SONODYNAMIC THERAPY

Abstract

The invention relates to microbubble complexes for use in methods of sonodynamic therapy which comprise a microbubble attached to or otherwise associated with one or more linking groups, each linking group being bound to at least one sonosensitising agent and at least one chemotherapeutic agent. It further relates to the microbubble complexes themselves and to pharmaceutical compositions which contain them. The invention is particularly suitable for the treatment of deep-sited tumors, in particular pancreatic cancer.

Claims

1. A microbubble complex which comprises a microbubble attached to or otherwise associated with one or more linking groups, each linking group being bound to at least one sonosensitising agent and at least one chemotherapeutic agent.

2. A complex as claimed in claim 1, wherein the sonosensitising agent and chemotherapeutic agent bound to any given linking group are not the same chemical entity.

3. A complex as claimed in claim 1 or claim 2, wherein each linking group is bound to or otherwise associated with the microbubble via a non-covalent linkage, e.g. via a biotin-avidin interaction.

4. A complex as claimed in any one of claims 1 to 3, wherein each linking group is bound to the sonosensitising agent and to the chemotherapeutic agent via covalent bonds.

5. A complex as claimed in any one of the preceding claims, wherein each linking group comprises an organic group comprising a chain of up to about 200 atoms, e.g. up to about 100 atoms.

6. A complex as claimed in any one of the preceding claims, wherein each linking group comprises a straight-chained or branched (preferably branched) C.sub.30-50 alkylene chain (preferably a C.sub.30-40 alkylene chain) optionally substituted by one or more groups selected from C.sub.1-3 alkyl, O(C.sub.1-3)alkyl, and OR (where R is H or C.sub.1-6 alkyl, preferably C.sub.1-3 alkyl, e.g. methyl); and in which one or more (preferably up to 10, e.g. from 4 to 9, or from 6 to 8) CH.sub.2 groups of the alkylene chain may be replaced by a group independently selected from O, CO, C(O)O, NR and NRCO (where each R is independently H or C.sub.1-6 alkyl, preferably C.sub.1-3 alkyl, e.g. methyl).

7. A complex as claimed in any one of the preceding claims, wherein each linking group comprises (e.g. is terminally substituted by) biotin or a biotin residue.

8. A complex as claimed in any one of the preceding claims, wherein each linking group is branched.

9. A complex as claimed in claim 8, wherein each linking group has three branches.

10. A complex as claimed in claim 9, wherein each linking group has the following structure: ##STR00020## wherein: L.sup.1, L.sup.2 and L.sup.3 are each independently (CH.sub.2)q in which q is an integer from 1 to 4, preferably 2; each R is independently either H or C.sub.1-6 alkyl (preferably C.sub.1-3 alkyl, e.g. CH.sub.3), preferably H; n is an integer from 2 to 10, preferably 4 to 8, more preferably 5 to 7, e.g. 6; p is an integer from 2 to 10, preferably 4 to 8, more preferably 5 to 7, e.g. 6; X is a functional group capable of binding to a microbubble or to a functionalised microbubble, preferably wherein X is biotin or a biotin residue which is capable of binding to an avidin-functionalised microbubble; * denotes the point of attachment of the linking group to a sonosensitising agent, a functionalised sonosensitising agent, or a residue of a sonosensitising agent; and ** denotes the point of attachment of the linking group to a chemotherapeutic agent, a functionalised chemotherapeutic agent, or a residue of a chemotherapeutic agent.

11. A complex as claimed in claim 9, wherein each linking group has the following structure: ##STR00021## wherein: X, * and ** are as defined in claim 10.

12. A complex as claimed in claim 9, wherein each linking group has the following structure: ##STR00022## wherein: L.sup.4, L.sup.5 and L.sup.6 are each independently (CH.sub.2).sub.t in which t is an integer from 1 to 4, preferably 2; each R is independently either H or C.sub.1-6 alkyl (preferably C.sub.1-3 alkyl, e.g. CH.sub.3), preferably H; r is an integer from 2 to 10, preferably 4 to 8, more preferably 5 to 7, e.g. 6; s is an integer from 2 to 10, preferably 4 to 8, more preferably 5 to 7, e.g. 6 or 7; X is a functional group capable of binding to a microbubble or to a functionalised microbubble, preferably wherein X is biotin or a biotin residue which is capable of binding to an avidin-functionalised microbubble; * denotes the point of attachment of the linking group to a sonosensitising agent, a functionalised sonosensitising agent, or a residue of a sonosensitising agent; and ** denotes the point of attachment of the linking group to a chemotherapeutic agent, a functionalised chemotherapeutic agent, or a residue of a chemotherapeutic agent.

13. A complex as claimed in claim 9, wherein each linking group has the following structure: ##STR00023## wherein: X, * and ** are as defined in claim 12.

14. A complex as claimed in claim 9, wherein each linking group has the following structure: ##STR00024## wherein: L.sup.7, L.sup.8 and L.sup.9 are each independently (CH.sub.2).sub.u in which u is an integer from 1 to 4, preferably 2; each R is independently either H or C.sub.1-6 alkyl (preferably C.sub.1-3 alkyl, e.g. CH.sub.3), preferably H; X is a functional group capable of binding to a microbubble or to a functionalised microbubble, preferably wherein X is biotin or a biotin residue which is capable of binding to an avidin-functionalised microbubble; * denotes the point of attachment of the linking group to a sonosensitising agent, a functionalised sonosensitising agent, or a residue of a sonosensitising agent; and ** denotes the point of attachment of the linking group to a chemotherapeutic agent, a functionalised chemotherapeutic agent, or a residue of a chemotherapeutic agent.

15. A complex as claimed in claim 9, wherein each linking group has the following structure: ##STR00025## wherein: X, * and ** are as defined in claim 14.

16. A complex as claimed in any one of the preceding claims, wherein the chemotherapeutic agent is selected from the following: antifolates (e.g. methotrexate); 5-fluoropyrimidines (e.g. 5-fluorouracil or 5-FU); cytidine analogues (e.g. gemcitabine); purine antimetabolites (e.g. mercaptopurine); alkylating agents (e.g. cyclophosphamide); non-classical alkylating agents (e.g. dacarbazine); platinum analogues (e.g. cisplatin); antitumour antibiotics (e.g. actinomycin D, bleomycin, mitomycin C); bioreductive drugs (e.g. mitomycin C, Banoxantrone (AQ4N)); anthracyclines (e.g. doxorubicin, mitoxantrone); topoisomerase I inhibitors (e.g. irinotecan); topoisomease II inhibitors (e.g. etoposide); antimicrotubule agents such as vinca alkaloids (e.g. vincristine), taxols (e.g. paclitaxel), and epothilones (e.g. ixabepiline); antioestrogens (e.g. tamoxifen); antiandrogens (e.g. biclutamide, cyproterone acetate); aromatase inhibitors (e.g. anastrazole, formestan); antiangiogenic or hypoxia targeting drugs (either naturally occuring, e.g. endostatin, or synthetic, e.g. gefitinib, lenalidomide); antivascular agents (e.g. cambretastatin); tyrosine kinase inhibitors (e.g. gefitinib, erlotinib, vandetanim, sunitinib); oncogene or signalling pathway targeting agents (e.g. tipfarnib, lonafarnib, naltrindole, rampamycin); agents targeting stress proteins (e.g. geldanamycin and analogues thereof); autophagy targeting agents (e.g. chloroquine); proteasome targeting agents (e.g. bortezomib); telomerase inhibitors (targeted oligonucleotides or nucleotides); histone deacetylase inhibitors (e.g. trichostatin A, valproic acid); DNA methyl transferase inhibitors (e.g. decitabine); alkyl sulfonates (e.g. busulfan, improsulfan and piposulfan); aziridines (e.g. benzodopa, carboquone, meturedopa, and uredopa); ethylenimines and methylamelamines (e.g. altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine); nitrogen mustards (e.g. chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard); nitrosureas (e.g. carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine); purine analogues (e.g. fludarabine, 6-mercaptopurine, thiamiprine, thioguanine); pyrimidine analogues (e.g. ancitabine, azacitidine, 6-azauridine, cannofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine); androgens (e.g. calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone); anti-adrenals (e.g. aminoglutethimide, mitotane, trilostane); and immune checkpoint inhibitors; and pharmaceutically acceptable salts, derivatives or analogues of any of these compounds.

17. A complex as claimed in claim 16, wherein the chemotherapeutic agent is an anti-metabolite, e.g. 5-fluorouracil or gemcitabine.

18. A complex as claimed in any one of the preceding claims, wherein the microbubble comprises a shell having incorporated therein one or more additional chemotherapeutic agents.

19. A complex as claimed in claim 18, wherein said one or more additional chemotherapeutic agents are as defined in claim 16 or claim 17, preferably wherein said additional chemotherapeutic agents are hydrophobic.

20. A complex as claimed in claim 18, wherein said additional chemotherapeutic agent is an anti-microtubule agent, e.g. a taxol such as paclitaxel.

21. A complex as claimed in any one of the preceding claims, wherein the microbubble comprises a shell which retains a gas, preferably a perfluorocarbon or oxygen.

22. A complex as claimed in any one of the preceding claims which comprises a microbubble having a diameter in the range of from 0.1 to 100 m.

23. A complex 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, e.g. polyethylene glycol (PEG).

24. A complex as claimed in any one of the preceding claims, wherein the microbubble is biotinylated and, optionally, further avidin-functionalised.

25. A complex as claimed in any one of the preceding claims, wherein the sonosensitising agent is selected from phenothiazine dyes (e.g. methylene blue, toluidine blue), Rose Bengal, porphyrins (e.g. Photofrin), chlorins, benzochlorins, phthalocyanines, napthalocyanines, porphycenes, cyanines and cyanine analogues (e.g. Merocyanine 540 and indocyanine green), azodipyromethines (e.g. BODIPY and halogenated derivatives thereof), acridine dyes, purpurins, pheophorbides, verdins, psoralens, hematoporphyrins, protoporphyrins and curcumins.

26. A complex as claimed in claim 25, wherein the sonosensitising agent is Rose Bengal, methylene blue, indocyanine green, or an analogue thereof, preferably Rose Bengal.

