WO2006034849A1

WO2006034849A1 - Antitumoral pharmaceutical compositions comprising a spisulosine and a cyclodextrin

Info

Publication number: WO2006034849A1
Application number: PCT/EP2005/010442
Authority: WO
Inventors: Monique W. J. Den Brok; Bastiaan Nuijen; Jos H. Beijnen; Consuelo Manada Del Campo
Original assignee: Pharma Mar, S.A., Sociedad Unipersonal
Priority date: 2004-09-27
Filing date: 2005-09-26
Publication date: 2006-04-06

Abstract

The present invention relates to a pharmaceutical composition, in particular to a composition comprising a spisulosine compound and a cyclodextrin or cyclodextrin derivative, and its use in the treatment of cancer. The pharmaceutical composition has long term stability upon storage and does not form gels or precipitates when diluted for administration for long periods of time.

Description

ANTITUMORAL PHARMACEUTICAL COMPOSITIONS COMPRISING A SPISULOSINE AND A CYCLODEXTRIN

FIELD OF THE INVENTION The present invention relates to a pharmaceutical composition, in particular to a composition comprising a spisulosine compound and a cyclodextrin or cyclodextrin derivative, and its use in the treatment of cancer.

BACKGROUND OF THE INVENTION In the effort to find antitumoral compounds from marine organisms, the spisulosines were first isolated from the marine clamp Spisula polynyma. See for example WO 99/52521 that describes spisulosine 285 (ES-285), ES-299 and ES-313 among other compounds. The spisulosines are long-chain, straight-chain allcanes having a amino group and a hydroxy group next to it. Preferably they are in positions 2 and 3 (2-amino-3- hydroxy-), although other positions are possible such as 3-amino-4-hydroxy-. The chain has from 14 to 24 carbons, for example it has 18 C for ES-285. More spisulosine compounds and derivatives thereof are described in WO 01/94357.

Spisulosine 285 has shown in vitro and in vivo cytotoxic activity against various tumor cell lines with selectivity for certain solid tumors (i.e. hepatocellular, prostate, and renal) and has been selected for further development. It is used in the form of one of its salts, ES-285.HC1 ((2S,3R)-2-amino-3-octadecanol hydrochloride), as an investigational anticancer agent. Cytotoxic compounds are usually administered to the patient as an intravenous infusion for a certain period of time and at certain intervals, in cycles. This requires the compound to be soluble and stable in water or a physiological solution such as a saline solution.

ES-285 has low aqueous solubility, even after pH adjustment. Besides the long alkyl chain, the aqueous solubility of ES-285 might be explained by the formation of an intramolecular hydrogen-bond between the hydroxyl and amine function, hindering ionization of the functional groups and leading to a much lower pKa than commonly seen for a primary amine. The solubility can be improved by the use of a salt instead of the free base. However, although adequate solubility in water can be reached for the spisulosine compounds or their salts, there is the additional problem that after a certain time there is a tendency to gel formation due to an aggregation of the molecules. In such a situation, a spisulosine solution will not be adequate for infusion, because times of infusion can go up to 24 or even 72 hours, and gel formation can occur over such periods of time.

In view of the potential of spisulosine compounds as antitumoral agents, there is a need to provide a formulation that solves the problem of stability in solution of these compounds. The formulation should be able to be freeze dried, reconstituted and further diluted with infusion fluid without any problems. Further, the formulation containing spisulosine compounds should be stable during long term storage. Importantly, the vehicle(s) used for the formulation should be non-toxic at the concentrations used for infusion. Finally, precipitation or gel formation of the drug substance upon intravenous injection, in particular during long time infusions, should be avoided, since this might cause inadequate dosing, thrombophlebitis and even the formation of emboli in blood capillaries.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a stable pharmaceutical composition of spisulosine compounds. It is another object to provide a pharmaceutical composition that does not precipitates and gelifϊes upon reconstitution and dilution for use, in particular for intravenous infusion. Another object of the present invention is to provide pharmaceutical compositions that do not provoke ulcerative damages, nor thrombophlebitis, at the site of injection.

