Sensitizer-peptide conjugate

Abstract

The present invention relates to a sensitizer-peptide conjugate for use in the treatment of cancer. In particular, the present invention relates to a sensitizer-peptide conjugate for use in the treatment of melanoma. The use of such sensitizer-peptide conjugate in photodynamic therapy or sonodynamic therapy is also disclosed. According to the present invention, there is provided a sensitizer-peptide conjugate for use in the treatment of cancer; wherein the sensitizer-peptide conjugate comprises at least one sensitizer and at least one peptide.

Claims

1. A method for the treatment of cancer in a subject in need thereof; the method comprising the step of administering a sensitizer-peptide conjugate comprising at least one sensitizer and at least one peptide to the subject, wherein the at least one sensitizer comprises Rose Bengal; and wherein the at least one peptide comprises a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

2. The method according to claim 1; wherein the at least one peptide comprises the amino acid sequence defined by SEQ ID NO. 6.

3. The method according to claim 1; wherein the at least one peptide comprises the amino acid sequence defined by SEQ ID NO. 2.

4. The method according to claim 1; wherein the sensitizer-peptide conjugate further comprises at least one linker between the at least one sensitizer and the at least one peptide.

5. The method according to claim 4; wherein the at least one linker comprises one or more polymers.

6. The method according to claim 4; wherein the at least one linker comprises the general formula —COOCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CO—.

7. The method according to claim 1; wherein the peptide comprises at least one chiral amino acid; wherein the at least one chiral amino acid is the L enantiomer.

8. The method according to claim 1; wherein the sensitizer-peptide conjugate is activated by a trigger.

9. The method according to claim 8; wherein the trigger is electromagnetic radiation.

10. The method according to claim 9; wherein the method is part of photodynamic therapy.

11. The method according to claim 8; wherein the trigger is sound.

12. The method according to claim 11; wherein the method is part of sonodynamic therapy.

13. The method according to claim 1; wherein the cancer is selected from the group consisting of breast cancer, pancreatic cancer, skin cancer, head and neck cancer, oesophageal cancer, bladder cancer, and prostate cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Embodiments of the present invention will now be described by way of non-limiting examples, and with reference to the accompanying drawings in which:

(2) FIG. 1 is a landscape-oriented representation of the structure of a sensitizer-peptide conjugate comprising Rose Bengal and a peptide having the amino acid sequence defined by SEQ ID NO. 2;

(3) FIG. 2 shows charts as detailed below for each part, wherein “m/z” refers to the mass-to-charge ratio. FIG. 2(a) is a chart showing a positive electrospray Mass Spectrum of all-L enantiomer of a Rose Bengal-peptide conjugate, wherein the peptide has the amino acid sequence defined by SEQ ID NO. 2. Multiple charged ions are denoted as M/2+1 and M/3+1. Calculated parent mass: 2721.2 Expected mass: 2721.6. FIG. 2(b) is a chart showing a positive mode electrospray ionisation mass spectrometry (ESI-MS) Mass Spectrum of all-L enantiomer of a peptide having the amino acid sequence defined by SEQ ID NO. 2; indicating the expected M+1 m/z and associated doubly charged daughter ion (expected mass-to-charge ratio=1626.1, observed mass-to-charge ratio=1625.8). FIG. 2(c) is a landscape-oriented chart showing a Matrix Assisted Laser Desorption/ESIIonization Time-of-Flight (MALDI-TOF) spectrum of the all L-enantiomer of a Rose Bengal-peptide conjugate, wherein the peptide has the amino acid sequence defined by SEQ ID NO. 2. Expected mass: 2721.6. FIG. 2(d) is a chart of 1,3-Diphenylisobenzofuran (DPBF) absorbance at 410 nm against time for solutions containing different substances and with or without ultrasound treatment as indicated by the key on the lower left of the chart. “RB” represents Rose Bengal, “Conjugate” represents a conjugate of Rose Bengal and a peptide having the amino acid sequence defined by SEQ ID NO. 2, “DPBF” represents 1,3-Diphenylisobenzofuran alone, “+US” represents ultrasound treatment;

