THERAPY

Abstract

The invention generally relates to sonodynamic therapy using microbubble-sonosensitiser complexes and, more specifically, to such therapy for the treatment of deeply-sited tumours and associated metastatic disease. In particular, the invention relates to a combination therapy in which sonodynamic treatment of deeply-sited tumours with microbubble-sonosensitiser complexes is combined with treatment using immune checkpoint inhibitors. It further relates to methods of sonodynamic therapy in which a sonodynamic-induced abscopal response modulates a systemic regression of metastatic disease. In such methods the abscopal response may be further enhanced by co-administration of an immune checkpoint inhibitor. The invention is particularly suitable for the treatment of pancreatic cancer (e.g. pancreatic ductal adenocarcinoma) and associated metastasis.

Claims

1. A microbubble-sonosensitiser complex for use in a method of sonodynamic therapy, wherein said method comprises simultaneous, separate or sequential use of an immune checkpoint inhibitor.

2. A complex for use as claimed in claim 1, wherein said microbubble-sonosensitiser complex comprises a microbubble attached to or otherwise associated with at least one sonosensitiser via a non-covalent linkage, e.g. via a biotin-avidin interaction.

3. A complex for use as claimed in claim 1 or claim 2, wherein said microbubble-sonosensitiser complex comprises at least one sonosensitiser selected from the group consisting of phenothiazine dyes (e.g. methylene blue, toluidine blue), Rose Bengal, porphyrins (e.g. Photofrin®), chlorins, benzochlorins, phthalocyanines, naphthalocyanines, 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.

4. A complex for use as claimed in claim 3, wherein said sonosensitiser is Rose Bengal, methylene blue, indocyanine green, or an analogue thereof, preferably Rose Bengal.

5. A complex for use as claimed any one of the preceding claims, wherein said microbubble-sonosensitiser complex further comprises at least one chemotherapeutic agent.

6. A complex for use as claimed in claim 5, wherein said microbubble-sonosensitiser complex is attached to or otherwise associated with a chemotherapeutic agent, preferably via a non-covalent linkage, e.g. via a biotin-avidin interaction, and/or wherein the microbubble comprises a shell having incorporated therein a chemotherapeutic agent.

7. A complex for use as claimed in claim 5 or claim 6, wherein said 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); topoisomerase II inhibitors (e.g. etoposide); antimicrotubule agents such as vinca alkaloids (e.g. vincristine), taxols (e.g. paclitaxel), and epothilones (e.g. ixabepilone); antioestrogens (e.g. tamoxifen); antiandrogens (e.g. bicalutamide, cyproterone acetate); aromatase inhibitors (e.g. anastrozole, formestane); antiangiogenic or hypoxia targeting drugs (either naturally occurring, e.g. endostatin, or synthetic, e.g. gefitinib, lenalidomide); antivascular agents (e.g. combretastatin); tyrosine kinase inhibitors (e.g. gefitinib, erlotinib, vandetanib, sunitinib); oncogene or signalling pathway targeting agents (e.g. tipifarnib, 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 uredepa); 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, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine); androgens (e.g. calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone); anti-adrenals (e.g. aminoglutethimide, mitotane, trilostane); immune checkpoint inhibitors (e.g. BMS-1001 and BMS-1166); immune response modifiers (e.g. imiquimod); and pharmaceutically acceptable salts, derivatives or analogues of any of these compounds.

8. A complex for use as claimed in claim 7, wherein the chemotherapeutic agent is an anti-metabolite, e.g. 5-fluorouracil or gemcitabine.

9. A complex for use as claimed in any one of claims 5 to 8, wherein the microbubble comprises a shell having incorporated therein an additional chemotherapeutic agent.

10. A complex for use as claimed in claim 9, wherein said additional chemotherapeutic agent is as defined in claim 7 or claim 8, preferably wherein said additional chemotherapeutic agent is hydrophobic.

11. A complex for use as claimed in claim 10, wherein said additional chemotherapeutic agent is an anti-microtubule agent, e.g. a taxol such as paclitaxel.