We have found that formulations of spisulosine compounds solving the above problems can be obtained by using a cyclodextrin or cyclodextrin derivative.

In one aspect, the invention is directed to a pharmaceutical composition comprising a compound according to formula I CH₃ (

wherein n is an integer selected from 11, 12, 13, 14, 15 or 16, R is -CH₃ , -CH₂CH₃ or -CH₂ CH₂CH₃, or a pharmaceutically acceptable acid-addition salt thereof; and a cyclodextrin or cyclodextrin derivative.

In a preferred embodiment the spisulosine compound is such that n is 13, 14 or 15, preferably 14.

In another embodiment R is preferably methyl or ethyl.

In another embodiment the compound is in the form of the hydrochloride salt.

In a most preferred embodiment the pharmaceutical composition comprises ES- 285. HCl, i.e the compound is ((2S,3R)-2-amino-3-octadecanol hydrochloride.

In one embodiment the cyclodextrin derivative is an etherified cyclodextrin. Preferably it is a β cyclodextrin.

In another embodiment the cyclodextrin is an hydroxyalkyl substituted β cyclodextrin, preferably 2-hydroxypropyl-β-cyclodextrin.

It is preferred that that the Molar degree of substitution of the 2-hydroxypropyl-β- cyclodextrin is comprised in the range of form 0.125 to 0.95 more preferably in the range of 0.2 to 0.80. Most preferred is a Molar degree of substitution of 0.60 to 0.65.

When the Molar degree of substitution of the 2-hydroxypropyl-β-cyclodextrin is comprised in the range of from about 0.5 to about 0.7, preferably from about 0.60 to about 0.65 a lower amount of 2-hydroxypropyl-β-cyclodextrin can be used with good results. In another embodiment the weight ratio of the amount of compound of formula I to the amount of cyclodextrin or cyclodextrin derivative is from about 1 : 15 to about 1 :45, preferably from about 1 : 15 to about 1 :25.

In a further embodiment the invention is directed to a pharmaceutical composition comprising ES-285.HC1 and 2-hydroxypropyl-β-cyclodextrin molar degree of substitution of about 0.65 in a weight ratio of about 1 :20.

In a preferred aspect, the pharmaceutical composition is in freeze-dried form.

In another aspect the invention is directed to a method for treatment of cancer comprising parenterally administering the pharmaceutical composition as defined above to a patient in need thereof.

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, we have found that cyclodextrins or cyclodextrins derivatives not only improves the solublity characteristics of the spisulosine compounds, but more important, they allow for the obtention of solutions stable over long periods of time and at the same time stable upon storage when in freeze dried form.

As summarized above, the present invention relates to novel pharmaceutical compositions of spisulosine compounds. The spisulosine compounds used in the present invention are represented by formula I:

wherein n is an integer selected from 11, 12, 13, 14, 15 or 16,

R is -CH₃, -CH₂CH₃ or -CH₂ CH₂CH₃, and their pharmaceutically acceptable acid- addition salt. Preferably n is 13, 14 or 15, compounds with such length of the alkyl chain have good properties in terms of biological activity.

When n is 14 and R is -CH₃ we have ES-285 which is the most preferred compound in view of its activity. Its hydrochloride salt, ES-285. HCl, is especially preferred:

ES-285.HC1

However, other compounds are also envisaged by the present invention, having shorter or longer chains, or having ethyl or propyl instead of methyl next to the amino group. Some representative compounds are:

NH₃Cl NH₃CI

NH₃CI

These compounds are readily available by known synthetic procedures, such as those described in US 6,107,520 and WO 01/94357. Different salts can be obtained by reacting the free base forms of these compounds with a stoichiometric amount of the appropriate acid in water or in an organic solvent or in a mixture of the two. Generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. Examples of such acid addition salts include mineral acid addition salts such as, for example, hydrobromide, hydroiodide, sulphate, nitrate, phosphate, and organic acid addition salts such as, for example, acetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulphonate and p-toluenesulphonate among other. Among the acid addition salts, the hydrochloride salt is preferred.

