CYCLODEXTRIN-BASED GAMMALINOLENIC ACID FORMULATION FOR TREATMENT OF BRAIN CANCER

Abstract

The present invention features a cyclodextrin-based gamma linolenic acid formulation for the treatment of brain cancer. More specifically, the invention relates to use of cyclodextrin analogs as carriers for gamma-linolenic acid and their pharmacological use. The present invention features GLA formulated as a cyclodextrin inclusion complex for the treatment of cancers of the brain, in particular glioblastoma multiforme and other gliomas, as well as metastatic cancers to the brain including leptomeningeal cancers. The inclusion complexes described herein take advantage of the ability of hydroxypropyl-beta-cyclodextrin (HPbCD), beta-cyclodextrin sulfobutyl ether (bCDSBE), and 2,6-dimethyl-beta-cyclodextrin (DMbCD) to complex with and subsequently solubilize GLA.

Claims

1. A cyclodextrin (CD) inclusion complex of gamma-linolenic acid (GLA), wherein the concentration of GLA is in the range of 1-20 mg/ml; the concentration of CD is in the range of 10-40% (w/v); wherein the cyclodextrin is selected from 2-hydroxypropyl-β-cyclodextrin; sulfobutylether-β-cyclodextrin, sodium salt; and 2,6-dimethyl-β-cyclodextrin; and the inclusion complex is in the form of a nanoemulsion.

2. The inclusion complex according to claim 1, wherein the CD:GLA molar ratio is in the range of about 2:1 to 8:1.

3. The inclusion complex according to claim 3, wherein the CD:GLA molar ratio is about 4:1.

4. The inclusion complex according to claim 1, wherein the nanoemulsion particle size is in the range of about 50 nm to 300 nm.

5. The inclusion complex according to claim 1, wherein said complex is in the form of a lyophilizate.

6. Use of the inclusion complex according to claims 1 for the manufacture of a medicament for the treatment of cancer.

7. The medicament according to claim 6, wherein said cancer is glioblastoma multiforme.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0018] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0019] FIG. 1 shows a Transmission Electron Microscope (TEM) photograph of a 10 mg/mL solution of GLA in 20% HPbCD.

[0020] FIG. 2 shows the inhibition of U87 cell viability with a GLA/HPbCD inclusion complex.

[0021] FIG. 3 shows the inhibition of U87 cell viability with a GLA/bCDSBE inclusion complex.

[0022] FIG. 4 shows the inhibition of U87 cell viability with a GLA/DMbCD inclusion complex.

[0023] FIG. 5 shows a photograph of tumor vs. control in an orthotopic tumor model using a GLA/CD formulation of the invention.

[0024] FIG. 6 and FIG. 7 show photomicrographs of tumor vs. control in an orthotopic tumor model using a GLA/CD formulation of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0025] Gamma-linolenic acid was obtained from the seeds of Borago officinalis that had been processed into borage oil, containing 22% GLA. This commercially available material was purified by methods known to those skilled in the art such that the resulting GLA purity was 98% or higher as analyzed by gas chromatography - flame ionization detection (GC-FID). Formulations used in the methods of the invention use GLA with this level of purity.

[0026] GLA was formulated as a cyclodextrin complex in either distilled water or in buffered solutions. Various cyclodextrins are useful for GLA formulation as indicated in the examples provided herein. Such cyclodextrins include 2,6-dimethyl-β-cyclodextrin (DMBCD), 2-hydroxypropyl-beta-cyclodextrin (HPbCD), or β-cyclodextrinsulfobutyl ether sodium salt (bCDSBE). In one example, 20 microliters of a 10 mg/mL GLA in 20% HPbCD solution was added to a carbon coated copper grid. The grid was air dried at room temperature and then viewed under Transmission Electron Microscope (Technai G2, Philips, Netherlands). The resulting photograph shows well dispersed droplets with average size of 200±85 nm. See FIG. 1.

[0027] Once mixed together to form a clear solution, water-soluble adduct can be lyophilized. Reconstitution of the lyophilizate yields as clear solution with the same properties as the formulation before lyophilization.

[0028] The following non-limiting examples are provided to further exemplify the invention.

Example 1. Formulation of GLA/CD Inclusion Complexes

(I) Preparation of 40% Cyclodextrin Solution

[0029] (a) 4.00 g of a cyclodextrin selected from 2,6-dimethyl-β-cyclodextrin (DMBCD), 2-hydroxypropyl-beta-cyclodextrin (HPbCD), or β-cyclodextrinsulfobutyl ether sodium salt (bCDSBE) was weighed into conical centrifuge tube. 0.01 M phosphate buffered saline (PBS, 5 mL at pH = 7.4) was added. The mixture was vortexed and/or sonicated until the solution became clear.

