SALICYLATE COMPOUND COMPOSITION

Abstract

A liquid composition contains a salicylate compound (e.g. aspirin), glycerin triacetate, saccharin. The salicylate compound is soluble in the composition, which is particularly suitable for oral, parenteral or pulmonary administration.

Claims

1.-40. (canceled)

41. A liquid composition comprising a salicylate compound, glycerin triacetate, and saccharin, wherein the salicylate compound is selected from the group consisting of aspirin, triflusal, diflunisal, salsalate and salicylic acid, wherein the composition comprises 0.5 to 7 wt % of the salicylate compound and 90 to 99 wt % of the glycerin triacetate.

42. The composition according to claim 41, wherein the salicylate compound is aspirin.

43. The composition according to claim 41, wherein the composition comprises 1 to 5 wt % of the salicylate compound.

44. The composition according to claim 41, wherein the composition comprises 0.5 to 3 wt % of the salicylate compound.

45. The composition according to claim 41, wherein the composition comprises 94 to 99 wt % of the glycerin triacetate.

46. The composition according to claim 41, wherein the composition comprises 95 to 97.5 wt % of the glycerin triacetate.

47. The composition according to claim 41, wherein the composition comprises 0.5 to 3 wt % of the salicylate compound and 94 to 99 wt % of the glycerin triacetate.

48. The composition according to claim 41, wherein the composition further comprises 0.1 to 3 wt % saccharin.

49. The composition according to claim 41, wherein the composition comprises 0 to 0.5 wt % water.

50. The composition according to claim 41, further comprising a flavoring agent.

51. The composition according to claim 50, wherein the flavoring agent is mint oil.

52. The composition according to claim 41, which is free of particulates.

53. The composition according to claim 41, which is designed for oral use.

54. The composition according to claim 41, which is formulated for intravenous or intra-arterial administration.

55. The composition according to claim 41, which is formulated for inhalation or insufflation administration.

56. A method of preparing a liquid composition according to claim 41, the method comprising admixing the salicylate compound, the glycerin triacetate, and saccharin, wherein the salicylate compound is selected from the group consisting of: aspirin, triflusal, diflunisal, salsalate and salicylic acid.

57. The method according to claim 56, wherein the salicylate compound is aspirin.

58. The method according to claim 56, further comprising admixing a flavoring agent.

59. A packaged article comprising the composition as defined in claim 41, which is sealed therein.

60. The packaged article according to claim 59, which is selected from the group consisting of a bottle, pipette, syringe, vial, sachet, stick shot, and liquid gel capsule.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0392] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0393] FIG. 1 is a plot of the normalized % of salicylic acid present in a test composition against the number of days elapsed since preparation of the composition, for a composition of 90 wt % glycerol+20EO and 10 wt % aspirin. The test was conducted at 25° C.

[0394] FIG. 2 is a plot of the normalized % of salicylic acid present in a test composition against the number of days elapsed since preparation of the composition, for a composition of 89 wt % giycerol+20EO, 10 wt % aspirin and 1 wt % saccharin. The test was conducted at 25° C.

[0395] FIG. 3 is a plot of the normalized % of salicylic acid present in a test composition against the number of days elapsed since preparation of the composition, for a composition of 96.5 wt % glycerin triacetate, 2.5 wt % aspirin and 1 wt % saccharin. The test was conducted at 25° C.

[0396] FIGS. 4A to 4E. Charts showing percentage cell death with varying concentration of drug (liquid aspirin (ASP); triacetin alone (ASP vehicle); temozolomide alone (TMZ)) in ex vivo patient glioblastoma cell lines (A) SEBTA 023, (B) SEBTA 003, (C) UP 029, (D) KNS42, (E) SNF188. Studies were conducted at 96 hours post-drug treatment and show the average (+/−StDev) of three experiments conducted in triplicate. Due to solubility issues, aspirin alone was not included in this study due to an inability to treat at comparable concentrations.