The pharmaceutical composition of the invention is further characterized because it comprises a cyclodextrin or cyclodextrin derivative.

Cyclodextrins are cone-shaped, cyclic oligosaccharides consisting of covalently (α- l,4)-linked α-D-glucopyranose rings with a relatively hydrophilic outer surface and lipophilic cavity. They have 6-12 glucose units, preferably 6-8. They are named α, β or γ cyclodextrins when the number of glucose units present is 6, 7 or 8 respectively.

Cyclodextrins are capable of improving various physicochemical properties such as solubility and stability of drug by forming inclusion complexes (Uekama K, Hirayama F, Irie T: Cyclodextrin drag carrier system. Chem Rev 98:2045-2076. 1998).

As used herein, the term "cyclodextrin derivative" means a cyclodextrine or mixtures thereof wherein hydrogen(s) of a part of or all hydroxyl groups at the 2-, 3- and 6- positions of glucose is (are) substituted by other functional groups, such as a dihydroxyalkyl group, a saccharide residue, a hydroxyalkyl group, a sulfonate group, a sulfoalkyl group, an alkyl group, alkanoyl group, acetyl group or benzoyl group.

The cyclodextrin or cyclodextrin derivative used in the present invention may be a commercially available one or can be produced by a method known per se. β cyclodextrins or derivatives thereof are preferred.

In one embodiment an etheriiϊed cyclodextrin derivative, is used. Good results are obtained with hydroxyalkyl substituted β cyclodextrins. Among them, 2-hydroxypropyl-β- cyclodextrin (HPBCD) is most preferred. It is a hydroxypropyl-derivative of β-cyclodextrin with high water solubility (>50% w/v) and low toxicity, see US 3,459,731 or Croft et al. Tetrahedron 39, 1417 (1983) for methods of manufacture. In fact it is a mixture of substituted cyclodextrins. It has been used for intravenous administration (Carpenter TO, Gerloczy A, Pitha J: Safety of parenteral hydroxypropyl beta-cyclodextrin. J Pharm Sd 84:222-225. 1995). For example, HPBCD has recently been commercialized in US and Europe for parenteral injection for itraconazole (Sporanox^®).

The average substitution degree (DS) refers to the average number of substituted 2- hydroxyls per β-cyclodextrin unit, whereas the Molar substitution degree (MS) refers to the number of hydroxypropyl groups per anhydroglucose unit. When 2-hydroxypropyl-B- cyclodextrin (HPBCD) is used in the compositions according to the present invention, it has preferably a MS in the range of 0.125 to 0.95, more preferably in the range of 0.2 to 0.80.

We have found that MS ranges from about 0.5 to about 0.7, in particular from about 0.60 to about 0.65 give good stability results and allow lower concentrations of HPBCD to be used.

The pharmaceutical composition is preferably obtained by dissolving a solution containing the spisulosine compound in the adequate solution of cyclodextrin or cyclodextrin derivative and stirring, normally at ambient temperature. After complete dissolution, if necessary the solution can be adjusted to the predetermined final weight with more solution of cyclodextrin or cyclodextrin derivative.