(4) FIG. 3 is a bar chart showing cell viability following treatment as indicated on the x-axis. “RB” represents Rose Bengal, “+light” represents photodynamic therapy, “C(KLAKLAK).sub.2” (SEQ ID NO. 2) represents a peptide having the amino acid sequence defined by SEQ ID NO. 2, “RB-C(KLAKLAK).sub.2” (SEQ ID NO. 2) represents a conjugate of Rose Bengal and a peptide having the amino acid sequence defined by SEQ ID NO. 2, “RB-D(KLAKLAK).sub.2” (SEQ ID NO. 1) represents the all-D enantiomer of a peptide having the amino acid sequence defined by SEQ ID NO. 1. For each treatment group on the x-axis, counting from left, 1.sup.st column=0 μM concentration, 2.sup.nd column=2.5 μM concentration; 3.sup.rd column=5 μM concentration, and 4th column=10 μM concentration. Light conditions used: white light (22.8 J/cm2). These results show photodynamic therapy using the conjugate of Rose Bengal and a peptide having the amino acid sequence defined by SEQ ID NO. 2 was significantly better than photodynamic therapy using Rose Bengal alone, or the conjugate of Rose Bengal and a peptide having the amino acid sequence defined by SEQ ID NO. 2 alone, or a peptide having the amino acid sequence defined by SEQ ID NO. 2+light;

(5) FIGS. 4 (a), (b) and (c) are bioluminescent images of B16-F10-Luc2 melanoma cell cultures in 96-well tissue culture plates. Far left wells were untreated and far right wells treated with light only. The middle three rows were treated with 2.5 μM, 5.0 μM or 10 μM of the treatment listed below, plus light. Cells were treated with either (a) a peptide having the amino acid sequence defined by SEQ ID NO. 2; (b) Rose Bengal; or (c) a conjugate of Rose Bengal and a peptide having the sequence defined by SEQ ID NO. 2. The light conditions used were: white light (22.8 J/cm.sup.2). FIG. 4 (d) shows a bar chart representing the mean bioluminescent intensity from the three repeats of each condition expressed relative to the control. Lightest grey=light only; 2.sup.nd lightest grey=2.5 μM concentration; 3.sup.rd darkest grey=5.0 μM concentration; and black=10 μM concentration. “C(KLAKLAK).sub.2” (SEQ ID NO. 2) represents a peptide having the amino acid sequence defined by SEQ ID NO. 2. “RB” represents Rose Bengal. “RB-C(KLAKLAK).sub.2” (SEQ ID NO. 2) represents a conjugate of Rose Bengal and a peptide having the amino acid sequence defined by SEQ ID NO. 2. “+light” represents photodynamic therapy. These results show a significant reduction in bioluminescence following photodynamic therapy using a conjugate of Rose Bengal and a peptide having the amino acid sequence defined by SEQ ID NO. 2, compared to photodynamic therapy using Rose Bengal or irradiating the peptide having the amino acid sequence defined by SEQ ID NO. 2 alone with light;

(6) FIG. 5 shows landscape-oriented bar charts representing MTT B16-F10-Luc2 melanoma cell viability assay results, wherein “rb” represents Rose Bengal; “+us” represents sonodynamic therapy; “klaklak” (SEQ ID NO. 5) represents a peptide having the amino acid sequence defined by SEQ ID NO. 2; “rb klaklak” (SEQ ID NO. 5) represents a conjugate of Rose Bengal and a peptide having the amino acid sequence defined by SEQ ID NO. 2 wherein all chiral amino acids are the L-enantiomer; “rb d klaklak” (SEQ ID NO. 5) represents a conjugate of Rose Bengal and a peptide having the amino acid sequence defined by SEQ ID NO. 2 wherein all chiral amino acids are the D-enantiomer; “US conditions” represents ultrasound conditions; and “DC” represents duty cycle;

