours 0.01  6 hours 0.04 30 minutes 0.06

(62) These results indicate that even brief exposure to cytotoxic concentrations may be sufficient for anti-tumour efficacy. Pharmacokinetic data suggest that HP-β-CD panobinostat in artificial CSF for CED-based delivery diffuses out from the catheter tip rapidly.

Example 4

Solubility and Stability of Panobinostat Formulations

(63) From literature study, it has been identified that panobinostat may be dissolved in DMSO/water mixed phases. In order to administer the drug directly to the brain of a patient via the CED approach, filtered artificial cerebrospinal fluid (CSF) must be used as a diluent.

(64) In the context of CED-based delivery, a drug solution is typically prepared on the day before the scheduled administration. If a patient is scheduled to receive a CED treatment on Monday, the drug solution would be prepared on Friday and stored in a refrigerator until Monday. Hence, a solution of panobinostat prepared in artificial CSF must be demonstrably stable for at least 48 hrs at 4° C.

(65) This study aimed to determine the concentrations of panobinostat that can be successfully reached in DMSO/aCSF mixtures and their stability at 4° C. and room temperature.

(66) A stock solution of panobinostat in DMSO was prepared at 70 mg/mL 0.2M≡200 mM.

(67) From this solution, a number of DMSO working solutions were prepared at 20 mM, 10 mM, 5 mM, 2 mM and 1 mM by dilution in DMSO.

(68) These solutions were diluted 50:50 with artificial CSF (aCSF) and gave the following results.

(69) TABLE-US-00003 After ½ dilution with aCSF to give . . . 100 mM 20 mM 10 mM 5 mM 2 mM 1 mM Soluble/homogeneous No No No Yes Yes Yes

(70) The 20 mM solution was diluted further with aCSF to give the following concentrations. After mixing by vortex, samples were centrifuged for 60 seconds.

(71) TABLE-US-00004 20 mM solution diluted with aCSF to give 10 mM 5 mM 2 mM Soluble/homogeneous No No No (pellet) (pellet) (pellet)

(72) The 10 mM solution was diluted further with aCSF to give the following concentrations. After mixing by vortex, samples were centrifuged for 60 seconds.

(73) TABLE-US-00005 20 mM solution diluted with aCSF to give 5 mM 2 mM 1 mM 0.5 mM Soluble/homogeneous Yes No No No (pellet) (pellet) (pellet)

(74) It is apparent that the DMSO:aCSF ratio is important. Useful working concentrations can be prepared between 5 mM and 1 mM, but only if the DMSO ratio is ˜50% v/v. For human in vivo use, presence of DMSO, particularly at a concentration as high as 50% v/v, is undesirable.

(75) By comparison, the HP-β-CD panobinostat adduct is soluble and stable in artificial CSF at 4° C. for at least 48 hours.

(76) Stock solutions of Panobinostat (10 mM) were made and dissolved in either DMSO or citrate buffer (pH 3). Samples were diluted to allow analysis by HPLC (to avoid overloading the system). Peak areas were measured for both solutions at time 0, 100 h and 150 h post preparation. Samples were stored at room temperature over the course of the analysis. Percentage change was calculated based on the peak area and compared to analysis at time zero. One replicate was analysed and 7.2 μl was injected (equivalent to 0.25 μg on column) for each sample. The concentration of the stock solutions assessed was 10 mM.

(77) Column: Ascentis Express Peptide, ES-C18, 10 cm×4.6 mm

(78) Mobile phases: 0.1% trifluoroacetic acid (TFA) in acetonitrile (organic) and 0.1% trifluoroacetic acid in water

(79) HPLC: Agilent 1260 infinity

(80) HPLC conditions used:

(81) TABLE-US-00006 Time Flow Water + Acetonitrile + (min) (ml) TFA TFA 0 1 90 10 2 1 90 10 8 1 0 100 9 1 90 10

(82) Results:

(83) At 100 hrs, the DMSO solution shows the Panobinostat concentration had decreased by 2.8%, whilst the citrate solution had decreased by 0.4%. Similarly, at 150 hrs the panobinostat in the DMSO solution had decreased by 4.4%, whereas the panobinostat in the citrate solution had decreased by only 1%.