12. A complex for use as claimed in any one of claims 1 to 11, wherein said method further comprises simultaneous, separate or sequential use of a microbubble-chemotherapeutic agent complex.

13. A complex for use as claimed in claim 12, wherein said microbubble-chemotherapeutic agent complex comprises a microbubble attached to or otherwise associated with at least one chemotherapeutic agent via a non-covalent linkage, e.g. via a biotin-avidin interaction.

14. A complex for use as claimed in claim 12 or claim 13, wherein said chemotherapeutic agent is as defined in claim 7 or claim 8.

15. A complex for use as claimed in any one of the preceding claims, wherein the microbubble comprises a shell which retains a gas, preferably oxygen gas.

16. A complex for use 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 μm.

17. 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, e.g. polyethylene glycol (PEG).

18. A complex for use as claimed in any one of the preceding claims, wherein said immune checkpoint inhibitor is an inhibitor of PD-1, PDL-1, CTLA-4, LAG-3 or TIM-3.

19. A complex for use as claimed in claim 18, wherein said immune checkpoint inhibitor is selected from the group consisting of nivolumab, pembrolizumab, spartalizumab, TSR-042, atezolizumab, avelumab, durvalumab, BMS-1001, BMS-1166, SB415286, ipilimumab, tremelimumab, and any combination thereof.

20. A complex for use as claimed in any one of the preceding claims, in which said complex is contacted with cells or tissues of a subject (e.g. a human patient) and, either simultaneously or sequentially, said cells or tissues are subjected to irradiation with ultrasound and/or light.

21. A complex for use as claimed in any one of the preceding claims in the treatment of cancer, metastasis or micrometastasis derived from said cancer, or in the treatment of circulating tumour cells (CTCs), preferably in the treatment of a deep-sited tumour, metastasis or micrometastasis derived from said tumour.

22. A complex for use as claimed in claim 21, 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, 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.

23. A complex for use as claimed in claim 21 for the treatment of pancreatic cancer or metastatic pancreatic cancer.

24. A product comprising a microbubble-sonosensitiser complex as defined in any one of claims 1 to 11 and an immune checkpoint inhibitor (e.g. as defined in claim 18 or claim 19) for simultaneous, separate or sequential use in a method of sonodynamic therapy.

25. A kit comprising the following components: (i) a microbubble-sonosensitiser complex as defined in any one of claims 1 to 11; and separately (ii) an immune checkpoint inhibitor (e.g. as defined in claim 18 or claim 19); optionally together with (iii) instructions for the use of said components in a method of sonodynamic therapy.

26. A pharmaceutical composition comprising a microbubble-sonosensitiser complex as defined in any one of claims 1 to 11 and an immune checkpoint inhibitor (e.g. as defined in claim 18 or claim 19), together with at least one pharmaceutical carrier or excipient.

27. A composition as claimed in claim 26 for use in therapy or for use as a medicament, preferably for use in a method of sonodynamic therapy.

28. A microbubble-sonosensitiser complex as defined in any one of claims 1 to 11 for use in a method of sonodynamic treatment of a metastatic disease, a micrometastatic disease or circulating tumour cells (CTCs), preferably metastatic pancreatic cancer.

29. A complex for use as claimed in claim 28, wherein said method of sonodynamic therapy comprises simultaneous, separate or sequential use of an immune checkpoint inhibitor (e.g. as defined in claim 18 or claim 19).

Description

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

[0161] FIG. 1 is a schematic representation of the MB-RB conjugate and the bilateral tumour model used in Example 3. Animals received a tail-vein injection of the MB-RB conjugate with ultrasound applied to the treated tumour (+) using the parameters described in Example 3. For the combined SDT/PDL-1 inhibitor experiment, the PDL-1 inhibitor was administered IP 2 hours before the MB-RB+US treatment.