[0030] (b) Additional PBS was added to bring the volume in each tube up to 10.0 mL and the solution was mixed well.

[0031] (c) The solution was filtered through a 0.22 .Math.m filter under sterile conditions and stored at ≤ 4° C.

(ii) Preparation of Gamma-linolenic Acid (GLA) Suspension in H.SUB.2.O

[0032] (a) 100 mg of GLA was placed into a 15 mL centrifuge tube.

[0033] (b) 4.9 mL of sterile water was added to the GLA and the tube was vortexed at high speed for 3 hours to produce a uniform suspension.

[0034] (c) Once the agitation of the suspension was complete, the GLA suspension was immediately apportioned into 2.0 mL Eppendorf tubes and mixed with various cyclodextrin analogs as indicated in Step (iii).

(iii) Dilution and Agitation of GLA Test Samples

[0035] GLA/cyclodextrin dilutions were prepared under sterile conditions by adding the appropriate amount of the 40% CD solution, 20 mg/mL GLA suspension, PBS, and/or water to a 2 mL Eppendorf tube as indicated in Table 1. The mixtures were vortexed until a clear appearance was observed (usually about 15 seconds) before use.

TABLE-US-00001 GLA/Cyclodextrin Dilutions Tube No. (contents) 40% CD Solution in PBS 20 mg/mL GLA in H.sub.2O PBS H.sub.2O 1 (10 mg/mL GLA/HPbCD, 0.005 M PBS) 500 .Math.L 500 .Math.L -- -- 2 (HPbCD control, 0.005 M PBS) 500 .Math.L -- -- 500 .Math.L 3 (10 mg/mL GLA/bCDSBE, 0.005 M PBS) 500 .Math.L 500 .Math.L -- -- 4 (Bcdsbe control, 0.005 M PBS) 500 .Math.L -- -- 500 .Math.L 5 (10 mg/mL GLA/DMbCD, 0.005 M PBS) 500 .Math.L 500 .Math.L -- -- 6 (DMbCD control, 0.005 M PBS) 500 .Math.L -- -- 500 .Math.L 7 (GLA control, 0.005 M PBS) -- 500 .Math.L 500 .Math.L --

Example 2. U87 Cell Viability Assay

(i) Preparation of 10 Mg GLA/mL in 20% Cyclodextrin Dilutions

[0036] Each of the cyclodextrin solutions from Table 1 were diluted in a 96 well plate with Eagle’s Minimum Essential Medium (EMEM) containing 10% fetal bovine serum (FBS) and 1% Pen/Strep as indicated in Table 2.

TABLE-US-00002 Dilution of GLA/CD Solutions for U87 Cell Assay S. No. Volume of medium (.Math.L) Test items or control Dilutions Concentration per dilution (.Math.g/mL) (2X) 1 1.86 mL 144 .Math.L - 720 2 150 - 150 .Math.L from 1 360 3 150 - 150 .Math.L from 2 180 4 150 - 150 .Math.L from 3 90 5 150 - 150 .Math.L from 4 45 6 150 - 150 .Math.L from 5 22.5 7 150 - 150 .Math.L from 6 11.3 8 150 - 150 .Math.L from 7 5.6 9 150 - 150 .Math.L from 8 2.8

(ii) Testing in U87 Cell Assay

[0037] U87 cells in 100 .Math.L of media were incubated individually in wells of a 96-well plate for 24 hours to ensure attachment. A 100 .Math.L aliquot from each of the test article or cyclodextrin control dilutions was then added to the appropriate test well and the plates were incubated for 72 hours in a 5% CO.sub.2 incubator at 37° C.

[0038] After the U87 cells were incubated in the presence of GLA/CD or control CD solutions, a CellTiter-Glo® Luminescent Cell Viability Assay from Promega was used to assess cell viability. Briefly, the CellTiter-Glo assay is a homogeneous method to determine the number of viable cells in culture based on quantitation of the ATP present, which is directly proportional to the number of metabolically active cells present in the culture medium. The assay relies on the properties of a thermostable luciferase, which generates a stable luminescent signal. The assay was performed as follows: [0039] (1) The plate was removed from the incubator and equilibrated at room temperature for about 30 minutes; [0040] (2) Cell media was carefully removed so as not to disturb the cells and replaced with 100 .Math.L of media that did not contain fetal bovine serum; [0041] (3) CellTiter-Glo® reagent (100 .Math.L) was added to each well; [0042] (4) The contents of the wells were mixed on an orbital shaker for 2 minutes to induce cell lysis; [0043] (5) The plate was incubated at room temperature for 10 minutes; and [0044] (6) The luminescence was recorded using a Perkin-Elmer EnVision multilabel reader.