27. A complex as claimed in any one of the preceding claims, wherein the microbubble is linked to one or more structures selected from: formula (9) as shown in Scheme 2 (Biotin-Gem-RB), formula (9) as shown in Scheme 4 (Biotin-Dox-RB), and formula (9) as shown in Scheme 5 (Biotin-Gem-RB).

28. A pharmaceutical composition comprising a complex as claimed in any one of claims 1 to 27, together with at least one pharmaceutical carrier or excipient.

29. A complex as claimed in any one of claims 1 to 27 or composition as claimed in claim 28 for use as in therapy or for use as a medicament.

30. A complex as claimed in any one of claims 1 to 27 or composition as claimed in claim 28 for use in a method of sonodynamic therapy.

31. A complex or composition for use as claimed in claim 30, in which said complex or composition is contacted with cells or tissues of a patient and, either simultaneously or sequentially, said cells or tissues are subjected to irradiation with ultrasound and/or light.

32. A complex or composition as claimed in claim 30 or claim 31 for use in the treatment and/or diagnosis of cancer, preferably in the treatment and/or diagnosis of a deep-sited tumour.

33. A complex or composition for use as claimed in claim 32, wherein said cancer is selected from the group consisting of sarcomas, including osteogenic and soft tissue sarcomas; carcinomas, e.g. head and neck, breast, lung, cerebral, bladder, thyroid, prostate, colon, rectum, pancreas, stomach, liver, uterine, hepatic, renal, prostate, cervical and ovarian carcinomas; lymphomas, including Hodgkin and non-Hodgkin lymphomas; neuroblastoma, melanoma, myeloma, Wilm's tumour; leukemias, including acute lymphoblastic leukaemia and acute myeloblastic leukaemia; astrocytomas, gliomas and retinoblastomas.

34. A complex or composition for use as claimed in claim 33, wherein said cancer is pancreatic cancer.

35. A kit comprising: (i) a microbubble complex as claimed in any one of claims 1 to 27 or composition as claimed in claim 28; and (ii) instructions for the use of (i) in a method of sonodynamic therapy and/or diagnostic imaging.

36. Use of a microbubble complex as claimed in any one of claims 1 to 27 in the manufacture of a medicament for use in a method of sonodynamic therapy and/or diagnostic imaging.

37. A method of sonodynamic therapy in which a microbubble complex as claimed in any one of claims 1 to 27 or a composition as claimed in claim 28 is administered to affected cells or tissues of a patient, and said cells or tissues are subjected to ultrasound irradiation and/or light.

38. A method of preparation of a microbubble complex as claimed in any one of claims 1 to 27, said method comprising the step of linking a microbubble, or suitably functionalised microbubble, to one or more linking groups, each linking group being bound to at least one sonosensitising agent and at least one chemotherapeutic agent.

39. A Gemcitabine-Biotin conjugate, e.g. a conjugate having the structure (4) in Scheme 1.

40. A Biotin-Gemcitabine-Rose Bengal conjugate, e.g. a conjugate having the structure (9) in Scheme 2 or the structure (9) in Scheme 5.

41. A Biotin-Doxorubicin conjugate, e.g. a conjugate having the structure (3) in Scheme 3.

42. A Biotin-Doxorubicin-Rose Bengal conjugate, e.g. a conjugate having the structure (9) in Scheme 4.

Description

[0182] The invention will now be described further with reference to the following non-limiting Examples and the accompanying drawings in which:

[0183] FIG. 1 shows the amount of Biotin-RB loaded onto the surface of MBs prepared using 0, 2.5 mg or 5 mg of paclitaxel (PTX).

[0184] FIG. 2 shows a plot of MB number against time for three batches of MB-RB prepared using 0 mg (black bar), 2.5 mg (dark bar) or 5 mg of paclitaxel (PTX) (light grey).

[0185] FIG. 3 shows a plot of cell viability for BxPC-3 cells treated with Gemcitabine (black) or Biotin-Gem (grey). Cell viability determined by MTT assay 48 h after incubation.

[0186] FIG. 4 shows a plot of cell viability for BxPC-3 cells treated with Gemcitabine or Biotin-Gem-RB. Cell viability determined by MTT assay 48 h after incubation.

[0187] FIG. 5 shows a plot of tumour growth against time for mice bearing ectopic Mia-Paca-2 tumours treated with (i) no treatment (CTRL) (ii) PTX-MB-Gem (iii) PTX-MB-RB and (iv) PTX-MB-Gem-RB. Tumours were exposed to ultrasound during administration of the MB conjugates while the PTX-MB-RB and PTX-MB-Gem-RB received a second ultrasound exposure 30 min following injection. Treatments were administered on days 0, 1 and 2.

[0188] FIG. 6 shows a schematic representation of PTX/GEM/RB-MB.

[0189] FIG. 7 shows the size distribution of PTX/GEM/RB-MB constructed from bright field and fluorescence microscope images.

[0190] FIG. 8 shows the results of an MTT assay comparing the efficacy of biotin-GEM-RB (squares) with gemcitabine hydrochloride (circles) in Panc-1 (a) BxPc-3 (b) and Mia-PaCa-2 (c) cell lines.

[0191] FIG. 9 shows the results of an MTT assay comparing the cell viability of Panc-1 spheroids following 48-hour incubation with of GEM/RB MBs (GEM/RB5M), PTX MBs (PTX6.6 M) and PTX/GEM/RB MBs (PTX6.6 M, GEM/RB5 M) in Panc-1 spheroids with (grey) and without (black) ultrasound exposure.

[0192] FIG. 10 shows the results of a Propidium Iodide (PI) assay comparing the cell viability of Panc-1 spheroids following 48-hour incubation with GEM/RB MBs (GEM/RB6.8 M), PTX MBs (PTX5M) and PTX/GEM/RB MBs (PTX5 M, GEM/RB6.8 M) with (grey) and without (black) ultrasound exposure.

[0193] FIG. 11 shows a plot of (a) % change in tumour volume and (b) average body weight for Mia-PaCa-2 tumour bearing mice treated with (i) no treatment (circles) (ii) O.sub.2MB-PTX/GEM/RB (squares) (iii) gemcitabine (triangles). The microbubble suspension (6.8610.sup.71.9910.sup.6 MB) was delivered as a 100 uL I.V injection (PTX2.440.37 mg/Kg, GEM0.50.04 mg/Kg, RB1.850.14 mg/Kg). Gemcitabine hydrochloride was dissolved in sterile PBS and administered as a 100 uL I.P injection (120 mg/Kg). Ultrasound treatment was delivered for 3.5 minutes at frequency of 1 MHz, an ultrasound power density of 3.5 W/cm.sup.2 and a duty cycle of 30% immediately after injection and 30 minutes following. Error bars representthe standard error.

[0194] FIG. 12 shows a plot of % change in tumour volume for mice treated with (i) no treatment (circles) (ii) O.sub.2MB-PTX/GEM/RB (squares) (iii) O.sub.2MB-PTX (triangles) (v) gemcitabine +PTX in cremophorEL (diamonds). The microbubble suspensions (O.sub.2MB-PTX/GEM/RB6.4710.sup.71.8610.sup.6 MBs, O.sub.2MB-PTX7.0110.sup.7+1.510.sup.6 MBs) were delivered as a 100 uL I.V. injection (O.sub.2MB-PTX/GEM/RBPTX3.3810.21 mg/Kg GEM0.820.06 mg/Kg RB3.020.24 mg/Kg, O.sub.2MBPTXPTX4.690.75 mg/Kg). Gemcitabine hydrochloride was dissolved in sterile PBS and administered as a 100 uL I.P. injection (120 mg/Kg). Paclitaxel was dissolved in 1 mL of ethanol, 1 mL of cremophor and 8 mL of sterile PBS and administered as a 100 uL I.V. injection. Ultrasound treatment was delivered for 3.5 minutes at frequency of 1 MHz, an ultrasound power density of 3.5 W/cm.sup.2 and a duty cycle of 30% immediately after injection and 30 minutes following. Error bars representstandard error of the mean.

[0195] FIG. 13 is a graph showing the relative loading of biotin-RB as the concentration of paclitaxel increases from 0 to 5mg.

[0196] FIG. 14 is a graph showing the relative stability of MB formulations loaded with either 0 M PTX (black bars), 250 M PTX (dark grey bars) or 700 M PTX (light grey bars) over a 3-hour period.

[0197] FIG. 15 is a schematic representation of a) O.sub.2MB-PTX/DOX and b) O.sub.2MB-PTX/RB.

[0198] FIG. 16 is a plot showing % survival fraction from a MCF-7 colony forming assay. Cells following treatment with PTX/Dox/RB alone (Drug combo) or following treatment with ultrasound (Drug Combo+US). After treatment cells were incubated for 8 days followed by fixation and staining. Crystal violet staining was quantified via Image J.

[0199] FIG. 17 is a plot showing cell viability of MCF-7 spheroids following treatment with (i) no treatment (ii) MB only (no drugs), (iii) PTX/Dox only (i.e. no MBs) or iv) PTX-MB-Dox/PTX-MB-Dox in the presence or absence of US (30 s, frequency of 1 MHz, 3.0 W/cm.sup.2, 50% duty cycle. Statistical significant of samples treated with US versus untreated sample: *p<0.05, **p<0.01, ***p<0.001. Statistical significance of iv versus iii: && p<0.01. Statistical significance of iv treated with US versus iv untreated with US: $$$ p<0.001. Error bars representthe standard error, n=3.

[0200] FIG. 18 is a plot showing relative PI fluorescence from fluorescence microscope images of MCF-7 spheroids treated with MB only (i.e. no MB); PTX/Dox only (i.e. no MBs); PTX-MB-Dox/PTX-MB-RB+/US (30 s, 1 MHz, 3.0 W/cm.sup.2, 50% duty cycle) and then stained with PI. Statistically significant of samples treated with US versus untreated sample: *p<0.05, **p<0.01, ***p<0.001. Statistically significance of iv versus iii: &&& p<0.001. Statistically significance of iv treated with US versus iv untreated with US: p<0.01. Error bars representthe standard error, n=3.