[0162] FIG. 2 shows a plot of % tumour growth against time for mice bearing bilateral KPC pancreatic tumours where the right-hand-side tumour was treated with the MB-RB conjugate+US (PRIMARY TUMOUR) while the left-hand-side tumour remained untreated (OFF-TARGET TUMOUR). [MB]=1.6×10.sup.9; [RB]=2.15±0.42 mg/kg. n=5; *p≤0.05, **p≤0.01 for comparison to untreated animals

[0163] FIG. 3 shows a plot of % tumour growth for the OFF-TARGET tumour in mice bearing bilateral KPC pancreatic tumours that received (i) no treatment; (ii) an IP injection of PDL-1 inhibitor; (iii) MB-RB conjugate+US at the primary tumour (SDT); and (iv) an IP injection of PDL-1 inhibitor 2 hours before treatment of the primary tumour with the MB-RB conjugate+US (PDL-1+SDT). [MB]=1.6×10.sup.9; [RB]=2.15±0.42 mg/kg; [PDL-1]=10 mg/kg. Animals treated on days 0, 4 & 7. *p≤0.05, **p≤0.01 for PDL-1+SDT v SDT.

[0164] FIG. 4 shows a plot of average tumour weight for tumours extracted from animals on day 11 following initial treatment.

[0165] FIG. 5 shows the fold change in expression of cytotoxic T-cells (CD45+ve, CD3+ve, CD8a+ve) extracted from tumours on day 11 following initial treatment.

[0166] FIG. 6 shows a schematic representation of PTX/GEM/RB-MB.

[0167] FIG. 7 is a schematic representation of a) O.sub.2MB-PTX/DOX and b) O.sub.2MB-PTX/RB.

EXAMPLES

Example 1—Synthesis of Biotin-Rose Bengal

[0168] ##STR00009##

[0169] Biotin functionalised Rose Bengal (4) was prepared as described in McEwan et al. (J Control Release. 2015; 203, 51-6).

Example 2—Synthesis of O.SUB.2.MB-Rose Bengal Conjugate

[0170] Avidin functionalised lipid stabilised microbubbles (MBs) were prepared by first dissolving 1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC) (4.0 mg, 4.44 μmol), 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 μmol) in chloroform to achieve a molar ratio of 82:9:9. The solvent was removed under vacuum at room temperature to yield a translucent film. The dried lipid film was reconstituted in 3 mL of PBS (pH 7.4±0.1):propylene glycol:glycerol (8:1:1 v/v) mixture and heated in a water bath at 80° C. under magnetic stirring for 30 min. The suspension was then sonicated using a Microson ultrasonic cell disrupter at an amplitude of 22% for 30 seconds. The headspace of the vessel was then filled with perfluorobutane (PFB) gas and the solution was sonicated at an amplitude of 90% for 30 seconds to form the MBs. The MBs were immediately placed on ice for 10 minutes followed by centrifugation at 700 rpm for 5 min and removal of the subnatant. An additional 2 mL of PBS (pH 7.4±0.1):propylene glycol:glycerol (8:1:1 v/v) mixture was added to the MB cake followed by an aqueous solution of avidin (500 μL, 5 mg/mL). The MB suspension was then stirred for 5 min on a rotary shaker (150 rpm, 0° C.) followed by centrifugation (700 rpm) to remove excess avidin.

[0171] To prepare Rose Bengal (RB) functionalised O.sub.2MBs (as shown in FIG. 1), a saturated solution of biotin-RB (4) (1 mL, 5 mg/mL) in PBS (5% DMSO) was added to avidin-functionalised MBs. The suspension was mixed for 5 min on a rotary shaker (150 rpm, 0° C.). The subnatant was removed and replaced with 2 mL of PBS (pH 7.4±0.1):propylene glycol:glycerol (8:1:1 v/v) mixture. This washing process was repeated 3 times to remove any unbound material. The MBs were then sparged with medical grade oxygen gas prior to use.