(7) FIG. 6(a) is a chart of tumour growth as a function of time for animals bearing orthotopic B16-Luc tumours and exposed to (i) no treatment (open squares) (ii) a conjugate of Rose Bengal and a peptide having the amino acid sequence defined by SEQ ID NO. 2 alone (open triangles) (iii) photodynamic therapy using Rose Bengal (open circles) and (iv) photodynamic therapy using a conjugate of Rose Bengal and a peptide having the amino acid sequence defined by SEQ ID NO. 2 (filled diamonds). FIG. 6(b) shows photographs of tumours excised 13 days following initial photodynamic therapy treatment using Rose Bengal (top) compared to photodynamic therapy treatment using a conjugate of Rose Bengal and a peptide having the amino acid sequence defined by SEQ ID NO. 2 (bottom). Results reveal significant reduction in tumour size for animals that underwent photodynamic therapy treatment using the a conjugate of Rose Bengal and a peptide having the amino acid sequence defined by SEQ ID NO. 2 relative to photodynamic therapy using Rose Bengal alone or the effect of the a conjugate of Rose Bengal and a peptide having the amino acid sequence defined by SEQ ID NO. 2 in the absence of light;

(8) FIG. 7 is a landscape-oriented line chart representing tumour growth as a percentage of the initial tumour size, over 8 days following treatment, for four different treatment groups and an untreated control; wherein “RB-Klaklak” (SEQ ID NO. 5) represents a conjugate of Rose Bengal and a peptide having the amino acid sequence defined by SEQ ID NO. 2; “+US” represents sonodynamic therapy; “+Light” represents photodynamic therapy; and “RB” represents Rose Bengal;

(9) FIG. 8 is a bar chart showing cell viability for breast cancer MCF-7 cells treated with either (i) Rose Bengal (RB) (ii) Rose Bengal+light (L+RB) (iii) Rose Bengal-C(KLAKLAK)2 (SEQ ID NO. 2) (RB-K2) (iv) Rose Bengal-C(KLAKLAK)2 (SEQ ID NO. 2)+light (L+RB-K2) (v) Rose Bengal-CKLAKLAK (SEQ ID NO. 6) (RB-K) (vi) Rose Bengal-CKLAKLAK (SEQ ID NO. 6)+Light (L+RB-K), wherein different bars reflect different concentrations (μM) as depicted in the figure inset;

(10) FIG. 9 is a bar chart showing cell viability for pancreatic cancer Panc-01 cells treated with either (i) Rose Bengal (RB) (ii) Rose Bengal+light (L+RB) (iii) Rose Bengal-C(KLAKLAK)2 (SEQ ID NO. 2) (RB-K2) (iv) Rose Bengal-C(KLAKLAK)2 (SEQ ID NO. 2)+light (L+RB-K2) (v) Rose Bengal-CKLAKLAK (SEQ ID NO. 6) (RB-K) (vi) Rose Bengal-CKLAKLAK (SEQ ID NO. 6)+Light (L+RB-K), wherein different bars reflect different concentrations (μM) as depicted in the figure inset; and

(11) FIG. 10 depicts the structure of RB-CKLAKLAK (SEQ ID NO. 6) (top) and positive electrospray mass spectrum of the RB-CKLAKLAK (SEQ ID NO. 6) (bottom).

EXAMPLES

(12) Embodiments of the present invention will now be described with reference to the following non-limiting examples:

(13) Materials

(14) In respect of each example described below: Rose Bengal was purchased from Sigma-Aldrich at the highest grade possible. All other chemicals were purchased from commercial sources at the highest possible purity and used as received. The B16-F10-Luc2 cell line was purchased from PerkinElmer. SCID and athymic nude mice were obtained from Envigo.