[0201] FIG. 19 shows: (a) Tumour growth delay plot in a MCF-7 Xenograft model following (1) no treatment (2) a mixed suspension (50 L) of O.sub.2MB-PTX/RB and O.sub.2MB-PTX/Dox delivered by IV with US applied to the tumour during injection (3) as for (2) but without US treatment (4) O.sub.2MB-PTX/Dox delivered by IV with US applied to the tumour during injection and (5) a Cremophor formulation containing free PTX and DOX. For MB treatments: [MB]=1.210.sup.9 MB/mL [PTX]=2.29 mg/kg; [DOX]=1.96 mg/kg and [RB]=5.34 mg/kg. For free PTX/DOX treatment in Cremophor: [PTX]=4.96 mg/kg and [Dox]=2.50 mg/kg. Error bars representSEM where n=5; and (b) Plot of animal weights recorded over the course of the experiments for each group.

EXAMPLE 1

Synthesis of Biotin-Gemcitabine (Biotin-Gem) and Biotin-Gemcitabine-Rose Bengal (Biotin-Gem-RB) Conjugates

1.1 Synthesis of Biotinylated Gemcitabine (Biotin-GEM)

[0202] Biotinylated Gemcitabine was synthesised according to scheme 1. The protocol is provided below.

##STR00011##

Synthesis of (2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-y0-4,4-difluoro-3 hydroxytetrahydrofuran-2-yl)methyl (2-(5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-ylkentanamido)ethyl) carbonate (4)

[0203] Compound 2 was prepared following a literature procedure (see McEwan et al. Biomaterials. 2016: 80, 20-32). To a DCM (10 mL) solution of 2 (0.28 g, 0.9 mmol), 4-nitrophenyl chloroformate (0.59 g, 2.9 mmol), DIPEA (0.50 g, 3.9 mmol) and a catalytic amount of pyridine were added at 0 C. and stirred for 24 hrs at room temperature. Then the reaction mixture was concentrated to dryness in vacuo. The crude residue containing 3 was dissolved in 20 mL DMF. To this solution, Gemcitabine (0.88 g, 2.9 mmol) in DMF (5 mL) and TEA (1 mL) were added and the mixture stirred for a further 24 hrs. After completion of reaction (monitored by TLC), excess diethyl ether (200 mL) was added to the reaction mixture and stirred for 45 min. The yellowish oil thus obtained was separated and washed three times with cold diethyl ether (50 mL3). The crude compound was purified by PTLC using DCM/MeOH (9:1) as eluent to afford the target compound 4 (0.12 g, 22% yield).

[0204] .sup.1H NMR (DMSO-d.sub.6): 7.99-7.91 (m, 3H, CH, NH.sub.2), 6.41-6.33 (m, 1H, CH), 6.12-6.01 (m, 3H, CH, NH X 2), 4.30-4.16 (m, 1H, CH), 4.19-4.12 (m, 3H, CH, CH.sub.2), 3.90-3.75 (m, 2H, CH.sub.2), 3.69-3.58 (m, 2H, CH.sub.2), 3.12-3.09 (m, 1H, CH), 2.93-2.88 (m, 2H, CH.sub.2), 2.83 (brs, 1H, OH), 2.82-2.77 (m, 2H, CH X2), 2.72 (brs, 1H, NH),2.49-2.04 (m, 2H, CH.sub.2), 1.49-1.28 (m, 6H, CH.sub.2 X 3).

[0205] .sup.13C NMR (DMSO-d.sub.6): 175.0 (CO), 166.3 (C), 165.5 (CO), 156.3 (CO), 156.1 (CO), 141.3 (CH), 125.3 (C), 95.2 (CH), 79.2 (CH), 67.2 (CH), 61.9(CH), 60.2 (OCH.sub.2), 55.5 (OCH.sub.2), 39.6 (CH), 37.8 (CH.sub.2), 35.2 (CH), 28.3 (CH.sub.2), 28.0 (CH.sub.2), 25.3 (CH.sub.2). ESI-MS: cald for C.sub.22H.sub.30F.sub.2N.sub.6O.sub.8S, 576.18; found 577.2 (M+H).

1.2 Synthesis of Biotin-Gem-RB

[0206] Biotin-Gem-RB was synthesised according to Scheme 2. The protocols for each intermediate are provided below.

##STR00012## ##STR00013##

Synthesis of N-(2-(bis(2-aminoethyl)amino)ethyl)-5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (3)

[0207] To a stirred solution of Biotin-NHS (0.5 g, 1.5 mmol) and TEA (catalytic amount) in anhydrous DMF (10 mL), a solution of tris(2-aminoethyl)amine (0.22 g, 1.5 mmol) in 5 mL of DMF was added. The reaction mixture was stirred at 0 C. under argon atmosphere. After 2 hr of stirring, another volume of TEA (catalytic amount) was added and the reaction mixture was allowed to stir overnight at room temperature. After completion of the reaction (by TLC), the excess DMF was removed under reduced pressure keeping the temperature below 45 C. and the white gummy liquid thus obtained was poured into excess diethyl ether (200 mL) and filtered. The crude product was purified by column chromatography on basic (TEA) silica gel (MeOH: DCM 1:9 to 3:7) to give 3 (0.33 g, 61% yield) as a white semi solid.

[0208] .sup.1H NMR (DMSO-d.sub.6): 7.94 (brs, 1H, NH), 6.42 (brs, 1H, NH), 6.35 (brs, 1H, NH), 4.49 (brs, 4H, NH.sub.2 X 2), 4.29 (s, 1H, CH), 4.12 (s, 1H, CH), 3.07-3.02 (m, 6H, CH.sub.2 X 3), 2.88-2.82 (m, 1H, CH), 2.44-2.06 (m, 10H, CH.sub.2 X 5), 1.59-1.48 (m, 4H, CH.sub.2 X 2), 1.47-1.29 (m, 2H, CH.sub.2).

[0209] ESI-MS: cald for C.sub.16H.sub.32N.sub.6O.sub.2S, 372.23; found 373.31 (M+H).

Synthesis of bis(2,5-dioxopyrrolidin-1-yl) 8,8-((((2-(5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl)azanediyl)bis(ethane-2,1-diyl))bis(azanediyl))bis(8-oxooctanoate) (5)

[0210] Compound 3 (0.5 g, 1.3 mmol) was dissolved in 10 mL anhydrous DMF in the presence of TEA (catalytic amount) and bis(2,5-dioxopyrrolidin-1-yl) octanedioate (4, 1 g, 2.7 mmol) was added. The reaction mixture was stirred at room temperature for 24 hrs under argon atmosphere. After completion of the reaction (by TLC), excess diethyl ether (200 mL) was added to the reaction mixture. The white precipitate thus obtained was filtered and washed three times with cold diethyl ether (50 mL3). The crude product was purified by column chromatography on basic (TEA) silica gel (MeOH: CHCl.sub.3 2:8 to 5:5 v/v) to give 5 (0.83 g, 71% yield) as a low melting white solid.

[0211] .sup.1H NMR (DMSO-d.sub.6): 7.94 (brs, 2H, NH X 2), 7.67 (brs, 1H, NH), 6.41 (brs, 1H, NH), 6.34 (brs, 1H, NH), 4.29 (s, 1H, CH), 4.12 (s, 1H, CH), 3.06-3.04 (m, 3H, CH and CH.sub.2), 2.88-2.72 (m, 6H, CH.sub.2 X 3), 2.71-2.63 (m, 8H, CH.sub.2 X 4), 2.45-2.34 (m, 6H, CH.sub.2 X 3), 2.20-2.06 (m, 10H, CH.sub.2 X 5), 1.60-1.21 (m, 22H, CH.sub.2 X 11). .sup.13C NMR (DMSO-d.sub.6): 172.5 (CO), 170.7 (CO), 163.1 (CO), 162.7 (CO), 61.4 (CH), 59.6 (CH), 55.8 (CH.sub.2), 53.9 (NCH.sub.2), 39.9 (CH.sub.2), 39.8(CH.sub.2), 39.6(CH.sub.2), 37.3(CH.sub.2), 36.2(CH.sub.2), 35.6(CH.sub.2), 31.2 (CH.sub.2), 28.7(CH.sub.2), 28.5(CH.sub.2), 25.8(CH.sub.2), 25.7(CH.sub.2), 25.6(CH.sub.2).

[0212] ESI-MS: cald for C.sub.40H.sub.62N.sub.8O.sub.12S, 878.4; found 901.3 (M+Na salt).

Synthesis of ((2R, 3R, 5R)-5-(4-amino-2-oxopyrimidin-1 (2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methyl 4,11,19-trioxo-15-(2-(5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl)-1-((2,3,4,5-tetrachloro-6-(6-hydroxy-2,4,5,7-tetraiodo-3-oxo-3H-xanthen-9-yl)benzoyl)oxy)-3,12,15,18-tetraazahexacosan-26-oate (9)

[0213] To a DMF (anhydrous, 10 mL) solution of 5 (0.4 g, 0.45 mmol) GMC-hydrochloride (8, 0.136 g, 0.45 mmol) and TEA (0.5 mL) were added at 0 C. and stirred for 24 hrs at room temperature under argon atmosphere. After completion of the reaction (monitored by GC-MS), Rose Bengal amine 7 (prepared according to McEwan et al. J. Control Release. 2015; 203, 51-56), (0.43 g, 0.45 mmol in DMF (5 mL)) and TEA (0.5 mL) were added to the reaction mixture and continued to stir for 24 hr. The progress of the reaction was monitored by mass spec analysis of the crude reaction mixture. After completion of the reaction, excess diethyl ether (200 mL) was added to the solution and stirred for 30 min. The pink red precipitate thus obtained was filtered and washes several times with cold diethyl ether (100 mL), ethyl acetate (100 mL), acetone-water mixture (10%, v/v, 100 mL) and finally with ethyl acetate-hexane mixture (50%, v/v, 100 ml) respectively to afford a pink red powder of compound 9 (0.26g, 30% yield).