[0172] The MB number was determined by withdrawing 10 μL samples of the MB conjugate and diluting in 90 μL of PBS (pH 7.4±0.1) followed by analysis using a haemocytometer (Bright-Line, Hausser Scientific, Horsham, Pa., USA).

Example 3—In Vivo Studies

[0173] General:

[0174] A mouse pancreatic cancer cell line (T110299), derived from a primary pancreatic tumour of a genetically modified KPC mouse model (Duewell et al., Oncoimmunology (2015) 14; 4(10): e1029698) was used to establish bilateral tumours in the dorsum of immunocompetent BALB/C mice in order to monitor effects at both treated and untreated tumours. When tumours reached a certain size (above 100 mm.sup.3), the mice were treated with a suspension of O.sub.2MB-RB administered via tail vein injection. The right-hand-side tumour was designated the treated tumour (or “primary tumour”) and the left-hand-side tumour the “off-target tumour”. During injection, low intensity ultrasound was directed at the target tumour to disrupt the bubbles and activate SDT. A second ultrasound treatment was administered 30 min after the injection to activate the Rose Bengal. These parameters induce microbubble inertial cavitation and maximise SDT mediated generation of ROS. Tumour growth was measured every 2 days and blood samples taken before and 1, 3 and 24 hours following treatment to determine any changes in immune response. Using the same animal model, mice were initially treated with an IP injection of a PDL-1 inhibitor. Two hours later, mice were treated with UTMD mediated SDT and monitored to determine the therapeutic benefit of the combined treatment.

[0175] UTMD Mediated SDT Treatment Using O.sub.2MB-RB Conjugate:

[0176] All animals employed in this study were treated in accordance with the licensed procedures under the UK Animals (Scientific Procedures) Act 1986. KPC cells were maintained in D-MEM high glucose, 10% Foetal Bovine Serum, 1% L-Glutamine and 1% non-essential amino acids. KPC cells (5×10.sup.5 per tumour) were sub-cutaneously injected in a 100 μl suspension of 1:1 Matrigel and media into the right and left flank of C57BL/6JOlaHsd mice. Bilateral tumours formed and were palpable within 1 week and measured using Vernier calipers. Tumour volume was calculated by (width×width×length)/2. When tumours reached an average size of 119 mm.sup.3±11.5, the animals were divided into 2 groups (n=5). Group 1 received no treatment and group 2 received a 100 μl tail intravenous injection of the O.sub.2MB-RB conjugate prepared in Example 2 (MB=1.6×10.sup.9; RB=2.15±0.42 mg/kg) and ultrasound was delivered to the right hand tumour (SDT; Primary Tumour) during injection and for a period of 3.5 minutes. Ultrasound was delivered using a Sonidel SP100 sonoporator (3.5 W cm.sup.−2, 1 MHz, 30% duty cycle, and PRF=100 Hz; PNP=0.48 MPa; MI=0.48; 3.5 minutes) (see FIG. 1). The left hand side tumour in group 2 was designated as the SDT; Off Target Tumour. Treatments were repeated on days 4 and 7. Results are presented in FIG. 2.

[0177] UTMD Mediated SDT Treatment Using O.sub.2MB-RB Conjugate in Combination with Immune Checkpoint Inhibitors:

[0178] PDL-1 inhibitor: InVivoMAb anti-mouse PDL-1 (B7-H1) —Clone: 10F.9G2; Catalog #BE0101 (supplier 2BScientific).

[0179] For MB mediated SDT+anti-PDL-1 treatment, bilateral tumours were established in a further four groups (n=5) of animals as described above. When tumours reached an average of 150 mm.sup.3±11.8, the animals were treated as follows: group 1 received no treatment; group 2 received an intraperitoneal injection of PDL-1 inhibitor (10 mg/kg), group 3 received a 100 μl tail intravenous injection of O.sub.2MB-RB conjugate (MB=1.6×10.sup.9; RB=2.15 t 0.42 mg/kg) and ultrasound was delivered to the primary tumour during injection and for a period of 3.5 minutes as described above. Group 4 received an intraperitoneal injection of PDL-1 inhibitor (10 mg/kg) and 2 hours later animals were treated with a 100 μl tail intravenous injection of O.sub.2MB-RB conjugate (MB=1.6×10.sup.9; RB=2.15±0.42 mg/kg) and ultrasound was delivered to the primary tumour during injection and for a period of 3.5 minutes. Tumours were measured daily for 11 days. Results are presented in FIG. 3. Treatments were repeated on days 4 and 7. On Day 11, animals were sacrificed, tumours were excised and the tumour weights recorded. Results are presented in FIG. 4.

