CANCER THERAPY WITH MICROBUBBLES

Abstract

The invention relates to a microbubble-chemotherapeutic agent complex comprising a microbubble carrying a combination of chemotherapeutic agents for use in a method of treating cancer in a patient, wherein said combination of chemotherapeutic agents comprises: (a) a 5-fluoropyrimidine or a derivative thereof; (b) irinotecan or a derivative thereof; and (c) a platinum-based chemotherapeutic agent or a derivative thereof; and wherein said method comprises simultaneous, separate or sequential administration of folinic acid or a derivative thereof. The invention is particularly suitable for use in the treatment of deep-sited tumours and associated metastatic disease, for example in the treatment of pancreatic cancer. The invention further relates to the microbubble- chemotherapeutic agent complexes themselves, to methods for their preparation and to pharmaceutical compositions which contain them, optionally in combination with folinic acid or a folinic acid derivative.

Claims

1. A microbubble-chemotherapeutic agent complex which comprises a microbubble carrying a combination of chemotherapeutic agents for use in a method of treating cancer in a patient, wherein said combination of chemotherapeutic agents comprises: (a) a 5-fluoropyrimidine or a derivative thereof; (b) irinotecan or a derivative thereof; and (c) a platinum-based chemotherapeutic agent or a derivative thereof; and wherein said method comprises simultaneous, separate or sequential use of folinic acid or a derivative thereof.

2. A complex for use as claimed in claim 1, wherein said 5-fluoropyrimidine is 5-fluorouracil (5-FU), 5-fluorouridine (5-FUR), capecitabine, carmofur, doxifluridine, tegafur, or a pharmaceutically acceptable salt thereof.

3. A complex for use as claimed in claim 1 or claim 2, wherein said combination of chemotherapeutic agents comprises irinotecan in the form of its free base.

4. A complex for use as claimed in any one of claims 1 to 3, wherein said platinum-based chemotherapeutic agent is cisplatin, oxaliplatin, carboplatin, satraplatin, picoplatin, tetraplatin, platinum-DACH, or a derivative thereof.

5. A complex for use as claimed in claim 4, wherein said platinum-based chemotherapeutic agent is oxaliplatin or Pt(DACH)(Ox)(OH).sub.2 (wherein DACH = 1,2-diaminocyclohexane, and Ox = oxalate).

6. A complex for use as claimed in any one of the preceding claims, wherein the microbubble has a diameter in the range of from 0.1 to 100 um.

7. A complex for use as claimed in any one of the preceding claims, wherein the microbubble comprises a shell which retains a gas selected from perfluorobutane, perfluoropropane, and oxygen.

8. A complex for use as claimed in any one of the preceding claims, wherein the microbubble has a shell comprising one or more phospholipids, each optionally linked to one or more polymers such as polyethylene glycol (PEG).

9. A complex for use as claimed in any one of the preceding claims, wherein one or more of said chemotherapeutic agents are attached to the microbubble via a non-covalent linkage, e.g. via a biotin-avidin-biotin interaction.

10. A complex for use as claimed in claim 9, wherein said platinum-based chemotherapeutic or derivative thereof is attached to the microbubble via a biotin-avidin-biotin interaction.

11. A complex for use as claimed in any one of the preceding claims, wherein one or more of said chemotherapeutic agents are attached to the microbubble via a covalent linkage.

12. A complex for use as claimed in claim 11, wherein said 5-fluoropyrimidine or derivative thereof (e.g. 5-FUR) and/or said platinum-based chemotherapeutic agent or derivative thereof (e.g. oxaliplatin or Pt(DACH)(Ox)(OH).sub.2) are attached to the microbubble via a covalent linkage.

13. A complex for use as claimed in claim 11, wherein each of said chemotherapeutic agents is attached to the microbubble via a covalent linkage, preferably wherein all of the following agents are attached to the microbubble via a covalent linkage: 5-FUR, irinotecan, and oxaliplatin or Pt(DACH)(Ox)(OH).sub.2.

