CRYSTALLINE LEUKOTRIENE B4

Description

Field of the Invention

[0001] The present invention relates to leukotriene B₄ and crystal forms thereof. The present invention furthermore relates to pharmaceutical formulations of said crystalline leukotriene B₄.

Background of the Invention

[0002] Leukotriene B₄, 5S,12R-dihydroxy-6,8,10,14(Z,E,E,Z)-eicosatetraenoic acid, is a twenty carbon tetra-unsaturated fatty acid and is a relatively unstable molecule. Isotonic aqueous solutions of leukotriene B₄ (LTB4) at pH 7.0-7.6, which are suitable for administration to humans and animals are stable for only short periods of time (weeks to months) when stored at temperatures ranging from 2°C to 25°C (and above 25°C). Indeed, LTB₄ agents are subject to oxidation, isomerization of double bounds (LTB₄ contains two cis and two trans double bounds), epimerization (LTB₄ contains two chiral centers), esterification (LTB₄ contains a carboxylic group), lactonization, among various possible structural alterations.

[0003] LTB₄ agents have great pharmaceutical utility, but their use as therapeutic agents in animals or human is problematic, because of their insufficient stability and shelf-life in solution at temperatures between 2°C to 25°C.

[0004] No formulations of solid LTB₄ have been reported. Indeed, free LTB₄ is known in the art to be an oily substance at room temperature (Kerdesky et al., J. Org. Chem., Vol. 58, 1993, 3516-3520, and US patent 4,873,024). Kobayashi et al., J. Org. Chem., Vol. 55, 1990, 5324-5335, report a melting point of 25-28 °C for solid leukotriene B₄. Crystalline LTB4 has not been reported in the art and no suggestion has been provided of a solid form of LTB4 suitable for solid formulations.

[0005] Given the potential of LTB4 agents as therapeutic agents for the prophylaxis and treatment of infections and cancer in humans and animals, it would be highly desirable to provide a novel form of LTB₄ suitable for solid pharmaceutical formulations.

Summary of the Invention

[0006] The inventors of the present invention have surprisingly found a novel crystalline form of LTB₄ that has a melting point as high as 79 °C. Accordingly, one aspect of the invention concerns leukotriene B₄ in a crystalline form, or a hydrate thereof. The high melting point of the novel crystalline form of leukotriene B₄ provides a particular advantage over the prior art for formulating pharmaceutical compositions of leukotriene B₄. Another aspect of the invention concerns a composition comprising crystalline leukotriene B₄, or a hydrate thereof, and a pharmaceutically acceptable carrier.

[0007] A third aspect of the invention concerns said crystal forms of leuktriene B₄ or said compositions thereof for use as a medicament.

Brief Description of the Figures

[0008] 

Figure 1 is a diffractogram of crystalline form A of leukotriene B₄, obtained using copper as an X-ray source.

Figure 2 is a diffractogram of crystalline form A of leukotriene B₄, obtained using chromium as an X-ray source.

Figure 3 is s DSC thermogram of crystalline form A of leukotriene B₄, wherein the scan has been performed from 30 to 350 °C.

Detailed Description of the Invention

[0009] The inventors of the present invention have surprisingly found that leukotriene B₄ may be crystallized from several solvent alternatives. Therefore, in one aspect, the present invention concerns crystalline leukotriene B₄, 5S,12R-dihydroxy-6,8,10,14(Z,E,E,Z)-eicosatetraenoic acid, or a hydrate thereof.

[0010] The X-ray powder diffractogram of one form of crystalline leukotriene B₄, referred to in the context of the present invention as Form A, is shown in Figure 1. The data have been obtained with copper as an X-ray source and the most significant peaks are found at 18.6 ± 0.2, 21.0 ± 0.2, 21.5 ± 0.2, 22.2 ± 0.2 and 23.5 ± 0.2 degrees two-theta (2θ). Accordingly, one embodiment relates to leukotriene B₄ Form A, which is characterized by X-ray powder diffraction peaks at 18.6 ± 0.2, 21.0 ± 0.2, 21.5 ± 0.2, 22.2 ± 0.2 and 23.5 ± 0.2 degrees two-theta with copper as an X-ray source, or a hydrate thereof. In a preferred embodiment, the leukotriene B₄ Form A is characterized by a powder X-ray diffraction pattern as substantially depicted in Figure 1. The intensity of the peaks may vary according to the method of measurement and the signal to noise ratio may also vary, but the angle of the peaks will only vary within the boundaries set by the measurement uncertainties of the equipment used for the measurements.