[0214] .sup.1H NMR (DMSO-d.sub.6): 7.95 (brs, 2H, NH.sub.2), 7.69 (s, 1H, CH, aromatic proton), 7.68 (s, 1H, CH, aromatic proton), 7.37(s, 1H, CH), 7.32 (brs, 4H, NH X 4), 6.89 (s, 1H, CH), 6.42 (brs, 1H, NH), 6.35 (brs, 1H, NH), 6.22 (d, J=5.5 Hz, 1H, CH), 6.13 (brs, 1H, NH), 5. 78-5.77 (m, 1H, CH), 5.19 (s, 1H, CH X 2), 4.9 (brs, 1H, OH), 4.30 (s, 2H, -OCH.sub.2), 4.13 (s, 2H, OCH.sub.2), 3.79-3.60 (m, 3H, CH, CH.sub.2), 3.39-3.32 (m, 2H, CH.sub.2), 3.07 (brs, 6H, N-NHCH.sub.23), 2.94-2.84 (m, 6H, NCH.sub.2 X 3), 2.81 (brs, 1H, OH), 2.45-2.46 (m, 3H, CH, CH.sub.2), 2.17-2.06 (m, 10H, CH.sub.2 X 5), 1.60-1.10 (m, 22H, CH.sub.2 X 11).

[0215] .sup.13C NMR (DMSO-d.sub.6): 171.8 (CO, C), 165.98 (CO), 163.2 (CO), 162.7 (CO), 159.3 (CH), 155.0 (CO), 150.5 (C), 145.8 (C), 141.2 (CH), 131.0 (C), 128.7 (C), 123.5 (C), 116.2 (C), 95.0 (CH), 80.9 (C), 69.3 (CH), 61.5 (C), 59.6 (CH.sub.2), 59.4 (CH), 55.8 (CH), 51.7 (CH.sub.2), 45.8 (CH.sub.2), 40.2 (CH.sub.2), 37.3 (CH.sub.2), 37.05 (CH.sub.2), 36.2 (CH.sub.2), 35.5 (CH.sub.2), 31.0 (CH.sub.2), 28.7 (CH.sub.2), 28.5 (CH.sub.2), 28.2 (CH.sub.2), 25.6 (CH.sub.2).

[0216] ESI-MS: cald for C.sub.63H.sub.72Cl.sub.4F.sub.2I.sub.4N.sub.10O.sub.15S, 1925.98; found 1925.90 (MH).

EXAMPLE 2

Preparation, Stability and Loading Efficiency of Avidin-Functionalised Paclitaxel (PTX) Loaded Microbubbles (MBs)

2.1 Preparation of Lipid Stabilised MBs with PTX Incorporated within the Shell (PTX-MB)

[0217] Avidin functionalised lipid stabilised microbubbles with PTX hydrophobically incorporated in the shell were prepared by first dissolving the lipids 1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC) (4.0 mg, 4.43 umol), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG(2000)) (1.35 mg, 0.481 mol) and DSPE-PEG (2000)-biotin (1.45 mg, 0.481 mol) in chloroform to achieve a molar ratio of 82:9:9. To this solution was added paclitaxel (5 mg, 5.86 mol) dissolved in chloroform. The solution was heated at 40 C. for 30 minutes until the chloroform had evaporated. The dried lipid film was reconstituted in 3 mL of Ungers solution (PBS, Glycerol, Propylene glycol (8:1:1 volume ratio)) and heated on a water bath at 75 C. for 30 minutes. The suspension was then sonicated using a Microson ultrasonic cell disrupter at an amplitude of 22% for 1 minute to fully incorporate the lipids with paclitaxel. The suspension was then sparged with PFB gas whilst sonicating the suspension at an amplitude of 89% for 1 min to form the microbubble suspension. The MBs were then cooled on ice for 10 minutes followed by centrifugation at 700 rpm for 5 min to remove the excess lipids/paclitaxel present in the liquid below the bubble cake. The cake was then washed with 2 mL of Ungers solution followed by the addition of an aqueous solution of avidin (500 L, 2.5 mg/mL). The suspension was then stirred for 5 min followed by centrifugation (700 rpm) to remove excess avidin. The MB cake was then washed again with 2mL of Ungers solution, centrifuged (700 rpm) and the MBs isolated.

2.2 Loading of Biotin-RB, Biotin-Gem and Biotin-Gem-RB onto the Surface of Avidin Functionalised PTX-MBs

[0218] A solution containing either Biotin-RB, Biotin-Gem, or Biotin-Gem-RB (500 L, 5 mg/mL), prepared in a DMSO: Ungers solution (10:90 v/v) was added to 2 mL of PTX-MBs (2.010.sup.8 MB/mL). The suspension was then mixed for 5 min using a rotary shaker followed by centrifugation (700 rpm) for 5 min to remove excess ligand. This coupling process was repeated one more time. The final microbubble cake was suspended in 2 mL of Ungers solution. The microbubbles were either used directly or oxygenated by sparging the suspension with oxygen gas for 2 min immediately prior to use.

2.3 Loading Capability of Avidin Functionalised PTX-MBs

[0219] To determine if the loading capacity of the avidin functionalised MBs was affected by the incorporation of PTX in the shell of the MB, three batches of PTX-MB were prepared using 0 mg, 2.5 mg or 5 mg of PTX during the MB manufacture process described in section 2.1. Each batch of MBs was then loaded with biotin-RB as described in section 2.2. The amount of Rose Bengal loaded on the surface of the MBs was then quantified using UV-Vis spectroscopy by purposely bursting a fixed amount of MBs and using a reference calibration curve. Each reaction was performed in triplicate. The reason biotin-RB was selected for use in this study was due to its inherent chromophore that made the quantification process more straight-forward.

[0220] The results from this study are shown in FIG. 1 and reveal no statistically significant difference in the loading of biotin-RB for any of the batches prepared.

[0221] These results indicate that the presence of PTX in the shell of the MBs does not affect the amount of biotinylated ligand attached to the surface.

2.4 Stability Determination of PTX-MBs

[0222] To determine the stability of PTX-MBs with a biotinylated payload attached to their surface, three batches of PTX-MB-RB MBs containing either 0, 2.5 or 5.0 mg PTX in the shell were prepared as described in section 2.3. Samples of the microbubbles (2 mL) from each batch were incubated at 37 C. for 3 hours and the MB number then counted at various time intervals (0 min, 10 min, 60 min, 120 min, and 180 min) using a haemocytometer.

[0223] The results from this study are shown in FIG. 2 and reveal no statistically significant difference in the MB number for either batch of PTX-MB-RB prepared. These results indicate that the stability of the PTX-MB-RB conjugates is unaffected by the presence of PTX in the shell of the MB.

EXAMPLE 3

Biological Testing of PTX-MB-RB, PTX-MB-Gem and PTX-MB-Gem-RB

3.1 Efficacy of Biotin-Gem and Biotin-Gem-RB in BxPC-3 Cells

[0224] To ensure the antimetabolite efficacy of Biotin-Gem and Biotin-Gem-RB were unaffected by chemical modification, the cytotoxicity of the conjugates was determined in the human pancreatic adenocarcinoma cell line BxPc-3. Cells were maintained in RPMI-1640 which was supplemented with 10% (v/v) foetal bovine serum in a humidified 5% CO.sub.2 atmosphere at 37 C. These cells were seeded into 96 well plates at a density of 5000 cells per well. The plates were then incubated for 24 hours followed by the addition of 100 uL of media spiked with either Gem, Biotin-Gem or Biotin-Gem-RB. The concentrations used for Biotin-Gem were 0, 0.1, 5, 10, 25, 50, 100 and 100 M while for Biotin-Gem-RB, concentrations of 0.01 M, 0.05 M, 0.1 M, 0.5 M, 1 M, 5 M and 10 M were investigated. Corresponding concentrations of Gem were used in each case. The cells were then further incubated for 48 hours before cell viability was determined using an MTT assay.

[0225] The results from this study are shown in FIGS. 3 and 4 and reveal no statistically significant difference in efficacy for Gemcitabine compared to Biotin-Gem or Biotin-Gem-RB over the concentration ranges tested. These results indicate that chemical modification of Gemcitabine in each of these ligands does not negatively impact its efficacy.

3.2 In Vivo Efficacy of Combined Paclitaxel/Gemcitabine/SDT Treatment Delivered Using PTX-MB-Gem-RB

[0226] MIA-Paca-2 cells were maintained in DMEM medium supplemented with 10% foetal calf serum and 1% penstrep in a humidified 5% CO.sub.2 atmosphere at 37 C. Cells (510.sup.6) were re-suspended in Matrigel and implanted subcutaneously into the rear dorsum of BALB/c SCID mice. All animals were treated humanely and in accordance with licenced procedures under the UK Animals (Scientific Procedures) Act, 1986. Once the tumours had reached an average volume of 150 mm.sup.3 animals were randomly assigned into 4 treatment groups (n=4 in each group). Animals were anaesthetised by intraperitoneal injection of Hypnorm:hypnovel:ice cold sterile water (1:2:1). Animals were treated with a 100 L suspension containing either PTX-MB-RB ([PTX]=878 M; [RB]=1179 M), PTX-MB-Gem ([PTX]=846 M; [Gem]=850 M) or PTX-MB-Gem-RB ([PTX]=742 M; [Gem]=489 M; [RB]=489 M) by intravenous injection into the tail vein. Ultrasound was applied directly to the tumour area using ultrasound gel to mediate contact during and for 3 min following injection at an ultrasound frequency of 1 MHz, an ultrasound power density of 3.5 Wcm.sup.1 and a duty cycle of 30% for 3.5 min. For the PTX-MB-RB and PTX-MB-Gem-RB groups, a second ultrasound treatment using the same parameters was also applied directly to the tumours after 30 min after injection. Treatments were administered on Days 0, 1, and 2. Tumour growth was monitored daily throughout the course of the treatment using callipers and tumour volume calculated using the equation: tumour volume=(W*H*L/2).

[0227] The results are shown in FIG. 5 and reveal a statistically significant reduction (p<0.001) in tumour growth for all treatment groups relative to the control. There was no significant difference in tumour growth for combined Paclitaxel-SDT (PTX-MB-RB) group compared to combined Paclitaxel-Gemcitabine (PTX-MB-Gem) treatment. However, there was a statistically significant difference in tumour growth (p<0.01) for combined Paclitaxel-Gemcitabine-SDT (PTX-MB-Gem-RB) treatment when compared to either combined Paclitaxel-SDT (PTX-MB-RB) or combined Paclitaxel-Gemcitabine (PTX-MB-Gem) treatment.