14. A complex for use as claimed in any one of claims 1 to 12, wherein one or more of said chemotherapeutic agents are incorporated into the shell of the microbubble, preferably wherein irinotecan is incorporated into the shell of the microbubble.

15. A complex for use as claimed in any one of the preceding claims, wherein said complex is delivered to affected cells or tissues of said patient and subjected to ultrasound irradiation whereby to rupture the microbubble.

16. A complex for use as claimed in any one of the preceding claims in the treatment of cancer or metastatic cancer, preferably in the treatment of a deep-sited tumour or metastasis derived from said tumour.

17. A complex for use as claimed in claim 16, wherein said cancer is a carcinoma, such as pancreatic adenocarcinoma (PAC) or metastatic pancreatic adenocarcinoma (mPAC).

18. A complex for use as claimed in any one of the preceding claims, wherein one or more of said chemotherapeutic agents are administered to said patient at a sub-therapeutic dosage, for example wherein each of said chemotherapeutic agents is administered at a sub-therapeutic dose.

19. A complex for use as claimed in any one of the preceding claims, wherein said patient has an Eastern Cooperative Oncology Group (ECOG) performance status (PS) which is greater than 1, for example an ECOG PS of 2 or greater.

20. A complex for use as claimed in any one of the preceding claims, wherein said patient suffers from hepatic or renal dysfunction.

21. A complex for use as claimed in any one of the preceding claims as a second-line treatment of cancer.

22. A microbubble-chemotherapeutic agent complex comprising a microbubble carrying a combination of the following chemotherapeutic agents: (a) a 5-fluoropyrimidine or a derivative thereof; (b) irinotecan or a derivative thereof; and (c) a platinum-based chemotherapeutic agent or a derivative thereof.

23. A complex as claimed in claim 22, wherein the microbubble is covalently linked to the 5-fluoropyrimidine or derivative thereof, covalently or non-covalently linked to the platinum-based chemotherapeutic agent or derivative thereof (e.g. linked via a biotin-avidin-biotin interaction), and has a shell in which irinotecan or a derivative thereof is embedded.

24. A method for the preparation of a microbubble-chemotherapeutic agent complex as claimed in claim 22 or claim 23, wherein said method comprises the following steps: (i) providing a lipid which is capable of forming a microbubble; (ii) optionally covalently linking one or more chemotherapeutic agents to said lipid whereby to form a functionalised lipid; (iii) preparing a microbubble from said functionalised lipid, optionally in the presence of one or more chemotherapeutic agents; and (iv) optionally linking the resulting microbubble to one or more chemotherapeutic agents via a non-covalent linkage, e.g. via a biotin-avidin-biotin interaction.

25. A pharmaceutical composition comprising a microbubble-complex as claimed in claim 22 or claim 23, and folinic acid or a derivative thereof, together with one or more pharmaceutically acceptable carriers or excipients.

Description

[0160] The invention will now be described further with reference to the following non-limiting Examples and the accompanying figures in which:

[0161] FIG. 1 shows a schematic of FIRINOX-loaded MBs and their constituents.

[0162] FIG. 2 shows (a) optical microscopy image of FIRINOX MBs; and (b) their size distribution.

[0163] FIG. 3 shows optical (grey panels) and fluorescence (black panels) microscopy images of Panc-01 3D spheroids treated with (+US) or without (-US) ultrasound treatment in the absence (a) or presence (b) of FIRINOX MBs. FIRINOX MBs contained 50 .Math.MIrinotecan, 90 .Math.M 5-fluorouracil and 48 .Math.M Oxaliplatin. Ultrasound conditions: Sonidel SP100 sonoporator with US gel used to mediate contact. Each well was treated with US for 30 secs using a frequency of 1 MHz, an US power density of 3.0 W/cm.sup.2 and a duty cycle of 50% (pulse frequency = 100 Hz). (c) plot of mean propidium iodide fluorescence intensity per micron of spheroid for each of the groups shown in (a) and (b).