In view of the price of cyclodextrin or cyclodextrin derivatives for pharmaceutical use and the possible toxicity problems they might generate, in one embodiment the weight ratio of the amount of compound of formula I to the amount of cyclodextrin or cyclodextrin derivative is from about 1 :10 to about 1 :50, more preferably from 1 :15 to about 1 :45, even more preferably from 1 :15 to about 1 :25, preferably about 1 :20. At such low concentrations the cyclodextrin or cyclodextrin provides the above mentioned advantages and does not increase the risk of toxicity. Surprinsingly, they also show long term stability, even up to 24 months. In another embodiment, to reduce the number of vials needed for treatment, a higher dose of spisulosine compound per vial is required. In such a case higher concentrations of spisulosine compound are needed, that can be reached with the use of the above mentioned cyclodextrins, in particular with those with a MS range from about 0.5 to about 0.7, preferably from about 0.60 to about 0.65, in higher concentrations. A concentration of about 20 mg ES-285 in about 40% HPBCD is preferred.

It is preferred that the pharmaceutical composition is in the freeze dried form, this allows long term storage and easier shipment. Well known freeze drying techniques can be used, the freeze and drying characteristics of the pharmaceutical composition are mainly governed by the cyclodextrin or cyclodextrin derivative, this being the major component of the solution.

The pharmaceutical compositions object of the present invention are prepared for parenteral use according to conventional techniques adopted in the preparation of pharmaceutical forms. Typically, a proper amount of the pharmaceutical composition, either as a dry powder or into a lyophilised form, is first dissolved in a small amount of reconstitution solution such as water for injection and then further diluted in a pharmaceutically acceptable solution for parenteral use, such as sterile water, 0.9% NaCl solution (normal saline) or aqueous dextrose solution, e.g. 5% dextrose in water for intravenous administration. Preferably a solution of 5% dextrose in water is used for dilution because it has shown to enhance the drug intrinsic solubility.

The pharmaceutical compositions of the invention are administered to a patient in need thereof for the treatment of cancer. The formulation, once diluted for administration is given in cycles. In the preferred application method, an intravenous infusion of the pharmaceutical compositions of the invention is given to the patients the first week of each cycle, the patients are allowed to recover for the remainder of the cycle. Dose delays and/or dose reductions and schedule adjustments are performed as needed depending on individual patient tolerance of treatments. Further information concerning the administration of chemotherapy can be found in DeVita, V. T. Jr. , Hellman, S. and Rosenberg, S.A. , Cancer: Principles and Practice of Oncology, 6^th. ed. , 2001, Lipincott, Philadelphia.

The formulations object of the present invention allow the administration of the spisulosine compounds either as a single agent or, alternatively, in combination with known anticancer treatments such as radiation therapy or another chemotherapeutic agent.

The following examples are intended to further illustrate the invention, they should not be interpreted as limiting the scope of the claims.

EXAMPLES

United States Pharmacopoeia (USP) grade 2-hydroxypropyl-B-cyclodextrin

(HPBCD, average Mw of 1399) with a molar degree of substitution of 0.65 was purchased from Roquette (Kleptose" HB, Lestrem, France) and European Pharmacopoeia (Ph.Eur) grade HPBCD with molar degrees of 0.60 and 0.93 were from Cerestar (Cavitron^® 82003 and 82004 respectively, Mechelen, Belgium). Throughout the experiments, the amounts of

HPBCD were corrected for water content. All chemicals were of analytical grade and used without further purification. Distilled water was used throughout.

MS-spectra were collected on a Sciex API 365 triple quadrupole LC/MS/MS spectrometer (Sciex, Thornhill, ON, Canada) equipped with an electrospray interface (ESI) ionization source operating in the positive ion mode. ES-285.HC1 drug substance in a concentration of 10 μg/mL in methanol was infused via continuous infusion. The product ion scan was obtained by mass-selecting the precursor ion from the Ql scan. ¹H NMR spectra were collected at room temperature on a Gemini 300 BB instrument (Varian Assoc, Palo Alto, CA; USA) operating at 300.1 mHz for ¹H. 1 mg of ES-285.HC1 drug substance was dissolved in deuterochloroform (CDCl₃) with 10% DMSO-J₆. Proton chemical shifts were determined relative to tetramethylsilane (TMS). The IR spectrum (400-4000 cm^"1) was obtained with a Model PU 9706 IR spectrophotometer (Philips Nederland B.V., Eindhoven, The Netherlands) using the potassium bromide (KBr) pellet technique. The pellet consisted of 1 mg ES-285.HC1 drug substance and 200 mg KBr. The ratio recording mode was auto-smooth and the scan time 8 minutes. UV/VIS spectra were recorded with a Model UV/VIS 918 spectrophotometer (GBC Scientific Equipment, Victoria, Australia). The spectrum of ES-285.HC1 drug substance in a concentration of 50 μg/mL in methanol was recorded from 800 to 200 nm.