[0397] FIG. 5. Representative microscopy images of treated ex vivo biopsy-derived cell lines. Images show non-treated (NT), liquid aspirin (“aspirin”) and triacetin only treatment. Contrast differences are the result of cell media pH changes following drug treatment and not due to cell confluence variation.

[0398] FIGS. 6A to 6C. (A) Chart showing percentage cell death in ex vivo biopsy-derived UP029 cells with varying concentration of drug (saccharin-only (Sac), liquid aspirin (Lqd Aspirin), triacetin-only, temozolomide-only (TMZ)). (B) and (C) Charts showing synergy analysis of ex vivo biopsy-derived UP029 cells following treatment with liquid aspirin, saccharin-only and triacetin-only. Synergy (of the liquid aspirin compound) was noted (in order to kill 30%, 50% and 70%) compared to each agent alone with the exception of triacetin-alone (at 90% reduced cell viability) at 96 hours post-treatment, where the highest dosages of triacetin alone was reducing overall cell viability.

[0399] FIGS. 7(A) to 7(F). Charts showing percentage cell death (MTS viability analysis) of control and biopsy-derived cell lines 96 hours post-treatment with varying concentration of drug (saccharin-only (Sac), liquid aspirin-only (Lqd Aspirin), triacetin-only, or temozolomide-only (TMZ)). (A) Non-neoplastic astrocytes (CC2565) included as a control, (B) SEBTA 003 (C) SEBTA 023 (D) KNS42 (E) SNF188 (F) SEBTA 025. Each plot is representative of 3 studies conducted in triplicate.

[0400] In this specification the following test method was used:

Aspirin Stability

[0401] Aspirin undergoes hydrolysis to salicylic acid and acetic acid. The aspirin and salicylic acid concentrations in the sample composition were determined for a minimum period of at least 200 days, preferably up to a maximum of 300 days. The composition was stored in a sealed glass vial in an oven at 25° C., and the concentration of aspirin and salicylic acid measured weekly. The glass vial was opened, a sample removed for testing every week and the glass vial resealed after purging with nitrogen. High performance liquid chromatography with UV detection was used. The conditions were as follows: [0402] Mobile phase: 40% of 1% acetic acid in water, 60% methanol. [0403] Column: Agilent Zorbax Eclipse XBD-C18. 4.6 mm×150 mm with 5 micron particle size. [0404] Column heater: 25° C. [0405] Sample concentration: 0.02 g made to 10 ml with mobile phase. [0406] Injection volume: 40 microlitre. [0407] Flow rate: 1 ml minute. [0408] Detection: UV at 280 nm.

[0409] The stability of the aspirin in the composition is defined as the aspirin degradation rate which was calculated as (i) the average % aspirin degradation (based on the original aspirin concentration) per day, and (ii) the average % aspirin degradation (based on the original aspirin concentration) per gram of composition per day.

[0410] The invention is illustrated by the following non-limiting examples.

EXAMPLES

Example 1

[0411] Food grade glycerin triacetate (ex Sigma Aldrich) was mixed with ethanol 40% w/v and passed through a fixed bed of activated earth. The solvent was then removed using vacuum distillation followed by steam distillation to levels below 1 ppm of ethanol in the glycerin triacetate.

Example 2

[0412] A composition was prepared by mixing 2.5 wt % aspirin (ex Sigma Aldrich), 96.5 wt % glycerin triacetate (produced in Example 1), and 1 wt % saccharin (ex Sigma Aldrich). The components were mixed in the appropriate ratios and sonicated to achieve complete solution. Microscopy showed that the solution was free of any particulate material. The aspirin stability in the composition was measured weekly as described above. The aspirin degradation after 277 days was 6.9% of the original amount present, which is equivalent to a degradation rate of 0.025%/day.