[0011] Depending on the wavelength of the X-rays, i.e. depending on the source of the X-rays, the scattering angle may vary. Hence, Form A may also be characterized by the peaks in a diffractogram obtained with chromium as an X-ray source. Accordingly, an alternative embodiment of the invention relates to leukotriene B₄ Form A, which is characterized by X-ray powder diffraction peaks at 8.8 ± 0.2, 10.7 ± 0.2, 31.4 ± 0.2, 32.2 ± 0.2 and 33.2 ± 0.2 degrees two-theta with chromium as an X-ray source, or a hydrate thereof. In a preferred embodiment, the leukotriene B₄ Form A is characterized by a powder X-ray diffraction pattern as substantially depicted in Figure 2. The intensity of the peaks may vary according to the method of measurement and the signal to noise ratio may also vary, but the angle of the peaks will only vary within the boundaries set by the measurement uncertainties of the equipment used for the measurements.

[0012] The dimensions of the unit cell of leukotriene B₄ Form A may be calculated on the basis of the X-ray data. The inventors have found the following unit cell dimensions:

a = 16.14 ± 0.01 Angstrom

b = 5.15 ± 0.01 Angstrom

c = 13.79 ± 0.01 Angstrom

[0013] In one embodiment the present invention relates to crystalline Form A of leukotriene B₄ having unit cell dimensions of a=16.14 ± 0.01 Angstrom, b=5.15 ± 0.01 Angstrom and c=13.79 ± 0.01 Angstrom.

[0014] Another characteristic of the novel crystalline forms is their high melting temperature. More specifically, a leukotriene B₄ crystal may be characterized by Differential Scanning Calorimetry (DSC). Figure 3 shows a DSC thermogram for leukotriene B₄ Form A. The endothermic phase transition peaks at about 79 °C. Therefore, in one embodiment, the present invention relates to leukotriene B₄ Form A having a peak in the range 75 to 85 °C in a DSC thermogram, preferably in the range 77 to 81 °C, more preferably around 79 °C, or a hydrate thereof. The peak in Figure 3 is quite wide. The width of the peak, without being bound by a particular theory, could be due to thermal decomposition of the relatively unstable LTB₄. In this particular example, the onset of said peak is at about 74°C.

[0015] As mentioned above, the novel crystalline forms of leukotriene B₄ provide advantages in formulating pharmaceutical compositions, in particular solid compositions. Accordingly, one aspect of the present invention relates to a pharmaceutical composition comprising the leukotriene B₄ in crystalline form, or a hydrate thereof, and a pharmaceutically acceptable carrier. In a preferred embodiment, the composition according to the present invention comprises leukotriene B₄ Form A as described above, or a hydrate thereof, and a pharmaceutically acceptable carrier.

[0016] The expression "pharmaceutically acceptable carrier", in the context of the present invention, is intended to include any carrier suitable for physiological and pharmaceutical usage. Such carrier is selected from the group consisting of water, buffered salt solutions, such as phosphate buffered saline (PBS), or sodium chloride solutions buffered with agents such as Tris, glycine or other amino acids, in particular basic amino acids, aqueous solution containing alcohol, such as ethanol, propylenglycol, propanediol, glycerol, or mannitol, as well as sugar solutions, such as glucose or lactose solutions, or a mixture of the various solvents mentioned. Furthermore, the expression "pharmaceutically acceptable carrier" may include inert diluents or fillers, such as sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate or sodium phosphate; granulating and disintegrating agents, for example, cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates or alginic acid; binding agents, for example, sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone or polyethylene glycol; and lubricating agents, including glidants and antiadhesives, for example, magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils or talc.

[0017] It was furthermore found that leukotriene B₄ is more stable at alkaline pH than at neutral or acidic pH. Accordingly, in a preferred embodiment, the composition according to the present invention comprises an alkaline reacting carrier. The expression "alkaline reacting carrier", in the context of the present invention, is intended to include an otherwise inert, pharmaceutically acceptable substance (or substances), which creates an alkaline "micro-pH" between 8.2 and 14, especially between 8.5 and 12.5, such as between 8.5 and 11.5, most preferably between 9.5 and 11.5, such as about 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, or 11.5, around each LTB₄ particle when water is adsorbed to the particles of the mixture or when water is added in small amounts to the mixture. In another preferred embodiment, the alkaline "micro-pH" ranges between 8.0 and 9.0, between 8.5 and 9.5, between 9.0 and 10.0, or between 10.0 and 11.5. Such substances creating said "micro-pH" can be chosen among, but are not restricted to substances such as the sodium, potassium, calcium, magnesium and aluminium salts of phosphoric acid, carbonic acid, citric acid or other suitable weak inorganic or organic acids; substances normally used in antacid preparations such as aluminium, calcium and magnesium hydroxides; magnesium oxide or composite substances, such as Al₂O₃.6MgO-CO₂-12H₂O,(Mg₆Al₂(OH)₁₆CO₃.4H₂O), MgO.Al₂O₃. 2SiO₂.nH₂O or similar compounds; organic pH-buffering substances such as trihydroxymethylaminomethane or other similar, pharmaceutically acceptable pH-buffering substances.