[0164] FIG. 4 shows (a) tumour growth delay and (b) Kaplan-Meier survival plot for animals treated in the following manner: (i) Group 1 received no treatment; Group 2 received a standard dose of FOLFIRINOX (folinic acid 100 mg/kg; 5-fluorouracil 50 mg/kg; Irinotecan 50 mg/kg and Oxilaplatin 5 mg/kg) by IV injection; Group 3 received folinic acid (100 mg/kg) and FIRINOX MBs containing 5-fluorouracil (2.1 mg/kg); Irinotecan (7.3 mg/kg) and Oxaliplatin (3.4 mg/kg) by IV injection; Group 4 received the same treatment as Group 3 but with ultrasound directed at the tumour to burst the MBs and release the drug payloads. (c) Graphical representation of the concentrations of the three chemotherapies used in the standard treatment and in the FIRINOX MBs.

[0165] FIG. 5 shows (a) tumour growth delay and (b) Kaplan-Meier survival plot for animals bearing subcutaneous HT-29 colon tumours treated with: (i) Group 1 received no treatment; Group 2 received a standard dose of FOLFRINOX; (ii) Group 3 received folinic acid and FIRINOX MBs; and Group 4 received the same treatment as Group 3 but with ultrasound directed at the tumour to burst the MBs and release the drug payloads. (c) Graphical representation of the concentrations of the three chemotherapies used in the standard treatment and FIRINOX MBs.

EXAMPLES

Example 1 - Preparation of FIRINOX-Loaded MBs and Characterisation

[0166] A single microbubble (MB) formulation carrying 5-flurouracil, irinotecan and oxaliplatin (FIRINOX) was produced by loading irinotecan (lRIN) hydrophobically into MB shells prepared from a 5-fluorouracil analogue functionalised lipid (F) and other lipids. The resulting FIRIN loaded MBs were attached to an oxaliplatin analogue (OX) using the biotin-avidin interaction to produce the FIRINOX loaded MBs.

1.0 Method

[0167] All materials were purchased from commercial sources with the exception of DBP-5 FU and Biotin-Ox which were chemically synthesised.

1.1 Synthesis of DBP-5FU (“FUR-Lipid”)

[0168] A CHCl.sub.3 solution (30 mL) of 1,2-didocosanoyl-sn-glycero-3-phosphocholine (DBPC) (500 mg) was added to a solution of Phospholipase D (PLDP) (10 mg) and 5-fluorouridine (720 mg) in sodium acetate buffer (200 mM pH 5.7 10 mL) containing CaCl.sub.2 (250 mM). The mixture was stirred at 45° C. for 6 hours, then a mixed solution of 2N HCl (5 mL), MeOH (20 mL) and CHCl.sub.3 (20 mL) was added, and the mixture was shaken. The separated organic layer was washed with H.sub.2O (2×10 mL) and then evaporated to dryness. The residue was purified by flash chromatography (silica gel CHCl.sub.3:MeOH 10:1 followed by 6:1) and fractions containing the desired product “FUR-lipid” combined and evaporated to dryness. .sup.1H NMR (500 MHz, CDCl.sub.3:CD.sub.3OD (2:1) d (ppm) 8.01 (d, 1H, CONHCO), 5.91 (br d, 1H, 1′(CH)), 5.20 (m, 1H, glycerol CH), 3.72-4.20 (m, 9H, 3′(CH), 2′(CH) 4′(CH), 5′(CH.sub.2) glycerol CH.sub.2, glycerol CH.sub.2OPO), 2.27 (m, 4H, 2x COCH.sub.2), 1.57 (m, 4H, 2×CH.sub.2), 1.23 (m, 72H, behenoyl CH.sub.2), 0.83 (t, 6H, 2×CH.sub.3). -ve mode MALDI-MS: Expected for C.sub.56H.sub.102O.sub.13N.sub.2P.sub.1F.sub.1 = 1060.71 Found 1059.48