EXAMPLE 4

Preparation, Characterisation and Biological Testing of Avidin Functionalised PTX-MBs Carrying Biotin-RB, Biotin-Gem and Biotin-Gem-RB

4.1 Loading of Biotin-RB, Biotin-Gem and Biotin-Gem-RB onto the Surface of Avidin Functionalised PTX-MBs

[0228] A saturated aqueous solution containing either Biotin-RB, Biotin-Gem or Biotin-Gem-RB (1 mL, 5 mg/mL) was added to 2 mL of PTX-MBs or PTX-free MBs (6.7210.sup.8 MB/mL). The suspension was mixed for 5 min (0 C.) followed by centrifugation (700 rpm) for 3 min to remove excess ligand. The MB cake was then washed a further 3 times with PBS solution. The final microbubble cake was suspended in 2 mL of PBS solution. The microbubbles were either used directly or oxygenated by sparging the suspension with oxygen gas for 2 min immediately prior to use. The final microbubble number was determined on a haemocytometer using an optical microscope. A schematic representation of the PTX/GEM/RB-MB is shown in FIG. 6.

4.2 Size Distribution Analysis of MB Formulation

[0229] Size distribution analysis was carried out using imageJ software. The bright field image was converted to 8-bit greyscale before an automated threshold strategy was applied to eliminate out of focus MBs. Particle diameter was then calculated relative to the scale bar present in the bright field image. FIG. 7 shows the size distribution histogram constructed from the image. The average microbubble diameter was 1.54 m.

4.3 In Vitro Efficacy

[0230] Human primary pancreatic adenocarcinoma cell lines BxPc-3, Panc-1 and Mia-PaCa-2 were maintained in RPMI 1640 medium which was supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% fetal bovine serum (FBS) and Dulbecco's Modified Eagle's Medium (DMEM) containing 1 g/L glucose and supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% fetal bovine serum (FBS) respectively in a humidified 5% CO.sub.2 atmosphere at 37 C. These cells were seeded into 96 well plates at a density of 5000 cells per well. The plates were then incubated for 24 hours followed by the addition of 100 L of media spiked with Gem or Biotin-Gem-RB at concentrations ranging from 0.001-1000 M. The cells were then further incubated for 48 hours before cell viability was determined by an MTT assay.

[0231] FIG. 8 shows the comparative efficacy of biotin-GEM-RB with commercially available gemcitabine HCl in the three pancreatic cancer cell lines: Panc-1 (a) BxPc-3 (b) and Mia-PaCa-2 (c) over a wide range of concentrations (0-1000 M). These results show that there is no decrease in efficacy following the modification of gemcitabine and there is also a significant increase in efficacy for Panc-1 cells above 10 M.

4.4 Culture of Panc-1 Spheroids

[0232] The human primary pancreatic carcinoma cell line PANC-1 was maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing 1 g/L glucose and supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% fetal bovine serum (FBS). Cells were incubated at 37 C. in a humidified atmosphere with 5% CO.sub.2. Spheroids were prepared by seeding 2000 cells (200 uL) into a pre-coated 96-well plate (60 uL 1.5% agarose per well). Spheroids were inspected daily and took 3 days to reach a treatable size.

4.5 In Vitro Cytotoxicity in Panc-1 Spheroids

[0233] Panc-1 spheroids were cultured as described above. The media in each well was replaced with either fresh drug-free media, PTX-MB (5uM), GEM/RB-MB (6.8 M) or PTX/GEM/RB-MB (PTX5 , M, GEM/RB6.8 M). Wells were then treated individually with ultrasound (Sonidel SP100 sonoporator, 30 s, frequency1 MHz, ultrasound power density3.0 W/cm.sup.2, duty cycle40%). Inspection of Panc-1 spheroids following 48-hour incubation with fresh media, GEM/RB MBs, PTX MBs and PTX/GEM/RB MBs, with and without ultrasound exposure, revealed a degradation in spheroid morphology in all groups treated with drug-loaded MBs following ultrasound exposure for 30 seconds.

4.6 Total Cell Viability Assay of Spheroids

[0234] Two days after initial treatment spheroids were washed as described above. A total of 5 spheroids/replicate from each condition was collected in an Eppendorf tube, washed with PBS and then incubated with trypsin for 15 min at 37 C. The resultant cellular suspension was then incubated for 3 hours with MTT (10 l in 100 l of media). The absorbance was then measured at 570-690 nm using FLUOstar Omega (BMG Labtech) plate reader.

[0235] Data is expressed as % of cell viability vs. untreated sample in FIG. 9. This shows a decrease in cell viability for all groups treated with MB formulations and a further decrease when these groups were treated with ultrasound for 30 sec. Spheroids treated with PTX/GEM/RB MBs+US performed the best with a decrease in cell viability of 67% compared with untreated controls.

4.7 Propidium Iodide Staining of Spheroids

[0236] Two days after initial treatment, Panc-1 spheroids were washed four times with PBS to remove excess drug and then incubated with a solution of PBS and Propidium Iodide (Invitrogen) with a final concentration of 100 g/mL. Spheroids were then incubated at RT for 40 min. After incubation spheroids were washed with PBS before being placed onto a glass microscope slide at which point live images were collected using a NIKON Eclipse E400 Phase contrast microscope in bright field (BF) and in fluorescence to visualise Propidium Iodide signal using 540 nm band pass excitation and 590 nm long pass emission filters, respectively. Spheroids treated with all MB formulations displayed an increased fluorescence intensity at 617 nm, and hence an increased uptake of propidium iodide (PI). Spheroids treated with either GEM/RB MBs or PTX/GEM/RB MBs showed a further increase in fluorescence intensity with the latter performing the best. A further increase in fluorescence intensity was evident in groups exposed to ultrasound for 30 sec following treatment. FIG. 10 shows an increase in PI uptake for all groups treated with MB formulations and a further increase when these groups were treated with ultrasound for 30 sec. Spheroids treated with PTX/GEM/RB MBs+US performed the best.

4.8 In Vivo Cytotoxicity Experiments

[0237] Mia-Paca-2 cells were maintained in DMEM medium and BxPc-3 cells were maintained in RPMI 1640 both supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% fetal bovine serum (FBS) in a humidified 5% CO.sub.2 atmosphere at 37 C. Cells (510.sup.6) were re-suspended in Matrigel and implanted subcutaneously into the rear dorsum of SCID (C.B-17/IcrHanHsd-Prkdcscid) mice. Tumours reached treatable size within 3 weeks. Tumour measurements were taken daily using callipers. Once the tumours had reached an average volume of 150mm.sup.3 animals were randomly assigned into treatment groups. Animals were anaesthetised by intraperitoneal injection of hypnorm:hypnovel:ice cold sterile water for injection (1:2:1). Animals were treated with 100 L of either O.sub.2MB-GEM-RB, O.sub.2MB-PTX-GEM-RB, Paclitaxel in cremophor EL by I.V. injection into the tail vein and gemcitabine hydrochloride by I.P. injection. Ultrasound was applied directly to the tumour immediately following injection at an ultrasound frequency of 1 MHz, an ultrasound power density of 3.5 Wcm.sup.1 and a duty cycle of 30% for 3.5 min. A second ultrasound treatment was applied directly to the tumour 30 min following. Tumour growth was monitored daily throughout the course of the treatment.

[0238] Gemcitabine hydrochloride was dissolved in sterile PBS and administered as a 100 uL I.P injection (120 mg/Kg). Ultrasound treatment was delivered for 3.5 minutes at frequency of 1 MHz, an ultrasound power density of 3.5 W/cm.sup.2 and a duty cycle of 30% immediately after injection and 30 minutes following.

[0239] The plots in FIG. 11(a) show that mice treated with PTX/GEM/RB MBs followed by ultrasound exposure had a statistically significant reduction in tumour growth. Tumour growth was controlled continually within this group throughout the 10-day experiment. The response shown by this group also compared favourably with mice treated with a clinical dose of the current standard of therapy for pancreatic cancer (gemcitabine). FIG. 11(b) shows that mice treated with PTX/GEM/RB MBs showed no significant reduction in body weight when compared to untreated controls indicating that not only is the treatment efficacious but also well tolerated. In contrast, mice treated with the clinical dose of gemcitabine showed a significant decrease in body weight of 12% at day 6.

[0240] The plots in FIG. 12 show that mice treated with PTX/GEM/RB MBs followed by ultrasound exposure had a statistically significantly decrease in rate of tumour growth. Tumour growth was controlled continually within this group throughout the 10-day experiment. The response shown by this group also compared favourably with mice treated with a clinical dose of the current standard combination therapy for pancreatic cancer (gemcitabine+paclitaxel).

4.9 Determination of the Concentration of PTX and Biotin-Gem-RB and Loading Capacity of RB

[0241] Three batches of PTX/RB MBs were prepared as described previously with 0, 2.5 or 5 mg of PTX added to the lipid film and with the same amount of biotin rose bengal added. After subsequent washing, the concentration of rose bengal was derived using standard UV spectrophotometry measuring at 550 nm. This experiment was done in triplicate. PTX concentration was determined by reverse phase HPLC using a Phenomenex C.sub.18 column (2504.6 mm, 5 m), a mobile phase consisting of acetonitrile:water (1:1 v/v), and a detection wavelength of 227 nm. The retention time of the analyte was 9 min. The loading of Biotin-Gem-RB was determined using UV spectrophotometry with an analyte absorbance wavelength of 560 nm.

[0242] FIG. 13 shows the relative loading of biotin-RB as the concentration of paclitaxel increases from 0 to 5 mg. This shows that as the concentration of paclitaxel loaded within the acyl chains of the phospholipid shell increases, the relative loading capacity for biotin-RB does not significantly decrease.

4.10 Stability of PTX-MBs

[0243] PTX-MBs were prepared as described previously with 0, 2.5 or 5 mg of PTX added to the lipid film. After subsequent washing the MB number was determined using a haemocytometer on an optical microscope. An initial reading was taken immediately after MB synthesis and then 10, 60, 120 and 180 min following. PTX concentration was determined using reverse phase HPLC as described previously.

[0244] The results in FIG. 14 show that as the concentration of PTX within the acyl chains of the phospholipid MBs increase, the relative stability of the formulation does not significantly decrease.