[0018] Any suitable type or mode of administration may be employed for providing a mammal, especially a human with an effective dosage of LTB₄. For example, oral, parenteral, intraduodenal, intrajejunal and topical may be employed. Dosage forms include tablets, capsules, powders, solutions, dispersions, suspensions, creams, ointments and aerosols.

[0019] In a preferred embodiment, the compositions of the present invention may be in an oral dosage form with an enteric coating. From the statement about the stability properties of LTB₄ above, it is obvious that it is advantageous that an oral dosage form of the said crystalline LTB₄ must be protected from contact with the acidic gastric juice in order to reach the small intestine without degradation.

[0020] The enteric coated preparations are resistant to dissolution in acid media and dissolve rapidly in neutral to alkaline media. The enteric coated dosage form is preferably characterized in the following way. Cores containing the crystalline LTB₄ mixed with alkaline reacting compounds are coated with two or more layers, in which the first layer/layers is/are soluble in water or rapidly disintegrating in water and consist(s) of non-acidic, otherwise inert pharmaceutically acceptable substances. This/these first layer/layers separates/separate the alkaline reacting core material from the outer layer, which is an enteric coating. The final, enteric coated dosage form is treated in a suitable way to reduce the water content to a very low level in order to obtain a good stability of the dosage form during long-term storage.

[0021] The crystalline LTB₄ is mixed with inert, preferably water soluble, conventional pharmaceutical constituents to obtain the preferred concentration of the active compound in the final mixture and with an alkaline reacting, otherwise inert, pharmaceutically acceptable substance (or substances), which creates a "micro-pH" as defined above, when water is adsorbed to the particles of the mixture or when water is added in small amounts to the mixture. Such substances can be chosen among substances such as the sodium, potassium, calcium, magnesium and aluminium salts of phosphoric acid, carbonic acid, citric acid or other suitable weak inorganic or organic acids; substances normally used in antacid preparations such as aluminium, calcium and magnesium hydroxides; magnesium oxide or composite substances such as Al₂O₃.6MgO CO₂.12H₂O, (Mg₆Al₂(OH)₁₆CO₃ 4H₂O MgO.Al₂O₃.2SiO₂.nH₂O, wherein n is not an integer and less than 2, or similar compounds; organic pH-buffering substances such as trishydroxymethylaminomethane or other similar, pharmaceutically acceptable pH-buffering substances.

[0022] The powder mixture is then formulated into small beads i.e. pellets or tablets, by conventional pharmaceutical procedures. The pellets or tablets are used as cores for further processing.

[0023] The alkaline reacting cores containing the crystalline LTB₄ must be separated from the enteric coating polymer(s) containing free carboxyl groups, which otherwise causes degradation of the crystalline LTB₄ during the coating process or during storage. The subcoating layer, (the separating layer), also serves as a pH-buffering zone in which hydrogen ions diffusing from the outside in towards the alkaline core can react with hydroxyl ions diffusing from the alkaline core towards the surface of the coated particles. The pH-buffering properties of the separating layer can be further strengthened by introducing in the layer substances chosen from a group of compounds usually used in antacid formulations such as, for instance, magnesium oxide, hydroxide or carbonate, aluminium or calcium hydroxide, carbonate or silicate; composite aluminium/magnesium compounds such as, for instance Al₂O₃.6MgO CO₂.12H₂O, (Mg₆Al₂(OH)₁₆CO₃,4H₂O), MgO.Al₂O₃.2SiO₂.nH₂O, wherein n is not an integer and less than 2, or similar compounds; or other pharmaceutically acceptable pH-buffering substances such as, for instance the sodium, potassium, calcium, magnesium and aluminium salts of phosphoric, citric or other suitable, weak, inorganic or organic acids.

[0024] The separating layer consists of one or more water soluble inert layers, optionally containing pH-buffering substances.