[0180] A single cell suspension was obtained from the tumours by homogenising the tumour, adding 5 ml of 4% FCS in RPMI and 160 μl (30 mg/ml) collagenase type II (Gibco, 17101-015) and 50 μl (2 μg/ml) DNAse and stirring for 15 minutes. A further 160 μl (30 mg/ml) collagenase was added and stirred for a further 15 minutes at room temperature. The suspension was filtered through a 100 μm filter, centrifuged at 1700 rpm for 5 minutes, re-suspended in 1 ml Red Lysis buffer and incubated for 10 minutes at room temperature. The remaining cells were centrifuged, the supernatant removed and washed twice in ice cold PBS. Cells were incubated in fluorochrome conjugated antibodies specific for CD45 (0.125 μg/test, eBioscience), CD3 (0.5 g/test, eBioscience), CD8a (0.25 μg/test, eBioscience). Red blood cells were removed using multi-species RBC lysis buffer as per the manufacturer's instructions (eBioscience). Cytotoxic T cells were identified as CD45+CD3+CD8a+ cells. Cells were acquired using a Gallios (Beckman Coulter) and analysed using FlowJo software. Results are presented in FIG. 5.

Example 4—Synthesis of Biotin-Gemcitabine (Biotin-Gem) and Biotin-Gemcitabine-Rose Bengal (Biotin-Gem-RB) Conjugates

4.1 Synthesis of Biotinylated Gemcitabine (Biotin-GEM)

[0181] Biotinylated Gemcitabine was synthesised according to scheme 2. The protocol is provided below.

##STR00010##

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

[0182] 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 mL×3). 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).

[0183] .sup.1H NMR (DMSO-d.sub.6): δ 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×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×2), 2.72 (brs, 1H, NH), 2.49-2.04 (m, 2H, CH.sub.2), 1.49-1.28 (m, 6H, CH.sub.2×3).

[0184] .sup.13C NMR (DMSO-d.sub.6): 175.0 (C═O), 166.3 (C), 165.5 (C═O), 156.3 (C═O), 156.1 (C═O), 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).

4.2 Synthesis of Biotin-Gem-RB

[0185] Biotin-Gem-RB was synthesised according to Scheme 3. The protocols for each intermediate are provided below.

##STR00011## ##STR00012##

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

[0186] 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.

[0187] .sup.1H NMR (DMSO-d.sub.6): δ 7.94 (brs, 1H, NH), 6.42 (brs, 1H, NH), 6.35 (brs, 1H, NH), 4.49 (brs, 4H, NH.sub.2×2), 4.29 (s, 1H, CH), 4.12 (s, 1H, CH), 3.07-3.02 (m, 6H, CH.sub.2×3), 2.88-2.82 (m, 1H, CH), 2.44-2.06 (m, 10H, CH.sub.2×5), 1.59-1.48 (m, 4H, CH.sub.2×2), 1.47-1.29 (m, 2H, CH.sub.2).

[0188] 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)

[0189] 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 mL×3). 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.

[0190] .sup.1H NMR (DMSO-d.sub.6): δ 7.94 (brs, 2H, NH×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.2×3), 2.71-2.63 (m, 8H, CH.sub.2×4), 2.45-2.34 (m, 6H, CH.sub.2×3), 2.20-2.06 (m, 10H, CH.sub.2×5), 1.60-1.21 (m, 22H, CH.sub.2×11).