1.2 Synthesis of Biotin-OX

[0169] (+)-Biotin N-hydroxysuccinimide ester (0.1 g, 0.293 mmol) in anhydrous DMSO (4 mL) was added to a suspension of cis,cis,trans-[Pt(DACH)(Ox)(OH).sub.2] (0.126 g, 0.305 mmol) in anhydrous DMSO (8 mL). The reaction was stirred at room temperature for 4 days under an argon atmosphere. A small amount of white solid was filtered. The yellow filtrate was concentrated using a DMSO trap to give a sticky yellow oil, to which acetone (40 mL) as added, precipitating a white solid. The suspension was stirred for 1 hr and subsequently the solid was filtered, washed, with acetone, diethyl ether and dried. Yield: 0.122 g (0.186 mmol, 60%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ: 8.63 (s, 1H, NH), 8.19 (s, 1H, NH), 7.86 (s, 1H, NH), 7.18 (s, 1H, NH), 6.38 (d, 2H, 3J = 16 Hz), 4.29 (t, 1H, 3J = 8 Hz), 4.11 (t, 1H, 3J = 8 Hz), 3.06 (dt, 1H, 3J = 8 Hz & 4J = 2 Hz), 2.79 (dd, 1H, 3J = 8 Hz & 4J = 4 Hz), 2.58 (d, 1H, 3J = 4 Hz), 2.54 (s, 2H), 2.16 (t, 2H, 3J = 8 Hz), 1.25 (m, 10H), 1.07 (m, 2H,) ppm.

[0170] .sup.195Pt NMR (86 MHz, DMSO-d.sub.6) δ: 1406.7 ppm.

[0171] EA calc. % for C.sub.18H.sub.30 N.sub.4O.sub.8PtS.1.5 H.sub.2O requires C, 31.58; H, 4.86; N, 8.18; S 4.68, found C, 31.60; H, 4.66; N, 7.84; S, 5.00 %.

[0172] ESI-MS: m/z ([M+H]+) 658.1 ([M+Na]+) 680.1.

1.3 Preparation of FIRINOX MBs

[0173] FlRINOX MBs were prepared by first dissolving DBP-5FU (4.0 mg, 3.77 .Math.mol), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol) -2000] (DSPE-PEG(2000)) (1.15 mg, 0.41 .Math.mol) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG (2000)-biotin) (1.24 mg, 0.41 .Math.mol) in chloroform to achieve a molar ratio of 82:9:9. To this solution was added Irinotecan free base (10 mg) dissolved in chloroform (100 .Math.L). The solvent was removed under vacuum at room temperature yielding a translucent film. The film was then reconstituted in 2 mL of a solution containing PBS, glycerol and proplyene glycol (8:1:1 vol ratio) and heated in a water bath at 80° C. for 30 min. The suspension was sonicated using a Microson ultrasonic cell disrupter at an amplitude of 22% for 1 min and then at an amplitude of 90% in a perfluorobutane (PFB) atmosphere for 30 sec. The MBs were then cooled on ice for 10 min followed by centrifugation at 100 rcf for 3 min and the liquid laying below the surface of the MB cake (infranatant) was removed. The MB cake was then washed a further 2 times with PBS (pH 7.4 ± 0.1) before being mixed for 5 min on ice with an aqueous solution of avidin (10 mg/mL) using an orbital shaker (150 rpm). The MBs were then centrifuged (100 rcf) for 3 min, the infranatant removed and the MB cake washed with PBS solution (2 mL, pH 7.4 ± 0.1) which was again removed following centrifugation. The MB cake was again reconstituted in PBS solution (2 mL, pH 7.4 ± 0.1), mixed for 5 min with an aqueous solution containing Biotin-Ox (1 mL, 5 mg/mL) and centrifuged (100 rcf) for 3 min. Following removal of the infranatant, the MB cake was then washed with PBS (2 mL, pH 7.4 ± 0.1), centrifuged and the MB cake isolated. This washing/centrifugation procedure was repeated twice further with the FIRINOX MB cake reconstituted in 2 mL of PBS. FIG. 1 shows a schematic of the FIRINOX-loaded MBs and their constituents.

1.4 Characterisation of FIRINOX-Loaded MBs

[0174] The FIRINOX-loaded MBs were characterised by optical microscopy and had a mean particle diameter of 1.14 .Math.m ± 1.17 .Math.m and a concentration of 6.33 × 10.sup.9 / mL (FIG. 2).