ES-285.HC1 content and purity of drug substance and final product were assayed by a validated, stability-indicating HPLC-UV method after derivatisation with phenylisothiocyanate (PITC) (Den Brok MWJ,et al.: "Development and validation of a liquid chromatography-ultraviolet absorbance detection assay using derivatisation for the novel marine anticancer agent ES-285.HC1 [(2S,3i?)-2-amino-3~octadecanol hydrochloride] and its pharmaceutical dosage form" J Chrom A 1020:251-258. 2003).

Residual water content of ES-285.HC1 final product was determined using a Model 658 KF Titrino apparatus (Metrohm, Herisau, Switzerland). Analyses were carried out in triplicate using the Karl Fischer titration method.

HPBCD content of ES-285.HC1 final product was determined indirectly via

UV/VIS spectrophotometric analysis based on the decreasing effect of HPBCD on the UV/VIS absorbance of phenolphthaleine in aqueous alkaline solutions (Frijlink HW, et al.: "Determination of cyclodextrins in biological fluids by high-performance liquid chromatography with negative colorimetric detection using post-column complexation with phenolphthalein", J Chromatogr 415:325-333. 1987). Calibration samples and quality control samples in the concentration of 35, 40, 50, 60, and 65 μg/mL HPBCD in 5 mM phosphate buffer containing 0.003 mM of phenolphthaleine were prepared from two separately weighed stock solutions. ES-285.HC1 lyophilised product was reconstituted with 5.0 mL of WfI and subsequently diluted with WfI to a theoretical HPBCD concentration of 50μg/mL. In the final dilution step, phenolphthaleine and phosphate buffer solution were added to obtain a concentration of 0.003 mM of phenolphthaleine and 5 mM phosphate buffer. UV absorbance was measured at 553 nm. Least-squares analysis of concentration, versus the measured absorption was applied.

Example 1: Solubility experiments

ES-285.HC1 was screened for solubility in various solvents at ambient temperature (+20-25°C). Solvents were chosen on the basis of current use and experience in clinical practice. Approximately 1 mg of ES-285.HC1 was weighed in a glass screw-capped test tube and subsequent solvent volumina of 100 μL, 1 mL, and 10 mL were added to the drug substance. After each addition the mixture was vigorously shaken for 30 seconds, placed in an ultrasonic bath for 15 minutes and examined visually under polarised light for complete dissolution. In this way, solubility of E S-285. HCl in the respective solvents was distributed over four ranges (s < 0.1 mg/mL; 0.1 mg/mL < s < 1 mg/niL; 1 < s < 10 mg/mL; s > 10 mg/mL). Solvent systems in which ES-285.HC1 visually dissolved were diluted 1 :1 v/v, 1 :2 v/v, 1 :10 v/v and 1:100 v/v with normal saline for infusion in glass test tubes. After gentle agitation, each of the dilutions was examined visually under polarised light over a one-day period for any sign of precipitation. The results of the solubility screening for ES- 285. HCl in various solvent systems are given in table 1.