(84) TABLE-US-00007 Percentage relative to t = 0 0 h 100 h 150 h DMSO 100 97.2 95.6 Citrate 100 99.6 99

(85) Without wishing to be bound by any particular theory, the present inventors believe that the relatively lower stability of panobinostat in DMSO compared with that in the citrate solution may be caused by the oxidising nature of DMSO and its action on panobinostat. In light of this, it would be desirable to formulate panobinostat in a substantially DMSO-free solution.

(86) A solution of HP-β-CD-Panobinostat in artificial CSF was prepared at an initial concentration of 40 μM panobinostat and several aliquots stored at 5° C. in upright sealed vials. Samples were taken at various time points (t=0, day 1, day 8, day 18 and day 22) and the panobinostat concentration measured by HPLC. The remaining panobinostat is expressed at a percentage of the starting concentration at t=0. The results were:

(87) TABLE-US-00008 Day Panobinostat conc. 0   100%  1 >99% 8 >99% 18 >99% 22 >99%

(88) At all of the time points measured, up to and including 22 days, the concentration of panobinostat was essentially unchanged: greater than 99% of the initial concentration. These results demonstrate that the HP-β-CD-Panobinostat formulated in artificial CSF is remarkably stable.

Example 5

Human DIPG Patient Treatment with HP-β-CD Panobinostat Via CED

(89) A single patient (5 year old female) with DIPG was treated by direct CED infusion of HP-β-CD-panobinostat in artificial CSF.

(90) The patient had a pre-implanted drug delivery system that incorporates a skull mounted transcutaneous port, which allows repeated administration of drug via CED.

(91) HP-β-CD-panobinostat in artificial CSF was prepared as described above. HP-β-CD-panobinostat at a concentration of 10 μM was infused/convected over a period of about 8 hours for a total infused volume of 12 mL distributed over four catheters. Consultant-led paediatric anaesthetic care was provided. Continuous physiological monitoring was present, and hourly assessment of neurological function was obtained. This included accurate recording of limb and cranial nerve function.

(92) The administration of HP-β-CD-panobinostat in artificial CSF via CED was found to be well-tolerated and no adverse events were observed.

Example 6

Lyophilisation of HP-β-CD-Panobinostat Adduct

(93) Two batches of the HP-β-CD-panobinostat adduct were produced according to the method set out in Example 1.

(94) The first batch was released and laid down at 4° C. for long-term stability testing. At day 42, visible particles were observed in many vials of the product (but not all). Analysis of these particles showed them to contain only inorganic salts (i.e. they did not contain either the cyclodextrin or the panobinostat compound). Analysis by Energy-dispersive X-ray spectroscopy (EDX) showed the presence of magnesium and phosphorus, hence it was concluded that the visible particles were magnesium phosphate crystals.

(95) Without wishing to be bound by any particular theory, the present inventors believe that the presence of an nanoparticulate form of cyclodextrin-adduct in artificial CSF (aCSF) may encourage nucleation of certain salts present in the aCSF. Artificial CSF contains a mixture of salts at varying concentrations. Some of these salts have intrinsically lower solubilities than others, hence are more likely to nucleate over time. The addition of a relatively large mass of cyclodextrin thought to be in an already-solid, nanoparticulate form, to the solution may well accelerate this nucleation.

(96) The present inventors theorised that an improvement could be brought about by introducing a lyophilisation step. That is, by minimising the time these lower solubility salts spend in solution with the HP-β-CD-panobinostat adduct, i.e. by reconstituting just before administration, the inventors expect to circumvent the problem of gradual crystallisation of inorganic salts.

(97) In the present example, the lyophilisation process involved cooling the samples containing HP-β-CD-panobinostat adduct to −52° C. under a vacuum (0.053 mbar) for around 2 hrs. However, other lyophilisation procedures are contemplated provided that they yield a substantially desiccated solid (e.g. water content below 10% w/w, below 5% w/w, or even below 2% w/w). The skilled person will be aware of numerous freeze-drying processes that will find use in connection with the present invention.

(98) The modified process for production of HP-β-CD-panobinostat adduct in artificial CSF, in which the HP-β-CD-panobinostat adduct is lyophilised for storage/shipping prior to reconstitution in aCSF at the time of delivery to give a ready-to-inject solution, is set forth below.

(99) Solution A: 0.1M Citric Acid in Water

(100) Citric acid (Sigma Aldrich C-0759; Lot #21K0042) (2.101 g) was weighed out and dissolved in 100 mL ultrapure water.

(101) Solution B: 0.1M Sodium Citrate in Water

(102) Sodium citrate (tribasic) (Sigma Aldrich C3434; Lot #1304640/41807230 (2.944 g) was weighed out and dissolved in 100 mL ultrapure water.