[0025] The separating layer(s) can be applied to the cores - pellets or tablets - by conventional coating procedures in a suitable coating pan or in a fluidized bed apparatus using water and/or conventional organic solvents for the coating solution. The material for the separating layer is chosen among the pharmaceutically acceptable, water soluble, inert compounds or polymers used for film-coating applications such as, for instance sugar, polyethylene glycol, polyvinylpyrollidone, polyvinyl alcohol, hydroxypropyl cellulose, hydroxymethyl cellulose or hydroxypropyl methylcellulose. The thickness of the separating layer is not less than 2 µm, for small spherical pellets preferably not less than 4 µm, for tablets preferably not less than 10 µm.

[0026] In the case of tablets another method to apply the coating can be performed by the drycoating technique. First a tablet containing the acid labile compound is compressed as described above. Around this tablet another layer is compressed using a suitable tableting machine. The outer, separating layer, consists of pharmaceutically acceptable, in water soluble or in water rapidly disintegrating tablet excipients. The separating layer has a thickness of not less than 1 mm. Ordinary plasticizers, pigments, titanium dioxide talc and other additives may also be included into the separating layer.

[0027] The enteric coating layer is applied on to the sub-coated cores by conventional coating techniques such as, for instance, pan coating or fluidized bed coating using solutions of polymers in water and/or suitable organic solvents or by using latex suspensions of said polymers. As enteric coating polymers can be used, for example, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, polyvinyl acetate phthalate, copolymerized methacrylic acid/methacrylic acid methyl esters such as, for instance, compounds known under the trade name Eudragit^R L 12,5 or Eudragit^R L 100, (Röhm Pharma) or similar compounds used to obtain enteric coatings.

[0028] The enteric coating can also be applied using water-based polymer dispersions, e.g. Aquateric (FMC Corporation), Eudragit^R L 100-55 (Röhm Pharma), Coating CE 5142 (BASF). The enteric coating layer can optionally contain a pharmaceutically acceptable plasticizer such as, for instance, cetanol, triacetin, citric acid esters such as, for instance, those known under the trade name Citroflex^R (Pfizer) phthalic acid esters, dibutyl succinate or similar plasticizers.

[0029] The amount of plasticizer is usually optimized for each enteric coating polymer(s) and is usually in the range of 1-20 % of the enteric coating polymer(s). Dispersants such as talc, colourants and pigments may also be included into the enteric coating layer.

[0030] Thus, the enteric coated preparation according to the invention consists of cores containing the crystalline LTB₄ mixed with an alkaline reacting compound. The cores are coated with a water soluble or in water rapidly disintegrating coating, optionally containing a pH-buffering substance, which separates the alkaline cores from the enteric coating. The sub-coated dosage form is finally coated with an enteric coating rendering the dosage form insoluble in acid media, but rapidly disintegrating/dissolving in neutral to alkaline media such as, for instance the liquids present in the proximal part of the small intestine, the site where dissolution is wanted.

[0031] LTB₄ has high potential as a therapeutic agent for the prophylaxis and treatment of infections and cancer in humans and animals. Accordingly, in another aspect, the invention relates to crystalline leukotriene B₄ according to the present invention or the pharmaceutical composition according to the present invention for use as a medicament.

Examples

[0032] The present invention will be further illuminated in the following, non-limiting examples.

Methods

[0033] Conditions for obtaining X-ray Powder Diffraction (XRPD) patterns:

Registration:	Photographic recording using a Guinier Hägg camera, corrected radius 50.1247 mm for Cu and corrected radius 50.0330 mm for Cr.
X-Ray source:	Strictly monochromatic CuKα1-radiation (λ = 1.5405 Å) and CrKα1-radiation (λ = 2.28975 Å), Instrumenttjänst AB, Sweden.
Evaluation:	Automatic laser scanner, University of Stockholm.

[0034] Conditions for obtaining Differential Scanning Calorimetry (DSC) thermograms:

Instrument:	DSC/822e Mettler Toledo
Software:	Mettler Toledo Stare System
Temperature range:	30.0 to 350.0 °C
Rate:	10.00 °C/min
N2 - flow:	80.0 mL/min

Example 1

Synthesis of Form A crystals

[0035] Leukotriene B₄ in ethanol solution (1.5% w/w) from Cascade Biochem Ltd. (Stock No: 31350, Lot. No:3468.B.05.1) was concentrated to dryness affording an oily residue, which was re-dissolved in t-butyl-methyl ether. Heptane was added at -70 °C to afford a colourless emulsion containing a small amount of precipitate. The emulsion was isolated and after about 10 minutes at room temperature a thin slurry with white crystalline precipitate was observed under a light microscope. The slurry was cooled to 0 °C and became thicker. The crystals were filtered off and dried at room temperature. The crystals were fibre-like with a thickness of less than 5 µm and a length of 40-80 µm.