Table 1: Solubility of ES-285.HC1 in various solvent systems

Solvent system Solubility (mg/mL)

Water for Injection (WfI) 0.1 < s < 1 ethanol absolute (EtOH) s > 10

Dimethyl Sulfoxide s > 10

N-methyl-2-pyrrolidone s > 10

Polyethylene glycol 300/EtOH/polysorbate 80 60/30/10% v/v/v s > 10

Cremophor EL/EtOH 50/50% v/v s > 10

Propylene glycol/EtOH/Wfl 40/10/50°/ό v/v/v 1 < s < 10

Wfl/EtOH/polysorbate 80 62.5/12.5/25% v/v/v 1 < s < 10

0.5% v/v polysorbate 80 in saline s < 0.1

10 mM anhydrous citric acid in WfI (pH 2.6) s < 0.1

10 mM TRIS buffer in WfI (pH 10.1 ) s < 0.1

40% w/v 2-hydroxypropyl-β-cyclodextrin (HPβCD) s ≥ 10

ES-285.HC1 is only very slightly soluble in water (0.3 mg/mL), but dissolves well in a number of organic solvents. Furthermore, ES-285.HC1 concentrations of > 10 mg/mL were reached in the co-solvent/surfactant systems 60/30/10% (v/v/v) polyethylene glycol 300/ethanol/polysorbate 80 (PET) and 50/50% (v/v) Cremophor EL/ethanol, and in 40% (w/v) HPBCD solubilised in water. However, upon dilution with normal saline up to 1 :100 (v/v), only ES-285.HC1 solubilised in PET and 40% (m/v) HPBCD did not precipitate over a 24-hour period. The maximal solubility of ES-285.HC1 in PET vehicle was found 12 mg/mL. The volume of this vehicle to be administered intravenously with ES-285.HC1 in the phase I clinical studies would be at least 6 mL based on the expected dose range, which was not considered to be appropriate. Thus HPBCD was selected as the vehicle of choice.

Although the solubilisation efficacy of HPBCD was shown sufficient, an attempt was made to increase the efficacy, in order to limit the amount of HPBCD needed to solubilise ES-285.HC1. An increase in cyclodextrin solubilisation efficacy is often accomplished with the addition of low concentrations of water-soluble polymers. Addition of the intravenously applicable pharmaceutical polymer polyvinylpyrrolidone (0.25% w/v), however, did not increase the solubility of ES-285.HC1 in a 20% (w/v) HPBCD solution.

Example 2: phase solubility diagram of ES-285.HC1 in HPBCD

A phase solubility diagram of ES-285.HC1 in HPBCD solution was generated according to the method of Higuchi and Connors ("Phase-solubility techniques", Adv Anal

Chem Instr 7:117-212. 1965). An excess amount of ES-285.HC1 was suspended in 1.0 mL of solutions containing 0, 5, 10, 20 and 40 % w/v HPBCD in glass screw-capped test tubes and subsequently shaken at room temperature until equilibrium. Experiments were conducted in duplicate. The resulting suspensions were centrifuged (4100 x g, 5 min), the supernatant filtered through a 0.45 μm membrane filter (Durapore syringe filter, Millipore,

The Netherlands) and analysed for ES-285.HC1 content. From the phase solubility diagram it can be seen that the aqueous solubility of ES-285.HC1 increases significantly with increasing concentrations of HPBCD, up to a factor 105 in HPBCD 40% w/v.

The stability constant (Ki_:1, in L/mole) was calculated from the initial linear portion of the phase solubility diagram, using equation 1 :

K₁, = ^slope (i)

So(I - slope) where S₀ represents the intrinsic solubility of the drug (in mole/L). The apparent stability constant determined from the linear portion of the phase solubility diagram was found to be 1045 L/mole.