[0191] .sup.13C NMR (DMSO-d.sub.6): 172.5 (C═O), 170.7 (C═O), 163.1 (C═O), 162.7 (C═O), 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).

[0192] 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)

[0193] 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 (0.26 g, 30% yield).

[0194] .sup.1H NMR (DMSO-d.sub.6): δ 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×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×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.2×3), 2.94-2.84 (m, 6H, NCH.sub.2×3), 2.81 (brs, 1H, OH), 2.45-2.46 (m, 3H, CH, CH.sub.2), 2.17-2.06 (m, 1011, CH.sub.2×5), 1.60-1.10 (m, 22H, CH.sub.2×11).

[0195] .sup.13C NMR (DMSO-d.sub.6): 171.8 (C═O, C), 165.98 (C═O), 163.2 (C═O), 162.7 (C═O), 159.3 (CH), 155.0 (C═O), 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).

[0196] ESI-MS: cald for C.sub.63H.sub.72Cl.sub.4F.sub.2I.sub.4N.sub.10O.sub.15S, 1925.98; found 1925.90 (M−H).

Example 5—Preparation of Avidin-Functionalised Paclitaxel (PTX) Loaded Microbubbles (MBs)

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

[0197] 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 μmol) and DSPE-PEG (2000)-biotin (1.45 mg, 0.481 μmol) in chloroform to achieve a molar ratio of 82:9:9. To this solution was added paclitaxel (5 mg, 5.86 μmol) 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 μL, 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 2 mL of Ungers solution, centrifuged (700 rpm) and the MBs isolated.

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

[0198] A solution containing either Biotin-RB, Biotin-Gem, or Biotin-Gem-RB (500 μL, 5 mg/mL), prepared in a DMSO:Ungers solution (10:90 v/v) was added to 2 mL of PTX-MBs (2.0×10.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.

Example 6—Preparation of Avidin Functionalised PTX-MBs Carrying Biotin-RB, Biotin-Gem and Biotin-Gem-RB

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

[0199] 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 (6.72×10.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.

Example 7—Preparation of Paclitaxel (PTX) Loaded Microbubbles (MBs) Carrying Biotin-Doxorubicin (Biotin-Dox) or Biotin-Rose Bengal (Biotin-RB) Conjugates

7.1 Reagents and Materials

[0200] 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. Phosphate 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.

7.2 Synthesis of Biotin-Dox

[0201] Biotinylated Doxorubicin (Biotin-Dox) (3) was synthesised according to scheme 4.

[0202] The protocol is provided below.

##STR00013##

[0203] 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 ml×3). 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.

[0204] .sup.1H NMR (DMSO-d.sub.6) δ: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.2—OH), 4.51 (brs, 2H, OH×2), 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×2), 3.10-3.00 (m, 4H, CH.sub.2×1, CH×2), 2.88-2.54 (m, 3H, CH.sub.2×1, CH), 2.20-2.00 (m, 1H, CH), 1.63-1.50 (m, 4H, CH.sub.2×2), 1.42-1.22 (m, 11H, CH.sub.3×1, CH.sub.2×4). .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.

[0205] ESMS (M−H]: calculated for C.sub.37H.sub.43I.sub.2N.sub.3O.sub.13S=769.25, found=767.9.

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

[0206] 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 μmol) 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 Propylene 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 (7.52×10.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. 7 is a schematic representation of a) O.sub.2MB-PTX/DOX b) O.sub.2MB-PTX/RB.

Example 8—Preparation of Biotin-Doxorubicin-Rose Bengal Conjugate

[0207] A tri-podal Biotin-Doxorubicin-Rose Bengal conjugate (Biotin-Dox-RB) was synthesised according to Scheme 5:

##STR00014## ##STR00015##

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

[0208] 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.