Example 3

[0413] The procedure of Example 2 was repeated except that the composition additionally contained 0.15 wt % of spearmint oil (ex Quinessence) (and correspondingly 0.15 wt % less of glycerin triacetate, i.e. 96.35 wt %). The aspirin degradation after 246 days was 5.7% of the original amount present, which is equivalent to a degradation rate of 0.023%/day.

Example 4

[0414] A composition was prepared by mixing 2.5 wt % aspirin (ex Sigma Aldrich), 96.5 wt % glycerin triacetate (produced in Example 1), and 1 wt % saccharin (ex Sigma Aldrich). The components were mixed in the appropriate ratios and sonicated to achieve complete solution. Microscopy showed that the solution was free of any particulate material. The aspirin stability in the composition was measured weekly as described above.

[0415] The “normalised % salicylic acid” was calculated by finding the % salicylic acid in the composition, as a percentage of the total % salicylic acid and aspirin in the composition, according to the formula

Normalised % salicylic acid=(% salicylic acid)/(% aspirin+% salicylic acid)

[0416] The results are shown in Table 1 below, and in FIG. 3.

TABLE-US-00001 TABLE 1 Day Normalised % salicylic acid 17 0.941483 24 1.108112 31 1.644392 38 1.763869 46 1.610604 61 1.932933 67 2.056293 74 2.043556 81 2.468435 88 2.647186 95 2.868206 102 3.043231 109 3.394404 116 3.499079 123 3.856439 137 4.496758 166 4.909486 186 5.350264

[0417] The observed 5.35% degradation after 186 days is equivalent to a degradation rate of 0.0288%/day.

[0418] Extrapolating from these results, the predicted shelf life limit (the point in time after initial preparation at which the normalised % salicylic acid reaches 10%) is 360 days.

Example 5

[0419] Compositions of aspirin with glycerin triacetate and saccharin were prepared according to the method of Example 2. Five compositions were prepared, with aspirin concentrations of 2.5 wt %, 3.0 wt %, 3.5 wt %, 4.0 wt % and 4.5 wt % respectively. Each composition also contained 1 wt % saccharin and the balance glycerin triacetate.

[0420] The compositions were tested for solubility at different temperatures. Compositions were observed to determine whether the aspirin had fully dissolved to provide a clear solution and whether the solution remained clear.

[0421] The results are shown in Table 2 below:

TABLE-US-00002 TABLE 2 Aspirin concentration Stable solubility at temperature (wt %) 25° C. 20° C. 15° C. 10° C. 4° C. 2.5 YES YES YES YES YES 3.0 YES YES YES YES NO 3.5 YES YES YES YES NO 4.0 YES NO NO NO NO

[0422] The results demonstrate stable solubilisation of aspirin in the composition of the present invention at 4 wt % concentration at 25° C. At a concentration of 2.5 wt % the aspirin was stably solubilised at temperatures down to 4° C.

[0423] The compositions of the invention are therefore particularly suitable for use in the treatment of cardiovascular and cerebrovascular disorders and cancer, where low doses of aspirin over extended periods are required.

[0424] For example, at 2.5 wt % aspirin, a 2 mL dose in a gel capsule would provide around 50 mg aspirin. A 1 mL dose would provide around 25 mg aspirin.

Comparative Example 1

[0425] A composition was prepared by mixing 10 wt % aspirin (ex Sigma Aldrich) and 90 wt % glycerol+20EO (glycerol ethoxylated with 20 mol equivalents of ethylene oxide). The components were mixed in the appropriate ratios and sonicated to achieve complete solution. Microscopy showed that the solution was free of any particulate material. The aspirin stability in the composition was measured weekly as described above.

[0426] The results are shown in Table 3 below, and in FIG. 1.

TABLE-US-00003 TABLE 3 Day Normalised % salicylic acid 0 1.4 6 1.3 14 1.9 20 2.2 27 2.6 34 3.7 41 4.2

[0427] The average degradation rate is 0.102%/day. Extrapolation from these results gives a predicted shelf life of 124 days.