Example 3: Cyclodextrin utility number The apparent stability constant was used to assess the feasibility of formulating ES- 285. HCl with HPBCD. The cyclodextrin utility number (U_CD) was calculated according to Rao VM and Stella VJ:"When can cyclodextrins be considered for solubilization purposes?" J Pharm Sci 92:927-932, 2003, via equation 2:

Where mo and ΓQ_CD represent the drug dose and workable amount of HPBCD in mg, respectively, and MW_D and MW_CD the molecular weights of drug and HPBCD, respectively. The U_CD was introduced by Rao and Stella as a guiding tool to determine, with minimum experimentation, if cyclodextrins might be the right choice as solubilisation enhancer for a given poorly water-soluble drug. When the dimensionless number, U_CD is found greater than or equal to one, solubilisation is adequately provided for by HPBCD. The U_CD for ES-285.HC1/HPBCD was calculated using a workable amount of HPBCD of 8 grams, which is the amount currently used in the marketed itraconazol formulation, the apparent stability constant of 1045 L/mole and an expected maximum ES-285.HC1 dose of 70 mg per day. The resulting U_CD value of 12.9 supports the use of HPBCD as potential vehicle for ES-285. HCl.

Example 4: HPβCD degree of substitution and concentration

Three different HPBCD commercial products were tested for possible use with ES- 285.HC1. Besides the USP grade Kleptose^® (with a molar degree of substitution of 0.65) used to construct the phase solubility diagram, Ph.Eur. grade Cerestar with molar degrees of substitution of 0.60 and 0.93 were tested. The HPBCD products with low molar degrees of subsitution (0.60 and 0.65) yielded visually stable solutions for ES-285. HCl in a concentration of 10 mg/mL in 20% (w/v) HPBCD. A visually stable solution was only obtained with the HPBCD product with a high molar degree of substitution (0.93) when increasing the HPBCD concentration to 40% (w/v). Therefore Kleptose^® HPBCD with a molar degree of substitution of 0.65 was selected for the development of ES-285.HC1 pharmaceutical final product.

The formulation solution selected for the manufacture of ES-285.HC1 final product was 10 mg/mL ES-285.HC1 in 20% (w/v) HPBCD (molar degree of substitution of 0.65; Kleptose) or 20 mg/ml ES-285 in 40% (w/v) HPBCD (molar degree of substitution of 0.65; Kleptose). These concentrations are below the maximal solubility of 19.2 ± 0.03 mg/mL observed at the given HPBCD concentrations. Using these concentrations, ES-285.HC1 dissolved within one hour, without the need for addition of excess drug, heating or sonification.

Example 5: Formulation Process

Freeze drying was selected as formulation approach for ES-285.HC1 25 mg and 50 mg/vial final products. ES-285.HC1 final product was aseptically prepared. The formulation solution contained 10 mg/mL ES-285.HC1, which was dissolved in 203^ (w/v) HPBCD with stirring at ambient temperature. After complete dissolution, the solution was adjusted to final weight with 20% (w/v) HPBCD and sterile filtered through a 0.22 μm Millipak 20 filter (Millipore, Milford, MA, USA). Subsequently, 2.5 mL (25 mg/vial final product) or 5.0 mL (50 mg/vial final product) aliquots were filled into 8 mL (25 mg/vial final product) or 20 mL (50 mg/vial final product) lyophilisation vials (Fiolax, Munnerstadter Glaswarenfabrik, Mϋnnerstadt, Germany). Platinum cured silicone tubing (Watson Marlow, Cheltenham, UK) was used during filtration and filling. Siliconized gray bromobutyl rubber stoppers (Type FM 157/1 , Helvoet Pharma NV, Alken, Belgium) were positioned on each vial. Two vials were equipped with thermocouples an all vials were loaded into a Model Lyovac GT 4 freeze-dryer (STERIS, Hϋrth, Germany) at ambient temperature and lyophilised. Vials were pneumatically closed, and retrieved afteT lifting the vacuum with sterile filtered nitrogen. The product was capped with aluminhαm caps (Bico Pharma GmbH, Neuss, Germany) and labelled.