[0209] .sup.1H NMR (DMSO-d.sub.6): δ 7.94 (brs, 1H, NH), 6.42 (brs, 1H, NH), 6.35 (brs, 1H, NH), 4.49 (brs, 4H, NH.sub.2×2), 4.29 (s, 1H, CH), 4.12 (s, 1H, CH), 3.07-3.02 (m, 6H, CH.sub.2×3), 2.88-2.82 (m, 1H, CH), 2.44-2.06 (m, 10H, CH.sub.2×5), 1.59-1.48 (m, 4H, CH.sub.2×2), 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).

8.2 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)

[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 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 mL×3). 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): δ 7.94 (brs, 2H, NH×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.2×3), 2.71-2.63 (m, 8H, CH.sub.2×4), 2.45-2.34 (m, 6H, CH.sub.2×3), 2.20-2.06 (m, 10H, CH.sub.2×5), 1.60-1.21 (m, 22H, CH.sub.2×11). .sup.13C NMR (DMSO-d.sub.6): 172.5 (C═O), 170.7 (C═O), 163.1 (C═O), 162.7 (C═O), 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).

8.3 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)

[0212] To a DMF (anhydrous, 10 mL) solution of 5 (0.4 g, 0.45 mmol) Doxorubicin-hydrochloride (8, 0.26 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 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).

[0213] .sup.1H NMR (DMSO-d.sub.6): 7.93 (s, 2H, Ar—CH), 7.90-7.85 (m, 2H, Ar—CH), 7.66 (brs, 5H, NH), 7.45 (s, 1H, 6.39 (brs, 11H, NH), 6.33 (brs, 1H, NH), 5.41-5.39 (m, 1H, CH), 5.19-5.18 (m, 4H, —CH.sub.2×2), 4.93-4.71 (m, 3H, CH×3), 4.55 (s, 3H, —OCH.sub.3), 4.27-3.90 (m, 4H, CH×4), 3.04-2.97 (m, 8H, CH.sub.2×4), 2.80-2.77 (m, 2H, CH.sub.2), 2.48-2.43 (m, 8H, CH.sub.2×4), 2.03 (brs, 12H, CH.sub.2×6), 1.43 (brs, 12H, CH.sub.2×6), 1.14-1.11 (m, 13H, CH.sub.2×5, CH.sub.3×1).

[0214] .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.

[0215] ESI-MS: cald for C.sub.81H.sub.90Cl.sub.4I.sub.4N.sub.8O.sub.22S, 2209.12; found 2208.02 (M−H).

Example 9—Alternative Method for the Synthesis of Biotin-Gemcitabine-Rose Bengal Conjugate

[0216] A Biotin-Gemcitabine-Rose Bengal (Biotin-Gem-RB) conjugate was synthesised according to Scheme 6:

##STR00016##

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

[0217] Compound 3 was synthesized according to the procedure described in Example 1.

9.2 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)

[0218] 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%).

[0219] .sup.1H NMR (DMSO-d.sub.6): δ 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.2×2, —CH×1), 2.36 (brs, 2H, —CH.sub.2), 1.23-1.17 (m, 18H, —CH.sub.2×9).

[0220] .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.

[0221] ESI-MS: cald for C.sub.22H.sub.33F.sub.2N.sub.3O.sub.8, 504.2; found 527.0 (M+Na salt).

9.3 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)

[0222] 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).

[0223] .sup.1H NMR (DMSO-d.sub.6): δ 7.86 (s, 2H, Ar—CH), 7.73 (d, J=7.0 Hz, 1H, CH), 7.39 (brs, 3H, NH×3), 6.40-6.34 (m, 2H, NH×2), 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×2), 3.86 (brs, 1H, OH), 3.81-3.50 (m, 6H, CH×2, CH.sub.2×2), 3.02-2.86 (m, 8H, CH.sub.2×4), 2.80-2.00 (m, 12H, CH.sub.2×6), 1.52-0.81 (m, 32H, CH.sub.2×16).

[0224] .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.

[0225] ESI-MS: cald for C.sub.66H.sub.79Cl.sub.4F.sub.2I.sub.4N.sub.9O.sub.15S, 1955.03; found 1956.5 (M+H).