(iii) Effect of GLA/CD Solutions of Cells Viability

[0045] As shown in FIG. 2, where TF is the test article (GLA/20% HPbCD) and VC is the vehicle control (20% HPbCD alone), theIC.sub.50of GLA/HPbCD= 117 .Math.g/mL (420 .Math.M). As shown in FIG. 3, where TF is the test article (GLA/20% bCDSBE) and VC is the vehicle control (20% bCDSBE alone), the IC.sub.50 of GLA/bCDSBE= 117 .Math.g/mL (420 .Math.M). And as shown in FIG. 4, where TF is the test article (GLA/20% DMbCD) and VC is the vehicle control (20% DMbCD alone), the IC.sub.50of GLA/DMbCD= 60 .Math.g/mL (216 .Math.M).

Example 3. Preparation of GLA/HPbCD Inclusion Complexes in Artificial Cerebrospinal Fluid

[0046] Artificial cerebrospinal fluid (aCSF) and HPbCD emulsions in aCSF are prepared as follows.

(i) Preparation of 20 mg/mL GLA in aCSF and in 20% HPbCD/aCSF

[0047] a) For 2x Solution A, the following amounts were dissolved in 250 mL sterile H.sub.2O:NaCl (8.66 g); KCl (0.224 g); CaCl.sub.2.sup..2H.sub.20 (0.206 g); and MgCl.sub.2.Math.6H.sub.2O (0.163 g).

[0048] b) For 2x Solution B, the following amounts were dissolved in 250 mL sterile H.sub.2O:Na.sub.2HPO.sub.4.Math.7H.sub.2O (0.214 g); andNaH.sub.2PO.sub.4.Math.H.sub.2O (0.027 g).

[0049] c) For 2x aCSF, Solutions A & B were combined in equal volumes.

(ii) Preparation of 40% HPbCD in 2x aCSF

[0050] a) 2.00 g of hydroxypropyl-β-cyclodextrin (HPbCD) was weighed out in a sterile graduated 15 mL conical centrifuge tube; and 2.0 mL of 2x aCSF was added and the suspension was vortexed until the HPbCD has been totally wetted.

[0051] b) Additional 2x aCSF (about 1.85 mL) was added portion-wise to the HPbCD solution up to the 5.0 mL graduation mark, vortexing /sonicating after each addition until all of the HPbCD went into solution.

[0052] c) The solution was filtered through a 0.22 .Math.m filter in a laminar flow hood.

Example 4. Efficacy of a GLA/HPbCD Formulation in an Orthotopic Tumor Model

[0053] C6 glioma cells were grown in DMEM cell culture medium containing 10% fetal calf serum and antibiotics (penicillin 50 U/ml, streptomycin 50 ug/ml). Cells in the exponential phase of growth were used, growing in 75 cm.sup.2 flasks in a humidified atmosphere of 5% CO.sub.2/95% air at 37° C.

[0054] A subcutaneous injection was made on the lateral side of the back above the hind leg where 100 .Math.L of Dulbecco’s phosphate-buffered saline (DPBS) containing 3 x 10.sup.6 C6 glioma cells were injected for the development of a flank tumor in Wistar rats. The animals were housed for 14-days during which the tumor size, weight of the animals were measured periodically. Following tumor development, the animals were randomly divided into 2 groups with 3 animals per group (n=3). A 100 .Math.L intratumoral injections of a 10 mg/ml of GLA/HPbCD/aCSF formulation was administered on alternate days for 14 days to one group along with vehicle control to the other group. The weight of the animals, food/water intake, and tumor volume was noted periodically. At the end of the experiment, the rats were euthanized and the tumor was excised for histopathological analysis. The efficacy of the GLA/HPbCD/aCSF formulation was assessed by reduction of the tumor volume and histological evidence of regression of the tumor.

[0055] As shown in FIG. 5, a significant tumor regression (75 +/- 3.2%) was noted. The detailed histological examination of tumor of TF021 test formulation showed the area of tumor regression was infiltrated by foamy macrophages and lymphocytes, which was not seen in the vehicle control VC021 (FIG. 6).