Comparative Example 2

[0428] A composition was prepared by mixing 10 wt % aspirin (ex Sigma Aldrich), 1% saccharin (ex Sigma Aldrich) and 89 wt % glycerol+20EO. The components were mixed in the appropriate ratios and sonicated to achieve complete solution. Microscopy showed that the solution was free of any particulate material. The aspirin stability in the composition was measured weekly as described above.

[0429] The results are shown in Table 4 below, and in FIG. 2.

TABLE-US-00004 TABLE 4 Day Normalised % salicylic acid 0 0.8 8 1.4 14 1.1 21 1.6 28 2.0 35 2.5

[0430] The average degradation rate is 0.0714%/day. Extrapolation from these results gives a predicted shelf life of 205 days.

[0431] The results of the above Examples and Comparative Examples are summarised in Table 5 below:

TABLE-US-00005 TABLE 5 Glycerin Glycerol + Spearmint Degradation Predicted Aspirin Triacetate 20EO Saccharin Oil rate shelf-life (wt %) (wt %) (wt %) (wt %) (wt %) (wt %/day) (days) Ex. 2 2.5 96.5 — 1.0 — 0.025 400 Ex. 3 2.5 96.35 — 1.0 0.15 0.023 435 Ex. 4 2.5 96.5 — 1.0 — 0.0288 360 Comp. 10 — 90 — — 0.102 124 Ex. 1 Comp. 10 — 89 1.0 — 0.0714 205 Ex. 2

[0432] It can be seen that the degradation rate of aspirin and thereby the shelf-life of the liquid composition is improved dramatically for compositions according to the invention. Shelf life of a year or longer is possible. A comparison of Comparative Examples 1 and 2 shows that the presence of saccharin provides a significant increase in stability. Furthermore, a comparison of Comparative Example 2 with Examples 2-4 reveals a dramatic improvement in stability when the composition includes glycerin triacetate.

[0433] The above examples illustrate the improved properties of a composition according to the present invention.

Example 6—an In Vitro Study to Investigate the Anti-Tumour Effects of Liquid Aspirin in Adult Glioblastoma, Paediatric High Grade Glioma and Medulloblastoma

Introduction

[0434] Soluble aspirins currently on the market are in fact dispersible and therefore still contain grains that sit on the gastric mucosa causing gastric side effects. The categorization as “soluble” is therefore not accurate. Alternative aspirin products are powders that quickly disperse in water. No truly shelf stable liquid formulation of acetylsalicylic acid (ASA) has been successfully produced, until now. The unique liquid ASA described herein (referred to in this Example as ‘liquid aspirin’) is expected to show a significant reduction in gastrointestinal side effects.

[0435] As described herein, liquid aspirin contains ASA and two excipients: glycerin triacetate (triacetin) and saccharin (Sac). All three ingredients are pharmaceutically approved and have been shown to have compelling anti-tumour properties.

[0436] Triacetin has been shown to significantly augment drug delivery across the blood brain barrier (BBB), suggesting that this combination could be highly effective against glioblastoma (GBM) [Van Tellingen et al., Overcoming the blood-brain tumor barrier for effective glioblastoma treatment. Drug Resist. Updat. 19, 1-12 (2015)].

[0437] Saccharin based compounds have been proposed as a new class of anti-cancer agent (Mahon et al., Saccharin: a lead compound for structure-based drug design of carbonic anhydrase IX inhibitors. Bioorg Med Chem 2015 Feb. 15; 23(4):849-54, incorporated herein by reference). Proescholdt et al. (‘Function of carbonic anhydrase IX in glioblastoma multiforme’, Neuro Oncol, 2012, Vol. 14, pp. 1357-1366) suggest that inhibition of carbonic anhydrase IX is a potential metabolic target for the treatment of glioblastoma patients.