Example 2 - In Vitro Cytotoxicity of Ultrasound Activated FIRINOX-Loaded Microbubbles in a Panc-01 3D Spheroid Model of Pancreatic Cancer

[0175] Cytotoxicity of ultrasound activated FIRINOX MBs prepared in Example 1 was determined in a Panc-01 3D spheroid model of pancreatic cancer.

2.1 Method

[0176] 96 well plates were coated with agarose solution (15 mg/ml in Dulbecco’s Modified Eagle’s Medium (DMEM) - low glucose, 60 .Math.L/well) and air-dried in a laminar-flow hood for 30 min. Panc-01 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing high glucose (4.5 g/L) which were supplemented with 10% (v/v) foetal bovine serum in a humidified 5% CO.sub.2 atmosphere at 37° C. 6×10.sup.3 Panc-01 cells were seeded in each well and placed in an incubator (37° C., 5% CO.sub.2) for 96 h to generate the spheroids. The spheroids were then treated with a PBS : medium (50:50 v/v) solution containing the FIRINOX MBs (50 .Math.M IR + 90 .Math.M FUR + 48 .Math.M OX) and selected wells treated with ultrasound delivered using a Sonidel SP100 sonoporator (1 MHz, 30 s, 3 W/cm.sup.2, duty cycle=50%, and PRF=100 Hz) for 30 secs from underneath the plate using ultrasound gel to mediate contact. Untreated spheroids and spheroids treated with ultrasound only were used for comparative purposes. Following treatment, the spheroids were incubated for a further 48 h when the medium was removed and spheroids then washed 3 times with PBS. The spheroids were then treated with a solution of propidium iodide in PBS (100 .Math.g/ml) and incubated for 30 min after which time the propidium iodide solution was removed and the spheroids washed 3 times with PBS. Micrographic images were recorded using a NIKON Eclipse E400 phase contrast microscope in bright field and fluorescence modes (540 nm band pass excitation and 590 nm long pass emission filters). Image J software was used to quantify propidium iodide fluorescence and it was expressed as a % of P.I. fluorescence intensity/.Math.m.sup.2, i.e. the propidium iodide fluorescence was normalized according to the area of the spheroid.

2.2 Results

[0177] The results are shown in FIG. 3 and revealed that spheroids treated with the FIRINOX MBs in combination with ultrasound were significantly smaller while the remaining cells were much brighter (propidium iodide staining indicating cell death) than spheroids treated with FIRINOX MBs alone (i.e. without ultrasound) or spheroids that remained untreated. These results suggest that the physical events that accompany the MB cavitation (bursting) help disperse the three chemotherapies much deeper into the spheroid matrix enhancing their cytotoxicity.

Example 3 - In Vivo Cytotoxicity of Ultrasound Activated FIRINOX-Loaded Microbubbles in Mice Bearing Subcutaneous KPC Pancreatic Tumours

[0178] Cytotoxicity of ultrasound activated FIRINOX MBs prepared in Example 1 was determined using C57 mice bearing ectopic KPC pancreatic tumours that were generated using the T110299 cell line derived from a KPC mouse strain (Duewell et al., 2015, Oncolmmunol. 4:10 e 1029698).