EXAMPLE 5

Preparation and Biological Testing of Paclitaxel (PTX) Loaded Microbubbles (MBs) Carrying Biotin-Doxorubicin (Biotin-Dox) or Biotin-Rose Bengal (Biotin-RB) Conjugates

5.1 Reagents and Materials

[0245] 1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG(2000)) and DSPE-PEG(2000)-biotin were purchased from Avanti Polar Lipids (Alabaster, Ala., USA). Oxygen gas was purchased from BOC Industrial Gases UK and perfluorobutane (PFB) was purchased from Apollo Scientific Ltd. Phosophate Buffered Saline (PBS) was purchased from Gibco, Life Technologies, UK. Glycerol and propylene glycol (1 kg, hydrolysed) were purchased from Sigma Aldrich (UK). Optical microscope images were obtained using a Leica DM500 optical microscope. Rose Bengal sodium salt, NHS-biotin, gemcitabine, MTT assay kit, avidin, chloroacetic acid, 4-dimethylaminopyridine (DMAP), hydroxybenzotriazole (HOBt), N,N-dicyclohexylcarbodiimide (DCC), anhydrous dimethylformamide (DMF), and ethanol were purchased from Sigma Aldrich (UK) at the highest grade possible. Biotin, di(N-succinimidyl) carbonate and 2-aminoethanol were purchased from Tokyo Chemical Industry UK Ltd.

5.2 Synthesis of Biotin-Dox

[0246] Biotinylated Doxorubicin (Biotin-Dox) (3) was synthesised according to scheme 3. The protocol is provided below.

##STR00014## ##STR00015##

[0247] To an ice cold solution of biotin-N-hydroxysuccinimide ester (1, 0.14 g, 0.41 mmmol) in DMF (10 ml) was added doxorubicin (2, 0.3 g, 0.41 mmol) under nitrogen atmosphere. After stirring for 30 min, triethylamine (0.5 ml, 2 mmol) was added to this reaction mixture and was allowed to stir for another 12 hrs at room temperature. The reaction was monitored by TLC (Merck Silica 60, HF 254, 20: 80 methanol-dichloromethane v/v). After completion of the reaction, excess diethyl ether (100 ml) was added to the reaction mixture. The red solid thus obtained was filtered and washed three times with diethyl ether (50 ml3). This red solid was then subjected to PTLC purification using methanol-dichloromethane (20:80, v/v) as an eluent to obtain 0.25 g (Yield=78%) of 3. An analytical sample was obtained from a recrystallization of this product from ethanol.

[0248] .sup.1H NMR (DMSO-d.sub.6):7.84 (d, J=7.5 Hz, 2H, aromatic), 7.58 (d, J=7.5 Hz, 1H, aromatic), 6.36 (s, 1H, NH), 6.29 (s, 1H, NH), 5.37 (brs, 1H, OH), 5.22 (brs, 1H, OH), 4.87 (s, 2H, CH.sub.2OH), 4.51 (brs, 2H, OH X2), 4.36-4.33 (m, 1H, CH), 4.25-4.22 (m, 1H, CH), 4.16-4.13 (m, 1H, CH), 3.99 (s, 3H, OCH.sub.3), 3.60-3.58 (m, 1H, CH), 3.55 (brs, 2H, OH X2), 3.10-3.00 (m, 4H, CH.sub.2 X1, CH X2), 2.88-2.54 (m, 3H, CH.sub.2 X 1, CH), 2.20-2.00 (m, 1H, CH), 1.63-1.50 (m, 4H, CH.sub.2 X 2), 1.42-1.22 (m, 11H, CH.sub.3 X 1, CH.sub.2X 4). .sup.13CNMR (DMSO-d.sub.6): 177.6, 176.9, 174.8, 166.4, 163.0, 161.2, 153.7, 152.7, 137.4, 132.4, 120.4, 119.4, 99.5, 97.8, 80.15, 75.1, 72.7, 66.4, 61.4, 59.5, 55.7, 47.8, 33.8, 31.9, 28.9, 28.8, 28.5, 28.4, 24.9, 19.8, 17.6, 17.1.

[0249] ESMS (MH]: calculated for C.sub.37H.sub.43I.sub.2N.sub.3O.sub.13S=769.25, found=767.9.

5.3 Preparation of Oxygen Carrying Microbubbles Loaded with PTX in the Shell and Either Biotin-Dox or Biotin-RB Attached to the MB Surface

[0250] Avidin-functionalised lipid-stabilised microbubbles with PTX hydrophobically incorporated in the shell were prepared by dissolving 1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC) (4.0 mg, 4.44 umol), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol) -2000] (DSPE-PEG(2000)) (1.35 mg, 0.481 umol) and DSPE-PEG (2000)-biotin (1.45 mg, 0.481 umol) in chloroform to achieve a molar ratio of 82:9:9. To this solution was added paclitaxel (5 mg, 5.86 mol) dissolved in chloroform (100 uL). The solvent was removed under vacuum at room temperature yielding a translucent film. The dried lipid film was reconstituted in 2 mL of a solution containing PBS, Glycerol and Proplyene glycol (8:1:1 volume ratio) and heated in a water bath at 80 C. for 30 minutes. The suspension was then sonicated using a Microson ultrasonic cell disrupter at an amplitude of 22% for 30 seconds to fully suspend paclitaxel. The suspension was then sparged with PFB gas whilst sonicating the suspension at an amplitude of 89% for 1 minute to form the microbubbles. The MBs were then cooled on ice for 10 minutes followed by centrifugation at 700 rpm for 3 min and removal of the subnatant to remove the excess lipids/paclitaxel. The cake was then washed a further 2 times before an aqueous solution of avidin (10 mg/mL) was added. The suspension was then stirred for 5 min (0 C.) followed by centrifugation (700 rpm) to remove unbound avidin. The MB cake was then washed again and suspended in PBS solution. A saturated aqueous solution containing either Biotin-Dox or biotin-RB (1 mL, 5 mg/mL) was added to 2 mL of PTX-MBs (7.5210.sup.8 MB/mL). The suspension was mixed for 5 min (0 C.) followed by centrifugation (700 rpm) for 3 min to remove excess ligand. The MB cake was then washed a further 3 times with PBS solution. The final microbubble cake was suspended in 2 mL of PBS solution. The microbubbles were oxygenated by sparging the suspension with oxygen gas for 2 min immediately prior to use. The final microbubble number was determined on a haemocytometer using an optical microscope. FIG. 15 is a schematic representation of a) O.sub.2MB-PTX/DOX b) O.sub.2MB-PTX/RB.

5.4 Colony Forming Assay to Determine the Cytotoxicity of Dox, PTX, SDT and Combinations of Each in MCF-7 Cells

[0251] In this study, the concentration of each individual drug used was intentionally sub-lethal, so that any benefit obtained by the combination treatment could easily be identified. In addition, as the action of ultrasound can influence the cellular uptake of drugs as a result of sonoporation, cells treated with Dox or PTX were also exposed to ultrasound, to control for any potential ultrasound mediated effects on cell viability as a result of sonoporation. Following treatment, cell viability was determined using a colony forming assay.

[0252] MCF-7 cells were seeded (510.sup.3) in a 96 well plate, 24 hours later cells were treated with free drug as a single treatment of PTX (1 nM), Dox (10 nM), RB (10 nM) or combination treatment for 3 hours followed by media replacement. Selected wells were treated with ultrasound delivered using a Sonidel SP100 sonoporator (1 MHz, 30 seconds, 3 Wcm.sup.2, duty cycle=50%, and PRF=100 Hz). The following day cells were pooled from 2 wells and seeded in a 6 well plate. Plates were placed in an incubator for 7 days. Media was removed from wells, fixation/staining solution was added at room temperature for 20 minutes. Fixative/staining solution contained: 0.5 g crystal violet (0.05%), 27 ml 37% formaldehyde, 100 ml 10PBS (X1), 10 mL methanol (1%) and 863 mL of distilled water. Solution was removed and washed under running water. Pictures were taking using a high-resolution camera and colony formation was analysed via Image J.

[0253] The results are shown in FIG. 16 and reveal no reduction in colony formation for cells treated with a combination of PTX, Dox and Rose Bengal (drug combo) in the absence of ultrasound compared to untreated cells. Treatment of cells with PTX+US, RB+US (i.e. SDT) or Dox+US reduced colony number by 7.3, 18.8 and 29.3% respectively compared to untreated cells, while cells treated with combined PTX, Dox and RB+US reduced in colony number by 44.0%. The lack of efficacy for the combined drug cocktail in the absence of US was not surprising as sub-lethal doses of the drugs were used. However, the significant improvement in efficacy following exposure to US suggests sonoporation effects improve the uptake of these drugs enabling a cytotoxic effect to be observed. The fact that the greatest reduction in cell viability was observed for the combined PTX, Dox and SDT treatment group indicates that these three treatments complement each other and improve the cytotoxic effect observed.

5.5 Preparation of MCF-7 Spheroids

[0254] The human breast cancer MCF-7 cell line was purchased from American Type Culture Collection (ATCC, Rockville, Md., USA). MCF-7 cells were cultured in DMEM medium supplemented with 10% FBS (Gibco), 100 g/ml streptomycin (Gibco) and maintained in a humidified 5% CO.sub.2 atmosphere at 37 C. 3D spheroids were generated by growing MCF-7 cells in Carrier Plate (ULA) from PerkinElmer. Briefly, 8000 MCF-7 were seeded in 100 l of media in each well. 24 h after the seeding, 100 l of media was added to each well and plates were incubated at 37 C. with 5% CO.sub.2 for 3 days to allow cell assembly. Media was changed every 3-4 days by removing 100 l of old media and replacing it with 100 l of fresh media.

5.6 Chemo-Sonodynamic Therapy Treatment of MCF-7 Spheroids Using MB-PTX-Dox MB-PTX-RBUltrasound

[0255] After 3 days of incubation, spheroids were divided into groups and treated according to the following conditions: untreated spheroids, spheroids treated only with MB (no drugs), spheroids treated with PTX/Dox only (i.e. no MB) ([PTX]=0.34 M, [DOX]=1 M), spheroids treated with a PTX-MB-Dox/PTX-MB-RB ([DOX]=1 M, [RB]=10 M). Where required, individual wells were then placed in direct contact with the emitting surface a Sonidel SP100 sonoporator with ultrasound gel used to mediate contact. Each well was treated with ultrasound (US) for 30 s, 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). At the end of each treatment plates were placed in incubator in a humidified 5% CO.sub.2 atmosphere at 37 C. for 3 h and then wells washed three times and fresh media added. An MTT assay (APPLICHEM LIFESCIENCE) was used to determine cell viability 48 h after the treatment. Briefly, five spheroids/replicate from each condition were collected in an Eppendorf tube, washed with PBS and then incubated with trypsin for 15 min at 37 C. The resultant cellular suspension was then incubated for 3 h with MTT (10 l n 100 l of media). The absorbance was then measured at 570-690 nm using FLUOstar Omega (BMG Labtech) plate reader. Data is expressed as % of cell viability vs. untreated sample.