[0438] Of the three components, aspirin has demonstrated the most potent anti-tumour effect, particularly against GBM. An initial in vivo study highlighted that the administration of aspirin into an established Fischer 344 rat glioma model (Aas, A. T., Brun, A., Blennow, C., Stromblad, S., & Salford, L. G. The RG2 rat glioma model. J. Neurooncol. 23, 175-183 (1995)) significantly inhibited the growth of differentiated malignant glioblastoma RG2 cells both when administered the day before tumour cell inoculation as well as in established rat glioblastoma tumours (Aas, A. T., Tonnessen, T. I., Brun, A., & Salford, L. G. Growth inhibition of rat glioma cells in vitro and in vivo by aspirin. J. Neurooncol. 24, 171-180 (1995)).

[0439] Prostaglandin E2 (PGE2) has been shown to have an important role in both immunosuppression and tumour growth. As a PGE2 inhibitor, aspirin has been shown to reduce in vitro tumour cell proliferation. Aspirin dosage studies were conducted, evaluating the effect of both high and low dose aspirin exposure on PGE2 synthesis in the in vitro C6 glioma model. These studies revealed that aspirin directly inhibited PGE2 synthesis in C6 cells and that critically, low-dose aspirin is as effective as high-dose aspirin in mediating this response (Hwang, S. L. et al. Effect of aspirin and indomethacin on prostaglandin E2 synthesis in C6 glioma cells. Kaohsiung. J. Med. Sci. 20, 1-5 (2004)).

[0440] Human A172 glioblastoma cells treated with aspirin induced significant apoptosis (programmed cell death) [Kim, S. R. et al. Aspirin induces apoptosis through the blockade of IL-6-STAT3 signaling pathway in human glioblastoma A172 cells. Biochem. Biophys. Res. Commun. 387, 342-347 (2009)]. The underlying mechanism for this response was a reduction in the level of phosphorylated signal transducer and activator of transcription 3 (STAT3), specifically pTyr-STAT3. STAT3 is a transcription factor that is required for survival of A172 cells. This conclusion was supported by measuring cyclin D1, XIAP, and Bcl-2 transcription that was notably attenuated after aspirin treatment (Kim et al., supra). Implicating STAT3 further, the expression and secretion of interleukin-6 (IL-6) (that induces STAT3 phosphorylation), was notably inhibited by aspirin treatment (Kim et al., supra).

[0441] Drawing from these findings, it is known that hypoxia can activate STAT3 and subsequently induce angiogenesis (the development of blood vessels) [Greten, F. R. & Karin, M. Peering into the aftermath: JAKi rips STAT3 in cancer. Nat. Med. 16, 1085-1087 (2010)]. In most solid malignancies, persistent STAT3 signalling is triggered by an autocrine-paracrine production of IL-6 that is noticeably higher in a hypoxic environment (Song, Y. Y. et al. STAT3, p-STAT3 and HIF-1alpha are associated with vasculogenic mimicry and impact on survival in gastric adenocarcinoma. Oncol. Lett. 8, 431-437 (2014)). Hypoxia is a hallmark of GBM, with tumours showing pseudopalisades of neoplastic cells surrounding areas of frank necrosis as well as signs of vascular proliferation. These areas of peri-necrotic hyper-cellularity have been well characterized and are not the result of increased proliferation. As one would predict, these regions have high levels of hypoxia-induced factor 1 alpha (HIF1α) expression, resulting in pro-angiogenic vascular endothelial growth factor (VEGF) secretion as well as elevated IL-6. This in turn drives vascular proliferation. However, the vessels that are generated in response to VEGF within this environment are severely malformed (Jain, R. K. Normalizing tumor microenvironment to treat cancer: bench to bedside to biomarkers. J. Clin. Oncol. 31, 2205-2218 (2013)). The result is deregulated vessel structure with gaps between endothelial cells and an absence of pericytes. Due to this malformation and inherent leakiness, the interstitial pressure is increased resulting in vascular stasis with corresponding exacerbation of hypoxia and increased microvascular thrombosis (Jain et al., supra). Strikingly, it has been shown that aspirin selectively suppresses inflammation, and specifically IL-6-induced T-helper cell 17. This mediates the down regulation of acetyl-STAT3 expression as well as blocking IL-17A-induced inflammation and IL-6 production. This reduction of IL-6 production will then result in a concomitant reduction in active (phosphorylated) STAT3.