3.1 Method

[0179] All animals employed in the study were treated humanely and in accordance with the licenced procedures under the UK Animals (Scientific Procedures) Act 1986. KPC cells were maintained in DMEM medium supplemented with 10% foetal calf serum. Cells (5 ×10.sup.5) were re-suspended in PBS and implanted into the rear dorsum of female C57 mice. Tumour formation occurred approximately 2 weeks after implantation and once tumours became palpable, dimensions were measured using Vernier callipers. Tumour measurements were taken every other day using calipers. Tumour volume was calculated using the equation: tumour volume = (length x width x height)/2. Once tumours reached approximately 100 mm.sup.3, animals were separated into the following groups: [0180] Group 1 - no treatment. [0181] Group 2 - a tail vein injection of FOLFIRINOX free drug, i.e. not on a MB. Oxaliplatin at a dose of 5.0 mg/kg was administered first and immediately followed by leucovorin (folinic acid) at a dose of 100 mg/kg, with the addition, after 30 minutes, of irinotecan at a dose of 50 mg/kg, then the treatment was immediately followed by 5-fluorouracil at a dose of 25 mg/kg, administered intravenously). [0182] Group 3 - leucovorin (folinic acid) at a dose of 50 mg/kg administered intravenously followed by FIRINOX MBs ([IRIN]=7.3±1.50 mg/kg, [OX]=3.35±0.37 mg/kg, [FUR]=2.09±0.19 mg/kg) injected intravenously with ultrasound applied to the tumour during and after injection for a total of 3.5 min. Ultrasound was administered using a Sonidel SP100 sonoporator (3.5 W/cm.sup.2, 1 MHz, 30% duty cycle, and PRF = 100 Hz; PNP = 0.48 MPa; M.I. = 0.48) and ultrasound gel used to mediate contact. [0183] Group 4 - the same treatment as for Group 3 but without ultrasound. Animals were treated on days 0, 3, 6, 8 and both the tumour volume and body weight measurements recorded at the indicated times.

3.2 Results

[0184] The results are shown in FIG. 4 and reveal a significant improvement in tumour growth delay for animals in Group 4 compared to the other three groups (FIG. 4a). In addition, the animals in Group 4 also survived much longer than those in the other three groups (FIG. 4b).

Example 4 - In Vivo Cytotoxicity of Ultrasound Activated FIRINOX-Loaded Microbubbles in Mice Bearing Subcutaneous HT-29 Colon Tumours

[0185] Cytotoxicity of ultrasound activated FIRINOX MBs prepared in Example 1 was determined in mice bearing subcutaneous HT-29 colon tumours.

4.1 Method

[0186] All animals employed in this study were treated humanely and in accordance with the licenced procedures under the UK Animals (Scientific Procedures) Act 1986. HT-29 cells were maintained in DMEM medium supplemented with 10% foetal calf serum. Cells (1 x10.sup.6) were re-suspended in 100 .Math.L of Matrigel® and implanted into the rear dorsum of female Balb/c SCID (C.B-17/IcrHan®Hsd-Prkdcscid) mice. Tumour formation occurred approximately 4 weeks after implantation and once tumours became palpable, dimensions were measured using Vernier callipers. Tumour measurements were taken every other day using calipers. Tumour volume was calculated using the equation: tumour volume = (length x width x height)/2. Once tumours reached approximately 100 mm.sup.3, animals were separated into the following groups: [0187] Group 1 - no treatment. [0188] Group 2 - an intraperitoneal injection of FOLFIRINOX free drug treatment (oxaliplatin at a dose of 2.5 mg/kg was administered first and after 2 hours followed by leucovorin (folinic acid) at a dose of 50 mg/kg, immediately followed by 5-fluorouracil at a dose of 25 mg/kg and irinotecan at a dose of 25 mg/kg). [0189] Group 3 - FIRINOX MBs ([IRIN]=2.95±2.04 mg/kg, [OX]=1.60±0.27 mg/kg, [FUR]=2.34±0.20 mg/kg) injection intravenously with ultrasound applied to the tumour during and after injection for a total of 3.5 min followed by an IP injection of leucovorin (folinic acid) at a dose of 50 mg/kg. Ultrasound was administered using a Sonidel SP100 sonoporator (3.5 W/cm.sup.2, 1 MHz, 30% duty cycle, and PRF = 100 Hz; PNP = 0.48 MPa; M.I. = 0.48) and ultrasound gel used to mediate contact. [0190] Group 4 - the same treatment as for Group 3 but without ultrasound applied to the tumour during treatment.

[0191] Animals were treated on days 0, 3, 7, 13, 17 and both the tumour volume and body weight measurements recorded at the indicated times.

4.2 Results

[0192] The results are shown in FIG. 5 and illustrate an improved tumour growth delay and survival advantage for animals receiving the treatment in accordance with the invention when compared to standard FOLFIRINOX treatment.