[0256] Moreover, at the end of each treatment, Propidium Iodide (PI) staining was performed in order to investigate cellular damage on spheroid crown. Briefly, spheroids were washed four times with PBS to remove excess of media and then incubated with a solution of PBS and PI (Invitrogen) with a final concentration of 100 g/ml. Spheroids were then incubated in the dark at RT for 40 min. At the end of incubation, spheroids were washed three times with PBS, to remove the excess of PI, and then live images were collected using a NIKON Eclipse E400 Phase contrast microscope in bright field and in fluorescence to see PI using 540 nm band pass excitation and 590 nm long pass emission filters, respectively. Moreover, fluorescence signal was evaluated with NIS-Elements BR 3.2 Imaging software, considering a total of 3 different spheroids per condition. Image J software was used to quantify PI fluorescence, expresses as % of PI fluorescence/m.sup.2. Furthermore, in order to investigate the effect of each treatment on spheroid morphology, volume was calculated for each spheroid by using the formula: volume=4/3r.sup.3.

[0257] As shown in FIG. 17, a statistically significant reduction of cells viability (p<0.05) was observed when 3D MCF-7 spheroids were exposed only to US treatment, or previously exposed to MB only or to PTX/Dox and then exposed to US. However, an increased reduction in cell viability was observed when MCF-7 3D spheroids were treated with a combination of PTX-MB-Dox/PTX-MB-Dox and then exposed to US (p<0.001), compared to spheroids treated only with PTX-MB-Dox/PTX-MB-Dox (p<0.001) and compared to spheroids treated with free drugs, i.e. PTX/Dox, and then exposed to US (p<0.01).

[0258] Results from the P.I. staining experiments revealed a slightly different trend from those observed in the MTT assay. P.I. is a DNA selective permeable dye that passes freely through the compromised plasma membranes of dead cells but does not permeate the membrane of living cells. In contrast to the MTT assay experiments, where a single cell suspension of the spheroid was analysed post-treatment, intact spheroids were examined following P.I staining. The bright field and fluorescent images from each treatment group were recorded with the fluorescence intensity quantified and plotted in FIG. 18. Bright red P.I. fluorescence was observed for spheroids treated with the O.sub.2MB-PTX-Dox/O.sub.2MB-PTX-RB+US group which was significantly more intense than any of the other groups. Surprisingly and in contrast to results from the MTT assay, the P.I. fluorescence intensity from the O.sub.2MB-PTX-Dox/O.sub.2MB-PTX-RB group in the absence of US was significantly more intense than that for the PTX/Dox+US group. It was also noticeable that the mean volume of spheroids treated with O2MB-PTX-Dox/O2MB-PTX-RB+US was significantly smaller than in any of the other groups including those spheroids treated with O2MB-PTX-Dox/O2MB-PTX-RB in the absence of US. Combined, the intense P.I. fluorescence and size reduction observed for spheroids treated with MB mediated chemo-sonodynamic therapy, in addition to the reduced cell viability observed from the MTT assay experiments, highlight the effectiveness of this approach in this particular model of breast cancer.

5.7 Cytotoxicity of Chemo-Sonodynamic Therapy In Vivo Using O.SUB.2.MB-PTX-Dox/O2MB-PTX-RBUltrasound

[0259] Subcutaneous MCF-7 tumours were established in recipient mice and a mixed suspension of the O.sub.2MB-PTX-Dox/O.sub.2MB-PTX-RB formulations administered by IV injection. During injection, ultrasound was positioned at the tumour to disrupt the MBs, release the payloads and activate SDT, where appropriate. To fully evaluate the effectiveness of the MB delivered treatments, a group of animals were also treated with a combination of free PTX/Dox (i.e. no MB attached).

[0260] All animals employed in this study were treated in accordance with the licenced procedures under the UK Animals (Scientific Procedures) Act 1986. Mia PaCa-2 cells (510.sup.6) in 100 L Matrigel were sub-cutaneously implanted into the rear dorsum of SCID (CB17/Icr-Prkdcscid/IcrIcoCrl) mice. Tumours started to form approximately 1-2 weeks after cell implantation. Once the tumour became palpable, dimensions were measured using Vernier callipers. Tumour volume was calculated using the equation tumour volume=(lengthwidthwidth)/2. Once tumours reached approximately 65mm.sup.34.20 animals were grouped and treatment commenced. Group 1 was untreated, group 2 a mixed suspension (50 L) of O.sub.2MB-PTX/RB and O.sub.2MB-PTX/Dox delivered by IV with US applied to the tumour during injection. Group 3 received the same MB treatment as group 2 but without US. Group 4 received O.sub.2MB-PTX/Dox delivered by IV with US applied to the tumour during injection and group 5 a cremaphor solution containing free PTX and DOX. Ultrasound was delivered using a Sonidel SP100 sonoporator (3.5 Wcm.sup.2, 1 MHz, 30% duty cycle, and PRF=100 Hz; PNP=0.48 MPa; MI=0.48) during and after injection (for a total of 3.5 min) with a second 3.5 min ultrasound exposure 30 min following injection. Treatments, tumour measurements and body weights were carried out and recorded once per week.

[0261] The tumour growth delay plot is shown in FIG. 19 and reveals a significant reduction in tumour volume for animals treated with PTX-MB-Dox/PTX-MB-RB+US, with tumours being 6.96% smaller when compared to their pre-treatment size, 25 days after the initial treatment. In contrast, tumours in animals treated with the same PTX-MB-Dox/PTX-MB-RB formulation in the absence of ultrasound grew by 43.15% over the same time period. These results suggest that Ultrasound Targeted Microbubble Destruction (UTMD) enables a greater proportion of drugs to be localised in the tumour yielding a significantly improved therapeutic effect. Indeed, the effect of PTX-MB-Dox/PTX-MB-RB+US was also significantly better that observed following treatment using the free PTX/Dox combination which grew by 24.77% at day 25, despite receiving a 16.8 and 98.4% increased dose of PTX and Dox respectively.

[0262] Combined, these results corroborate the in vitro efficacy results and highlight the effectiveness of MB delivered chemo-sonodynamic therapy as a targeted treatment for breast cancer. In addition to the improved efficacy offered by this approach, the treatment was also well tolerated with the body weight of animals in the MB treated groups mapping closely to that of untreated animals. In contrast, there was a 12.1% drop in body-weight for animals treated with free PTX/Dox over the course of the experiment. This reduction in body weight most likely results from toxicity exhibited by the free drugs or the Cremophor EL vehicle required to deliver PTX. Cremophor EL is known to produce undesirable side-effects and while poorly tolerated, is necessary to enable the dispersion of hydrophobic PTX in aqueous solution. Therefore, the ability to avoid the necessity of such a toxic vehicle by incorporating PTX within the MB shell is an added advantage.

EXAMPLE 7

Preparation of Biotin-Doxorubicin-Rose Bengal Conjugate

[0263] A tri-podal Biotin-Doxorubicin-Rose Bengal conjugate (Biotin-Dox-RB) was synthesised according to Scheme 4:

##STR00016## ##STR00017##

7.1 Synthesis of N-(2-(bis(2-aminoethyl)amino)ethyl)-5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (3)

[0264] To a stirred solution of Biotin-NHS (0.5 g, 1.5 mmol) and TEA (catalytic amount) in anhydrous DMF (10 mL), a solution of tris(2-aminoethyl)amine (0.22 g, 1.5 mmol) in 5 mL of DMF was added. The reaction mixture was stirred at 0 C. under argon atmosphere. After 2 hr of stirring, another volume of TEA (catalytic amount) was added and the reaction mixture was allowed to stir overnight at room temperature. After completion of the reaction (by TLC), the excess DMF was removed under reduced pressure keeping the temperature below 45 C. and the white gummy liquid thus obtained was poured into excess diethyl ether (200 mL) and filtered. The crude product was purified by column chromatography on basic (TEA) silica gel (MeOH: DCM 1:9 to 3:7) to give 3 (0.33 g, 61% yield) as a white semi solid.

[0265] .sup.1H NMR (DMSO-d.sub.6): 7.94 (brs, 1H, NH), 6.42 (brs, 1H, NH), 6.35 (brs, 1H, NH), 4.49 (brs, 4H, NH.sub.2 X 2), 4.29 (s, 1H, CH), 4.12 (s, 1H, CH), 3.07-3.02 (m, 6H, CH.sub.2 X 3), 2.88-2.82 (m, 1H, CH), 2.44-2.06 (m, 10H, CH.sub.2 X 5), 1.59-1.48 (m, 4H, CH.sub.2 X 2), 1.47-1.29 (m, 2H, CH.sub.2). ESI-MS: cald for C.sub.16H.sub.32N.sub.6O.sub.2S, 372.23; found 373.31 (M+H).

7.2 Synthesis of bis(2,5-dioxopyrrolidin-1-yl) 8,8-((((2-(5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl)azanediyl)bis (ethane-2,1-diyl))bis(azanediyl))bis(8-oxooctanoate) (5)

[0266] Compound 3 (0.5 g, 1.3 mmol) was dissolved in 10 mL anhydrous DMF in the presence of TEA (catalytic amount) and bis(2,5-dioxopyrrolidin-1-yl) octanedioate (4, 1 g, 2.7mmol) was added to it. The reaction mixture was stirred at room temperature for 24 hrs under argon atmosphere. After completion of the reaction (by TLC), excess diethyl ether (200 mL) was added to the reaction mixture. The white precipitate thus obtained was filtered and washed three times with cold diethyl ether (50 mL3). The crude product was purified by column chromatography on basic (TEA) silica gel (MeOH: CHCl.sub.3 2:8 to 5:5 v/v) to give 5 (0.83 g, 71% yield) as a low melting white solid.