Example 1

Preparation of Rose Bengal-C(KLAKLAK).SUB.2 .(SEQ ID NO. 2) and Rose Bengal-C(KLAKLAK) (SEQ ID NO. 6) Conjugates

(15) The Rose Bengal-C(KLAKLAK).sub.2 (SEQ ID NO. 2) and Rose Bengal-C(KLAKLAK) (SEQ ID NO. 6) conjugates were prepared by first synthesising the C(KLAKLAK).sub.2 (SEQ ID NO. 2) or C(KLAKLAK) (SEQ ID NO. 6) peptide using Fmoc solid phase peptide synthesis on Rink Amide resin. In parallel, a carboxylic acid derivative of Rose Bengal was also prepared by reacting Rose Bengal with 1-bromooctanoic acid following a literature procedure (Fowley C et al. 2012). This carboxylic acid derivative was added to the N-terminus of C(KLAKLAK).sub.2 (SEQ ID NO. 2) or C(KLAKLAK) (SEQ ID NO. 6) while still on the resin using standard peptide coupling reagents (i.e. HOBt/TBTU). The conjugate was then cleaved from the resin and purified using preparative reverse phase HPLC. Product formation was confirmed using MALDI-TOF and positive electrospray mass spectrometry.

(16) RB-C(KLAKLAK).sub.2 (SEQ ID NO. 2) Conjugate: Chirality represents the structural difference between all-Levorotatory (L) and all-Dextrorotatory (D) form of RB-(KLAKLAK).sub.2 (SEQ ID NO. 1). The all-D enantiomer is postulated to be proficient in evading the body's immune-system and digestive enzymes, preventing proteolytic degradation and increasing the amount of active drug at the tumour site. Here, the all-L conjugate was modified to include an additional cysteine (C) amino acid to provide a further site of attachment via a Michael addition reaction if necessary. An all-D enantiomer RB-(KLAKLAK).sub.2 (SEQ ID NO. 1) was also prepared but did not possess cysteine functionality. The all-L enantiomer C(KLAKLAK).sub.2 (SEQ ID NO. 2) peptide i.e. minus RB was also prepared as a control.

(17) TABLE-US-00001 TABLE 1 The amino-acid sequence and differences of the two enantiomers of the conjugate. Amino- In text Acid Conjugate Abbreviation Chirality Length Amino acid sequence SEQ ID RB-C(KLAKLAK).sub.2 All-L Levorotatory 15 Cys-Lys-Leu-Ala-Lys- SEQ ID  Leu-Ala-Lys-Lys-Leu- NO: 2 Ala-Lys-Leu-Ala-Lys C(KLAKLAK).sub.2 All-L Levorotatory 15 Cys-Lys-Leu-Ala-Lys- SEQ ID  Leu-Ala-Lys-Lys-Leu- NO: 2 Ala-Lys-Leu-Ala-Lys RB-(KLAKLAK).sub.2 All-D Dextrorotatory 14 Lys-Leu-Ala-Lys-Leu- SEQ ID  Ala-Lys-Lys-Leu-Ala- NO: 1 Lys-Leu-Ala-Lys RB-C(KLAKLAK) All-L Levorotatory  8 Cys-Lys-Leu-Ala-Lys- SEQ ID  Leu-Ala-Lys NO: 6

(18) Synthesis of the all-L enantiomer of Rose Bengal-C(KLAKLAK).sub.2 (SEQ ID NO. 2) conjugate: The C(KLAKLAK).sub.2 (SEQ ID NO. 2) peptide was synthesised via a standard Fmoc solid phase peptide synthesis (SPPS) procedure using Rink amide MBHA resin. Piperidine was used for Fmoc de-protection while HOBt and HBTU were used to activate the amino acids. Carboxylic acid functionalised Rose Bengal (RB-COOH) was prepared following a literature procedure (Costley et al. 2017). This was attached to the N-terminus of the peptide while on the resin to produce the conjugate. The RB-C(KLAKLAK).sub.2 (SEQ ID NO. 2) conjugate was cleaved using a TFA:water:EDT:TIPS (94:2.5:2.5:1 v/v) cleavage cocktail and the crude peptide purified using RP-HPLC (C.sub.18 column, solvent A: 89.9% H.sub.2O, 10% ACN, 0.1% TFA; solvent B: 99.9% ACN, 0.1% TFA, gradient of 75% to 2% solvent A over a period of 60 minutes). Fractions (containing the peak of interest) were combined and freeze dried to afford the desired product as a red solid.