Example 6: Stability upon storage

Stability of ES-285.HC1 final product of Example 5 was evaluated for each, dosage unit (25 mg/vial and 50 mg/vial), according to ICH guidelines (ICH QlA R "Stability testing guidelines: stability testing of new drug substances and products", London (CPMP/ICH/2736/99), EMEA). Accelerated testing was conducted in the dark in a model CCM-0/125 climate chamber (P. Selecta, Barcelona, Spain) set at 25 ± 2°C/6O ± 5% relative humidity (RH). Long term testing was conducted at the designated storage condition of +5°C ± 3°C, in the dark. Samples were taken at different points in time and analysed for appearance, reconstitution characteristics (time until complete dissolution, pH after reconstitution), and residual water content (n=2). Furthermore, samples were subjected to HPLC analysis to determine content and chromatographic purity (n=3). Table 2 shows the results obtained for the stability studies up to 24 months.

Table 2 Stability of ES-285.HC1 25 mg/vial and 50 mg/vial final product (mean with standard deviation in parentheses).

ES-285.HC1 25 mg/vial and ES-285.HC1 50 mg/vial final product proved stable for 6 months at accelerated storage conditions and for 24 months at the designated long term storage condition. No major changes were observed in any of the parameters.

Claims

1. A pharmaceutical composition comprising a compound according to formula I

NH₂ wherein n is an integer selected from 11, 12, 13, 14, 15 or 16, R is -CH₃, -CH₂CH₃ Or-CH₂ CH₂CH₃, or a pharmaceutically acceptable acid-addition salt thereof; and a cyclodextrin or cyclodextrin derivative.

2. The pharmaceutical composition according to claim 1, characterized in that n is 13, 14 or 15.

3. The pharmaceutical composition according to claim 1, characterized in that n is 14 and R is CH₃.

4. The pharmaceutical composition according to any of claims 1-3, characterized in that the compound is in the form of the hydrochloride salt.

5. The pharmaceutical composition according to claim 1, characterized in that the compound is ((2S,3R)-2-amino-3-octadecanol hydrochloride.

6. The pharmaceutical composition according to any of claims 1-5, characterized in that the cyclodextrin derivative is an etherified cyclodextrin.

7. The pharmaceutical composition according to claim 6, characterized in that the etherified cyclodextrin is a β cyclodextrin.

8. The pharmaceutical composition according to claim 7, characterized in that the cyclodextrin is an hydroxyalkyl substituted β cyclodextrin.

9. The pharmaceutical composition according to claim 8, characterized in that the cyclodextrin is 2-hydroxypropyl-β-cyclodextrin.

10. The pharmaceutical composition according to claim 9, characterized in that the Molar degree of substitution of the 2-hydroxypropyl-β-cyclodextrin is comprised in the range of form 0.125 to 0.95 more preferably in the range of 0.2 to 0.80.

1 1. The pharmaceutical composition according to claim 10, characterized in that the Molar degree of substitution of the 2-hydroxypropyl-β-cyclodextrin is comprised in the range of from about 0.5 to about 0.7, preferably from about 0.60 to about 0.65.

12. A pharmaceutical composition comprising ES-285.HC1 and 2-hydroxypropyl-β- cyclodextrin having a Molar degree of substitution of about 0.65.

13. The pharmaceutical composition according to any of claims 1-12, characterized in that the weight ratio of the amount of compound of formula I to the amount of cyclodextrin or cyclodextrin derivative is from about 1 :10 to about 1 :50, preferably from about 1 :15 to about 1:45.

14. The pharmaceutical composition according to claim 13, characterized in that the weight ratio of the amount of compound of formula I to the amount of cyclodextrin or cyclodextrin derivative is from about 1 : 15 to about 1 :25.

15. The pharmaceutical composition according to any of claims 1-15 in freeze-dried form.

16. A method for treatment of cancer comprising parenterally administering the pharmaceutical composition as defined in any of claim 1-15 to a patient in need thereof.

17. Use of a pharmaceutical composition as defined in any of claims 1-15 in the manufacture of a medicament for the treatment of cancer.