(103) Solution C: 0.1M Citrate Buffer

(104) Solution A (82 mL) and solution B (18 mL) were added to a 250 mL Duran flask and mixed briefly to form [C]. The pH of the citrate buffer was measured and found to be pH3.0.

(105) Solution D: 10 mM Panobinostat solution in citrate buffer (25 mL)

(106) Concentration=10 mM=0.01M

(107) Volume=25 mL=0.025 L

(108) Therefore number of moles required=2.5×10.sup.−4

(109) Molecular weight of Panobinostat=349.43 g/mol

(110) Therefore mass of Panobinostat required=82.36 mg

(111) Thus, Panobinostat free base (81.0 mg) was weighed out and dissolved in solution C (24.59 mL).

(112) The mixture was shaken on an orbital shaker for 10 minutes to give a clear, colourless solution [D].

(113) Solution E: 100 mg/mL 2-Hydroxypropyl-β-Cyclodextrin in Water (20 mL)

(114) 2-Hydroxypropyl-β-cyclodextrin has an average molecular weight of 1460 g/mol. For simplicity it was prepared as follows.

(115) Concentration=100 mg/mL. 2-Hydroxypropyl-β-cyclodextrin (Sigma Aldrich H107; Lot #048K0672) (2.004 g) was weighed out and dissolved in 20.04 mL ultrapure water. The mixture was shaken on an orbital shaker for 10 minutes to give a clear, colourless solution [E].

(116) Solution F: Panobinostat-2-Hydroxypropyl-β-Cyclodextrin Adduct (5 mM)

(117) Equal volumes of solutions D (2 mL) and E (2 mL) were mixed in a Falcon tube. The clear, colourless solution was vortexed briefly to homogenise.

(118) Solution G: Panobinostat-2-Hydroxypropyl-β-Cyclodextrin Adduct (3.125 mM)

(119) To produce a neutral solution (˜pH7), a sample of [F] (1 mL) was mixed with NaOH.sub.(aq) (0.6 mL, 0.2M) to give a clear, colourless solution (1.6 mL in volume).

(120) Lyophilised Solution G: 2 mL of adduct Solution G was lyophilised by cooling to −52° C. under a vacuum (0.053 mbar) for around 2 hrs to produce a solid cake. The lyophilised solid may then be stored as necessary, e.g. for shipping to a site of delivery.

(121) Solution H: Sterile solution of Panobinostat-2-Hydroxypropyl-β-Cyclodextrin Adduct in aCSF (Artificial Cerebrospinal Fluid) (40 μM)

(122) The lyophilised solid cake from 2 mL of solution G above was reconstituted by adding 10 mL of aCSF to give a 40 μM solution of Panobinostat in aCSF. This solution may be further diluted according to the dosage or volume that is required for the treatment.

Example 7

Long-Term Storage Stability of HP-β-CD-Panobinostat Adduct

(123) The long-term stability of the HP-β-CD-panobinostat adduct was assessed by measuring panobinostat concentration and percentage impurities at intervals from t=0 to a total of 24 weeks stored at −25° C.-−15° C. (data not shown), 2° C.-8° C. (data not shown), and 15° C.-25° C. (FIGS. 3 and 4).

(124) As shown in FIG. 3, panobinostat concentration was found to be very stable over the 24-week period at 15° C.-25° C. The equivalent concentrations plots for −25° C.-−15° C. and 2° C.-8° C. also showed a similar profile with measured concentration remaining within the range 38 μM to 45 μM.

(125) Possible degradation of panobinostat over time at 15° C. to 25° C. was assessed by measuring peaks either side of the main panobinostat compound peak by HPLC (see RRT 0.86 and RRT 1.12 in FIG. 4). Total impurities as a percentage are also shown on FIG. 4. The FIG. 4 plots show that impurities remained at a very low level (around 1% or less) for the 24-week duration of the study. Very similar results were seen for storage at −25° C.-−15° C. and 2° C.-8° C. over the same time period.

(126) Taken together, the panobinostat concentration and impurity measurements indicate that the HP-β-CD-panobinostat adduct is stable even at room temperature over a period of at least up to 24 weeks.

(127) All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

(128) The specific embodiments described herein are offered by way of example, not by way of limitation. Any sub-titles herein are included for convenience only, and are not to be construed as limiting the disclosure in any way.