[0442] More recently, it has been shown that aspirin represses the transcriptional activity of the β-catenin/TCF protein complex. As a consequence of this, aspirin directly inhibits GBM proliferation and invasion as well as inducing apoptosis (Jin, T., George, F., I, & Sun, J. Wnt and beyond Wnt: multiple mechanisms control the transcriptional property of beta-catenin. Cell Signal. 20, 1697-1704 (2008)). The results presented within this study suggest that aspirin exerts its anti-neoplastic action by suppressing the β-catenin/TCF signalling pathway in GBM. This is particularly striking as recent data has highlighted that FoxM1 promotes the development and progression of GBM by regulating key factors involved in cell division, epithelial to mesenchymal transition (EMT), invasion, angiogenesis and upregulation of the Wnt/β-catenin signalling network (Wang, Z., Zhang, S., Siu, T. L., & Huang, S. Glioblastoma multiforme formation and EMT: role of FoxM1 transcription factor. Curr. Pharm. Des 21, 1268-1271 (2015)). A deregulated Wnt/β-catenin network has been reported in GBM and it has been suggested that this could also constitute a therapeutic target (Zhang, K. et al. ICAT inhibits glioblastoma cell proliferation by suppressing Wnt/beta-catenin activity. Cancer Lett. 357, 404-411 (2015)).

Experimental Method and Results

[0443] Many of the established glioma cell lines, such as C6, A172, U87, U373 and U251 have been grown/passaged in research laboratories around the world for many years and, as a consequence, display a high degree of cellular heterogeneity rendering them increasingly dissimilar to their original primary/early passage cultures and, indeed, from the biopsy from which they were derived.

[0444] To address this issue, we used a panel of patient-derived (adult and paediatric) ex vivo GBM low passage cell cultures, which have been characterized at the molecular level, including DNA fingerprinting (Prof. G. Pilkington, Brain Tumour Research Centre, University of Portsmouth, UK). These cells are truly representative of the patient GBM and, as a result, any anti-tumour effect observed will have clinical relevance. This extensive cell culture bio-bank allows screening of novel anti-GBM therapeutics prior to in vivo studies and clinical trials.

[0445] It is also critical to compare these novel therapeutics against the currently prescribed frontline chemotherapeutics. Consequently, our studies also include temozolomide, which is a standard of care treatment for GBM.

[0446] Non-neoplastic astrocytes provide a control element within these studies. These are non-cancerous and any proposed therapeutic will preferably have selective anti-cancer action (i.e. not kill normal, non-tumor, cells).

[0447] Viability studies were conducted to compare the effect of liquid aspirin (ASP), triacetin (ASP vehicle), and temozolomide (TMZ) in five adult GBM ex vivo cell lines from our panel. Results are shown in FIGS. 4A to 4E. Liquid aspirin showed notably better induction of cell death in all GBM cells tested.

[0448] Synergy studies were also conducted to directly address the specific anti-tumour efficacy of each of aspirin, triacetin and saccharin (Sac) as individual components and the triple-formulation of aspirin, triacetin and saccharin (liquid aspirin). We have significant experience conducting this type of analysis and importantly can differentiate between additive effects versus a synergistic response (Hallden, G. et al. Novel immunocompetent murine tumor models for the assessment of replication-competent oncolytic adenovirus efficacy. Mol. Ther. 8, 412-424 (2003); Cheong, S. C. et al. E1A-expressing adenoviral E3B mutants act synergistically with chemotherapeutics in immunocompetent tumor models. Cancer Gene Ther. 15, 40-50 (2008)). FIGS. 6B and 6C show synergy of liquid aspirin in order to kill 30%, 50% and 70% of UP 029 cells compared to use of each agent alone with the exception of triacetin-alone at 90% reduced cell viability, where the highest dosages of triacetin alone reduced overall cell viability.