Variation of solvents

[0036] It was found that a variation of different solvents could be used for obtaining the crystals. In general it was found that addition of an anti-solvent (solvent wherein LTB₄ is only slightly soluble or insoluble) to a solution of LTB₄ would provide a crystalline precipitate. Table 1 below summarizes the solubility of LTB₄ in various solvents.

Table 1

Very soluble	Slightly soluble or insoluble
Methanol	Toluene
Ethanol	Water
Isopropyl alcohol	Heptane
Acetone
Methyl isobutyl ketone
Ethyl acetate
Isopropyl acetate
Acetonitrile
t-butyl-methyl ether
Dichloromethane
Tetrahydrofuran

[0037] It is envisioned that any combination of a solvent from the left column in combination with a solvent from the right column will result in a crystalline precipitate. In particular solvent combinations selected from the group consisting of ethanol/water, t-butyl-methyl ether/heptane, t-butyl-methyl ether/toluene, acetone/toluene, ethanol/toluene, isopropyl alcohol/toluene and tetrahydrofuran/toluene, which have all been shown to provide LTB₄ crystals in a manner similar to the process described for t-butyl-methyl ether/heptane above, are contemplated as being part of the present invention.

Characterization of the crystals

[0038] A diffractogram with Cu as an X-ray source performed on crystals obtained from ethanol/water is shown in Figure 1. The two-theta values corresponding to Figure 1 are summarized in Table 2:

Table 2

Cu radiation	Cr radiation
10.703	8.774
13.895	10.277
14.408	10.669
16.474	15.923
17.078	20.683
17.859	21.478
17.951	24.574
18.210	25.493
18.585	26.619
20.317	26.790
20.582	27.216
20.949	27.768
21.513	30.403
22.161	30.825
22.539	31.367
23.497	31.967
23.915	32.208
24.306	33.223
24.861	33.782
26.043	35.254
26.241	35.833
26.596	36.498
27.197	37.358
27.695	38.068
27.825	38.861
28.446	39.158
29.552	39.465
31.216	39.987
32.071
33.312
33.997
34.100
34.740

[0039] A diffractogram with Cr as an X-ray source is shown for the same crystals in Figure 2. The two-theta values corresponding to Figure 2 are also summarized in Table 2.

[0040] A DSC thermogram for the crystals obtained from ethanol/water is shown in Figure 3.

Claims

1. Leukotriene B₄ Form A, which is characterized by X-ray powder diffraction peaks at 18.6 ± 0.2, 21.0 ± 0.2, 21.5 ± 0.2, 22.2 ± 0.2 and 23.5 ± 0.2 degrees two-theta with copper as an X-ray source, having a peak in the range 75 to 85°C in a DSC thermogram.

2. A solid pharmaceutical composition comprising the leukotriene B₄ Form A according to claim 1 and a pharmaceutically acceptable carrier.

3. The leukotriene B₄ Form A according to claim 1 or the pharmaceutical composition according to claim 2 for use as a medicament.

Ansprüche

1. Leukotrien-B₄ Form A, gekennzeichnet durch Peaks der Röntgen-Pulverdiffraktometrie bei 18,6 ± 0,2, 21,0 ± 0,2, 21,5 ± 0,2, 22,2 ± 0,2 und 23,5 ± 0,2 Grad 2-Theta mit Kupfer als Röntgenquelle mit einem Peak im Bereich von 75 bis 85 °C in einem DSC-Thermogramm.

2. Feste pharmazeutische Zusammensetzung, umfassend die Leukotrien-B₄ Form A nach Anspruch 1 und ein pharmazeutisch verträglicher Träger.

3. Leukotrien-B₄ Form A nach Anspruch 1 oder die pharmazeutische Zusammensetzung nach Anspruch 2 zur Verwendung als Medikament.

Revendications

1. Leucotriène B₄ de forme A, caractérisé par des pics de diffraction X sur poudre à angles deux thêta de 18,6 ± 0,2, 21,0 ± 0,2, 21,5 ± 0,2, 22,2 ± 0,2 et 23,5 ± 0,2 degrés avec une source de rayons X en cuivre, présentant un pic dans la plage de 75 à 85 °C dans un thermogramme DSC.

2. Composition pharmaceutique solide comprenant le leucotriène B₄ de forme A selon la revendication 1 et un véhicule pharmaceutiquement acceptable.

3. Le leucotriène B₄ de forme A selon la revendication 1 ou la composition pharmaceutique selon la revendication 2 destinés à être utilisés comme médicament.

Drawing