(19) Synthesis of the all L-enantiomer of C(KLAKLAK).sub.2 (SEQ ID NO. 2): The same procedure as described above for the synthesis of the all-L enantiomer was followed, except the peptide was cleaved from the resin before the addition of Rose Bengal. The HPLC purification conditions used were the same.

(20) Synthesis of the all-D enantiomer of the Rose Bengal-(KLAKLAK).sub.2 (SEQ ID NO. 1) conjugate: The same procedure as described above for the synthesis of the all-L enantiomer was followed, except D-amino acids were used and the cysteine residue not included. The HPLC purification conditions were the same.

(21) Synthesis of the all L-enantiomer of Rose Bengal-C(KLAKLAK) (SEQ ID NO. 6): The same procedure as described above for the synthesis of the all-L enantiomer was followed, except the peptide was cleaved from the resin before the addition of Rose Bengal. The HPLC purification conditions used were the same.

Example 2

The Ability of the Rose Bengal-C(KLAKLAK).SUB.2 .(SEQ ID NO. 2) Conjugate to Generate ROS

(22) The ability of the Rose Bengal-C(KLAKLAK).sub.2 (SEQ ID NO. 2) conjugate to generate reactive oxygen species (ROS) upon exposure to low intensity ultrasound was determined using the chromogenic ROS probe 1,3-diphenylisobenzofuran (DPBF). DPBF has an intense absorbance band centred at 410 nm in its native furan form but is readily bleached by ROS to the corresponding di-ketone. This conversion to the di-ketone is accompanied by a loss in absorbance at 410 nm that can be used to determine the amount of ROS produced. An EtOH:H.sub.2O (50:50 v/v) (10 mL) solution was prepared containing Rose Bengal or Rose Bengal-C(KLAKLAK).sub.2 (SEQ ID NO. 2) (0.5 μM) and 1,3-diphenylisobenzofuran (DPBF) (20 μM). The solutions were then irradiated for 30 min with ultrasound using a sonoporator (Sonidel SP 100 sonoporator; frequency 1 MHz, power density 3.0 W/cm.sup.2, 50% duty cycle, pulse repetition frequency 100 Hz). Aliquots were taken every 5 minutes and the absorbance at 410 nm was recorded using a ultraviolet-visible spectrometer (Cary 50 ultraviolet-visible (UV-Vis) spectrometer). Control experiments in the absence of drug (i.e. DPBF+stimulus) were also performed for comparative purposes (n=3). The results are shown in FIG. 2(d) and show a significant reduction in DPBF absorbance for both Rose Bengal, or Rose Bengal-C(KLAKLAK).sub.2 (SEQ ID NO. 2), treated with ultrasound relative to the controls indicating efficient ROS production in the ultrasonic field. In addition, the almost identical profile observed for both Rose Bengal and Rose Bengal-C(KLAKLAK).sub.2 (SEQ ID NO. 2) suggests the presence of the peptide does not inhibit ultrasound-induced ROS production by the sensitizer.

Example 3

The Effect of PDT Using a Rose Bengal-C(KLAKLAK).SUB.2 .(SEQ ID NO. 2) Conjugate on B16 Melanoma Cells

(23) Murine melanoma B16-F10 Luc-2 cells were maintained Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% foetal bovine serum (FBS) and 1% penicillin streptomycin. The cells were incubated at 37° C. in a humidified 5% (v/v) CO.sub.2 atmosphere. Cells were seeded at a concentration of 4×10.sup.3 cells per well in 96-well tissue culture plate for 24 hours. The medium was removed from each well and the cells incubated for 3 h with drug (100 μL) at concentrations of 2.5 μM, 5.0 μM and 10 μM in media. The drug solution was then removed and selected wells exposed to white light for 1 min (22.8 J/cm.sup.2). Fresh media was then added to each of the wells and cell viability determined 24 h later using a MTT assay. Each experiment was repeated on 3 separate occasions with an n=6 on each occasion.