[0449] We also confirmed that liquid aspirin demonstrates anti-cancer specificity, i.e. is not toxic to non-neoplastic astrocytes. Results are shown in FIG. 7A to 7F which show liquid aspirin to have markedly better cell killing ability than triacetin or temozolomide; liquid aspirin typically having at least one order of magnitude greater potency in inducing cell death than triacetin or temozolomide. Liquid aspirin had significantly less toxicity when added to non-neoplastic cells (FIG. 7A). Indeed, the associated toxicity noted within the astrocyte cell line could be associated with triacetin-alone (which demonstrated a similar MTS-viability profile).

REFERENCES

[0450] The following are incorporated herein by reference: [0451] 1. Lan et al. “Antitumor effect of aspirin in glioblastoma cells by modulation of β-catenin/T-cell factor-mediated transcriptional activity”, J Neurosurg, 2011, Vol. 115, pp. 780-788; [0452] 2. M. W. Brown, ‘Characterisation Of The Effects Of Chronic Aspirin Treatment On The Viability And Proliferation Of Stage 4 Glioblastoma Cells’, Diffusion: the UCLan Journal of Undergraduate Research, Vol. 6, Issue 2, December 2013; [0453] 3. Aas et al., ‘Growth inhibition of rat glioma cells in vitro and in vivo by aspirin’, Journal of Neuro-Oncology, 1995, Vol. 24, Issue 2, pp. 171-180; [0454] 4. Ning et al., ‘Overexpression of S100A9 in human glioma and in-vitro inhibition by aspirin’, European Journal of Cancer Prevention, 2013, Vol. 22, Issue 6, pp. 585-595; [0455] 5. Hwang et al., ‘Effect of aspirin and indomethacin on prostaglandin E2 synthesis in C6 glioma cells’, Kaohsiung J Med Sci, 2004, Vol. 20, pp. 1-5; [0456] 6. Okada et al., ‘Integration of epidemiology, immunobiology, and translational research for brain tumors’, Ann N Y Acad Sci, 2013, Vol. 1284, pp. 17-23; [0457] 7. Rothwell et al., ‘Effect of daily aspirin on long-term risk of death due to cancer: analysis of individual patient data from randomised trials’, Lancet, 2011, Vol. 377, pp. 31-41; [0458] 8. K. D. Rainsford, ‘Aspirin and Related Drugs’, 2004 (book); [0459] 9. Douthwaite and Lintott, ‘Gastroscopic observations of the gastric mucosa after use of aspirin’, Lancet, 1938, Vol. 232, pp. 1222-1225; [0460] 10. Hurst and Lintott, ‘Aspirin as a cause of hematemesis’, Guy's Hosp Rep, 1939, p. 173; [0461] 11. D. J. Levy ‘An aspirin tablet and gastric ulcer’, N Engl J Med, 2000, Vol. 343, No. 12, p. 863 (photograph); [0462] 12. Rainsford et al. ‘Electronmicroscopic observations on the effects of orally administered aspirin and aspirin-bicarbonate mixtures on the development of gastric mucosal damage in the rat’, Gut, 1975, Vol. 16(7), pp. 514-527; [0463] 13. Jaiswal et al. IUPHAR 9th Int. Conference of Pharmacology, 1984; [0464] 14. Liversage et al. ‘Drug particle size reduction for decreasing gastric irritation and enhanced absorption of Naproxen in rats’, Int J Pharm, 1995, Vol. 125(2), pp. 309-313; [0465] 15. Gyory et al. ‘Effect of particle size on aspirin-induced gastrointestinal bleeding’, Lancet, Vol. 292, No. 7563, pp. 300-302; [0466] 