[0267] .sup.1NMR (DMSO-d.sub.6): 7.94 (brs, 2H, NH X 2), 7.67 (brs, 1H, NH), 6.41 (brs, 1H,

[0268] NH), 6.34 (brs, 1H, NH), 4.29 (s, 1H, CH), 4.12 (s, 1H, CH), 3.06-3.04 (m, 3H, CH and CH.sub.2), 2.88-2.72 (m, 6H, CH.sub.2 X 3), 2.71-2.63 (m, 8H, CH.sub.2 X 4), 2.45-2.34 (m, 6H, CH.sub.2 X 3), 2.20-2.06 (m, 10H, CH.sub.2 X 5), 1.60-1.21 (m, 22H, CH.sub.2 X 11). .sup.13C NMR (DMSO-d.sub.6): 172.5 (CO), 170.7 (CO), 163.1 (C=0), 162.7 (CO), 61.4 (CH), 59.6 (CH), 55.8 (CH.sub.2), 53.9 (NCH.sub.2), 39.9 (CH.sub.2), 39.8(CH.sub.2), 39.6 (CH.sub.2), 37.3 (CH.sub.2), 36.2 (CH.sub.2), 35.6 (CH.sub.2), 31.2 (CH.sub.2), 28.7 (CH.sub.2), 28.5 (CH.sub.2), 25.8 (CH.sub.2), 25.7 (CH.sub.2), 25.6 (CH.sub.2). ESI-MS: cald for C.sub.40H.sub.62N.sub.8O.sub.12S, 878.4; found 901.3 (M+Na salt).

7.3 Synthesis of 26-(((2S,3S,4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)tetrahydro-2H-pyran-4-yl)amino)-4,11,19,26-tetraoxo-15-(2-(5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl)-3,12,15,18-tetraazahexacosyl 2,3,4,5-tetrachloro-6-(6-hydroxy-2,4,5,7-tetraiodo-3-oxo-3H-xanthen-9-yl)benzoate (9)

[0269] To a DMF (anhydrous, 10 mL) solution of 5 (0.4 g, 0.45 mmol) Doxorubicin-hydrochloride (8, 0.26g, 0.45 mmol) and TEA (0.5 mL) were added at 0 C. and stirred for 24 hrs at room temperature under argon atmosphere. After completion of the reaction (monitored by GC-MS), Rose Bengal amine 7 (prepared separately according to literature procedure), 0.46 g, 0.45 mmol} in DMF (5 mL) and TEA (0.5 mL) were added to the reaction mixture and continued to stir for 24 hrs. The progress of the reaction was monitored by GC-MS analysis of the crude reaction mixture. After completion of reaction, excess diethyl ether (200 mL) was added to the solution and stirred for 30 min. The dark red precipitate thus obtained was filtered and washes several times with cold diethyl ether (100 mL), ethyl acetate (100 mL), acetone-water mixture (10%, v/v, 100 mL) and finally with ethyl acetate-hexane mixture (50%, v/v, 100 ml) respectively to afford a red powder of compound 9 (0.28 g, 28% yield).

[0270] .sup.1H NMR (DMSO-d.sub.6):7.93 (s, 2H, ArCH), 7.90-7.85 (m, 2H, ArCH), 7.66 (brs, 5H,

[0271] NH), 7.45 (s, 1H, 6.39 (brs, 1H, NH), 6.33 (brs, 1H, NH), 5.41-5.39 (m, 1H, CH), 5.19-5.18 (m, 4H, CH.sub.2 X 2), 4.93-4.71 (m, 3H, CH X 3), 4.55 (s, 3H, OCH.sub.3),4.27-3.90 (m, 4H, CH X 4), 3.04-2.97 (m, 8H, CH.sub.2 X 4), 2.80-2.77 (m, 2H, CH.sub.2), 2.48-2.43 (m, 8H, CH.sub.2 X 4), 2.03 (brs, 12H, CH.sub.2 X 6), 1.43 (brs, 12H, CH.sub.2 X 6), 1.14-1.11 (m, 13H, CH.sub.2 X 5, CH.sub.3 X 1).

[0272] .sup.13C NMR (DMSO-d.sub.6): 220.1, 177.3, 172.5, 172.2, 171.5, 168.9, 163.2, 162.7, 157.3, 156.6, 154.6, 153.7, 150.3, 138.5, 136.5, 135.6, 133.0, 110.7, 101.9, 97.6, 96.3, 89.2, 79.3, 76.7, 66.8, 63.5, 61.4, 60.5, 59.6, 57.5, 55.8, 53.9, 51.9, 40.4, 40.2, 37.4, 36.5, 36.2, 35.6, 31.2, 28.7, 28.4, 28.3, 25.7.

[0273] ESI-MS: cald for C.sub.81H.sub.90Cl.sub.4I.sub.4N.sub.8O.sub.22S, 2209.12; found 2208.02 (MH).

EXAMPLE 8

Alternative Method for the Synthesis of Biotin-Gemcitabine-Rose Bengal Conjugate

[0274] A Biotin-Gemcitabine-Rose Bengal (Biotin-Gem-RB) conjugate was synthesised according to Scheme 5:

##STR00018## ##STR00019##

8.1 Synthesis of N-(2-(bis(2-aminoethyl)amino)ethyl)-5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (3)

[0275] Compound 3 was synthesized according to the procedure described in Example 1.

8.2 Synthesis of 7-(((((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)carbonyl)oxy)heptanoic acid (6)

[0276] To a DCM (10 mL) solution of 4 (1 g, 4.6 mmol), 4-nitrophenyl chloroformate (2.79 g, 13.8 mmol), DIPEA (2.38 g, 18.4 mmol) and a catalytic amount of pyridine were added at 0 C. and stirred for 5 h at room temperature. Then the reaction mixture was concentrated in vacuo. The crude residue was dissolved in DMF (10 mL). To this solution, GMC hydrochloride (4.1 g, 13.8 mmol) in DMF (5 mL) and TEA (2 mL) were added and continued to stir for 24 h. The progress of the reaction was monitored by GC-MS analysis of the crude reaction mixture. After completion of reaction, excess diethyl ether (200 mL) was added to the reaction mixture. The yellowish oil thus obtained was separated and purified by flash chromatography using MeOH/CHCl.sub.3 (5%, v/v) as eluent. Compound 6 was isolated as a sticky yellow liquid. (1.5 g, Yield=64.3%).

[0277] .sup.1H NMR (DMSO-d.sub.6): 10.5 (brs, 1H, COOH), 7.63 (d, J=7.5 Hz, 1H, CH), 7.41 (brs, 2H, NH.sub.2), 6.20 (d, J=7.5 Hz, 1H, CH), 5.18 (brs, 1H, CH), 3.71-3.55 (m, 5H, CH.sub.2X2, CHX1), 2.36 (brs, 2H, CH.sub.2), 1.23-1.17 (m, 18H, CH.sub.2 X 9).

[0278] .sup.13C NMR (DMSO-d.sub.6): 172.1, 166.0, 155.1, 154.9, 153.6, 123.5, 95.2, 95.0, 80.8, 69.1, 68.8, 33.6, 29.4, 29.3,29.2, 28.9, 28.8, 25.5, 25.4.

[0279] ESI-MS: cald for C.sub.22H.sub.33F.sub.2N.sub.3O.sub.8, 504.2; found 527.0 (M+Na salt).

8.3 Synthesis of 1-((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-3,16,24-trioxo-20-(2-(5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl)-2,4-dioxa-17,20,23-triazahentriacontan-31-yl 2,3,4,5-tetrachloro-6-(6-hydroxy-2,4,5,7-tetraiodo-3-oxo-3H-xanthen-9-yl)benzoate (9)

[0280] EDCl, HCl (0.4 g, 2.0 mmol), and DIPEA (0.45 g, 3.5 mmol) were added to a solution of the acid 6 (0.34 g, 0.6 mmol), compound 3 (0.25 g, 0.6 mmol) and HOBt (0.27 g, 2.0 mmol) in anhydrous DMF (50 mL) and stirred for 24 hrs at room temperature under nitrogen. The progress of the reaction was monitored by GC-MS analysis of the crude reaction mixture. After completion of reaction, RB-Octanoic Acid, 8 (prepared according to the literature procedure, 0.8 g, 0.7 mmol) in 5 mL of DMF was added to the reaction mixture followed by a catalytic amount of DIPEA and continued to stir for 24 h at room temperature. After completion of reaction, excess diethyl ether (200 mL) was added to the reaction mixture and stirred for 30 min. The pink red precipitate thus obtained was filtered and was washed several times with cold diethyl ether (100 mL), ethyl acetate (100 mL), acetone-water mixture (10%, v/v, 100 mL) and finally with ethyl acetate-hexane mixture (50%, v/v, 100 ml) respectively to afford a pink red powder of compound 9 (0.63 g, 45% yield).

[0281] .sup.1H NMR (DMSO-d.sub.6): 7.86 (s, 2H, ArCH), 7.73 (d, J=7.0 Hz, 1H, CH), 7.39 (brs, 3H, NH X 3), 6.40-6.34 (m, 2H, NH X2), 6.03 (s, 1H, CH), 5.72 (d, J=7.0 Hz, 1H, CH), 4.22 (s, 1H, CH), 4.05 (brs, 3H, CH.sub.2, CH X2), 3.86 (brs, 1H, OH), 3.81-3.50 (m, 6H, CH X 2, CH.sub.2 X 2), 3.02-2.86 (m, 8H, CH.sub.2 X 4), 2.80-2.00 (m, 12H, CH.sub.2 X 6), 1.52-0.81 (m, 32H, CH.sub.2 X 16).

[0282] .sup.13C NMR (DMSO-d.sub.6): 172.6, 171.6, 169.9, 168.9, 166.0, 163.2, 162.7, 155.1, 141.5, 141.1, 123.5, 98.9, 96.4, 95.0, 83.9, 80.8, 70.2, 69.1, 68.9, 68.7, 61.4, 59.6, 59.2, 54.2, 54.0, 48.9, 46.0, 38.0, 37.3, 36.1, 35.5, 31.1, 28.9, 28.6, 28.3, 25.7.

[0283] ESI-MS: cald for C.sub.66H.sub.79 Cl.sub.4F.sub.2I.sub.4N.sub.9O.sub.15S, 1955.03; found 1956.5 (M+H).