(24) For the bioluminescent assay, imaging was undertaken using a Xenogen IVIS-100 bioluminescence/fluorescence optical imaging system. Treatments were performed as described above. D-Luciferin at 1.5% w/w was then added to treated wells and bioluminescence intensity was quantified 5 min later using the IVIS-100 system and Living Image Software as per protocol (Caliper LifeSciences). Regions of interest (RoI) were drawn around the wells to evaluate the intensity of signal emitted and expressed photons/second (p/s).

(25) The RB-C(KLAKLAK).sub.2 (SEQ ID NO. 2) conjugate (which is a conjugate of Rose Bengal and a peptide having the amino acid sequence defined by SEQ ID NO. 2) effectively killed B16-F10-Luc2 melanoma cells as part of PDT. The RB-C(KLAKLAK).sub.2 (SEQ ID NO. 2) L enantiomer had a more potent cytotoxic effect than the RB-C(KLAKLAK).sub.2 (SEQ ID NO. 2) D enantiomer as part of PDT in this experiment, suggesting that stereochemistry may be a factor.

(26) These results are presented in FIG. 3.

Example 4

The Effect of SDT Using a Rose Bengal-C(KLAKLAK).SUB.2 .(SEQ ID NO. 2) Conjugate on B16 Melanoma Cells

(27) Murine melanoma B16-F10 Luc-2 cells were maintained Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% foetal bovine serum (FBS) and 1% penicillin streptomycin. The cells were incubated at 37° C. in a humidified 5% (v/v) CO.sub.2 atmosphere. Cells were seeded at a concentration of 4×10.sup.3 cells per well in 96-well tissue culture plate for 24 hours. The medium was removed from each well and the cells incubated for 3 hours with drug (100 μL) at concentrations of 2.5 μM, 5.0 μM and 10 μM in media. The drug solution was then removed and selected wells exposed to white light for 1 min (22.8 J/cm.sup.2). Fresh media was then added to each of the wells and cell viability determined 24 hours later using a MTT assay (a colorimetric assay). Each experiment was repeated on 3 separate occasions with an n=6 on each occasion.

(28) For the bioluminescent assay, imaging was undertaken using a Xenogen IVIS-100 bioluminescence/fluorescence optical imaging system. Treatments were performed as described above. D-Luciferin at 1.5% w/w was then added to treated wells and bioluminescence intensity was quantified 5 minutes later using a small animal in vivo imaging system (IVIS-100 system) and image analysis software (Living Image Software; Caliper LifeSciences) as per the manufacturer's protocol. Regions of interest (RoI) were drawn around the wells to evaluate the intensity of signal emitted and expressed photons/second (p/s).

(29) The RB-C(KLAKLAK).sub.2 (SEQ ID NO. 2) conjugate (which is a conjugate of Rose Bengal and a peptide having the amino acid sequence defined by SEQ ID NO. 2) effectively killed B16-F10-Luc2 melanoma cells as part of SDT.

(30) These results are presented in FIG. 5.

Example 5

Effect of PDT Using a Rose Bengal-(KLAKLAK).SUB.2 .(SEQ ID NO. 1) Conjugate on B16 Melanoma Tumour Growth in a Mouse Model