16. M. I. Grossman et al. ‘Fecal Blood Loss Produced By Oral And Intravenous Administration Of Various Salicylates’, Gastroenterology, 1961, Vol. 40, pp. 383-388; [0467] 17. Kevin et al., ‘Acute effect of systemic aspirin on gastric mucosa in man’, Digestive Diseases and Sciences, 1980, Vol. 25, Issue 2, pp 97-99; [0468] 18. Cooke et al., ‘Failure of intravenous aspirin to increase gastrointestinal blood loss’, British medical journal, 1969; Vol. 3(5666), pp. 330-332; [0469] 19. Wallace et al., ‘Adaptation of rat gastric mucosa to aspirin requires mucosal contact’ Am J Physiol, 1995, Vol. 268, G134-8; [0470] 20. Cryer et al. ‘Effects of low dose daily aspirin therapy on gastric, duodenal and rectal prostaglandin levels and on mucosal injury in healthy humans’, Gastroenterology, 1999, Vol. 117, pp. 17-25; [0471] 21. Lichtenberger et al., ‘Where is the evidence that cyclooxygenase inhibition is the primary cause of nonsteroidal anti-inflammatory drug (NSAID)-induced gastrointestinal injury? Topical injury revisited’, Biochemical Pharmacology, 2001, Vol. 61, pp. 631-637; [0472] 22. Ligumsky M et al., ‘Aspirin can inhibit gastric mucosal cyclo-oxygenase without causing lesions in the rat’, Gastroenterology, 1983, Vol. 84, pp 756-61; [0473] 23. Zhao et al., ‘Clinical Research Feasibility of intravenous administration of aspirin in acute coronary syndrome’, Journal of Geriatric Cardiology, 2008, Vol. 5 No. 4, pp. 212-216; [0474] 24. Mashita et al., ‘Oral but not parenteral aspirin upregulates COX-2 expression in rat stomachs: a relationship between COX-2 expression and PG deficiency’, Digestion, 2006, Vol. 73(2-3), pp. 124-32; [0475] 25. Hall, S L; Lorenc, T (1 Feb. 2010). “Secondary prevention of coronary artery disease”. American family physician 81 (3): 289-96; [0476] 26. Fengming Lan et al., Antitumor effect of aspirin in glioblastoma cells by modulation of β-catenin/T-cell factor-mediated transcriptional activity. J Neurosurg 115:780-788, 2011; [0477] 27. Morris et al., Effects of Low-Dose Aspirin on Acute Inflammatory Responses in Humans. The Journal of Immunology. 2009; 183:2089-2096; [0478] 28. Gasic G I, et al. Lancet. 1972; 2:932-937; [0479] 29. Tsen et al., Triacetin-based acetate supplementation as a chemotherapeutic adjuvant therapy in glioma. Int J Cancer. 2014 March 15; 134(6):1300-1310; [0480] 30. Long et al., ‘Acetate Supplementation Induces Growth Arrest of NG2/PDGFRa-Positive Oligodendroglioma-Derived Tumor-Initiating Cells’, PLoS One, 2013, e80714; [0481] 31. Mahon et al., Saccharin: a lead compound for structure-based drug design of carbonic anhydrase IX inhibitors. Bioorg Med Chem 2015 Feb. 15; 23(4):849-54; [0482] 32. Proescholdt et al. (‘Function of carbonic anhydrase IX in glioblastoma multiforme’, Neuro Oncol, 2012, Vol. 14, pp. 1357-1366; [0483] 33. Buck M L et al., ‘Use of Aspirin in children with cardiac disease’, Pediatr Pharm, 2007, Vol. 13(1); [0484] 34. Garcia Albeniz. X et al., ‘Aspirin for the prevention of colorectal cancer’, Best Pract Res Clin Gastroenterol, 2011 August, Vol. 25(0), pp. 461-472.