(31) In these studies all animals were treated humanely and in accordance with licensed procedures under the UK Animals (Scientific Procedures) Act 1986. B16-F10-Luc2 cells (3×10.sup.5) maintained as described in Example 3, were re-suspended in 100 μL of PBS and implanted into the rear dorsum of SCID mice. Four days after implantation tumour measurements were taken using calipers and tumour volume calculated from the geometric mean diameter using the equation: tumour volume=4πR.sup.3/3. The animals were then randomly distributed into four groups (n=5): Group 1 involved untreated animals; Group 2 treated with the RB-(KLAKLAK).sub.2 (SEQ ID NO. 1) conjugate and no light; Group 3 treated with the RB-(KLAKLAK).sub.2 (SEQ ID NO. 1) conjugate and light; Group 4 treated with RB and light. Following induction of anaesthesia (intraperitoneal injection of Hypnorm/Hypnovel), a 100 μL aliquot of PBS containing 100 μM RB or the RB-(KLAKLAK).sub.2 (SEQ ID NO. 1) conjugate was administered by tail vein injection. Group 3 and Group 4: 30 minutes later tumours were exposed to white light for 3 min (68.4 J/cm.sup.−2). After treatment, animals were allowed to recover from anaesthesia and tumour volume was monitored for 12 days with animals retreated using the same conditions on days 1, 2, 7 and 9. The % increase in tumour volume was calculated employing the pre-treatment measurements for each group. On day 13, the animals were euthanized and the tumours were removed for analysis.

(32) The RB-C(KLAKLAK).sub.2 (SEQ ID NO. 2) conjugate (which is a conjugate of Rose Bengal and a peptide having the amino acid sequence defined by SEQ ID NO. 2) effectively reduced B16-F10-Luc2 melanoma tumour growth in mice as part of PDT or SDT. Use of the RB-C(KLAKLAK).sub.2 (SEQ ID NO. 2) conjugate as part of PDT inhibited tumour growth to a greater extent than use of the RB-C(KLAKLAK).sub.2 (SEQ ID NO. 2) conjugate as part of SDT in this experiment.

(33) These results are presented in FIG. 6.

Example 6

PDT and SDT Treatment of Ectopic B16 Melanoma Tumours in SCID Mice Using the Rose Bengal-(KLAKLAK).SUB.2 .(SEQ ID NO. 1) Conjugate

(34) B16-F10-Luc2 cells (3×10.sup.5) maintained as described in Example 3, were re-suspended in 100 μL of PBS and implanted into the rear dorsum of SCID mice. Four days after implantation tumour measurements were taken using calipers and tumour volume calculated from the geometric mean diameter using the equation: tumour volume=4πR.sup.3/3. The animals were then randomly distributed into four groups (n=5): Group 1 involved untreated animals; Group 2 treated with the RB-(KLAKLAK).sub.2 (SEQ ID NO. 1) conjugate and no light; Group 3 treated with the RB-(KLAKLAK).sub.2 (SEQ ID NO. 1) conjugate and light; Group 4 treated with the RB-(KLAKLAK).sub.2 (SEQ ID NO. 1) conjugate and SDT, and Group 5 treated with RB and light. Following induction of anaesthesia (intraperitoneal injection of Hypnorm/Hypnovel), a 100 μL aliquot of PBS containing 100 μM RB or the RB-(KLAKLAK).sub.2 (SEQ ID NO. 1) conjugate was administered by tail vein injection. Group 3 and Group 5: 30 minutes later tumours were exposed to white light for 3 min (68.4 J/cm.sup.−2). Group 4: on the same day tumours were exposed to ultrasound for 3.5 min (power: 3 W cm-2; Frequency 1 MHz; Duty cycle=30%; Pulse repetition 100 Hz). After treatment, animals were allowed to recover from anaesthesia and tumour volume was monitored for 12 days with animals retreated using the same conditions on days 1, 2, 7 and 9. The % increase in tumour volume was calculated employing the pre-treatment measurements for each group. On day 13, the animals were euthanized and the tumours were removed for analysis.

(35) The RB-(KLAKLAK).sub.2 (SEQ ID NO. 1) conjugate (which is a conjugate of Rose Bengal and a peptide having the amino acid sequence defined by SEQ ID NO. 1) effectively reduced B16-F10-Luc2 melanoma tumour growth in mice as part of PDT and SDT, although PDT was superior using this model system.

(36) These results are presented in FIG. 7.

Example 7

The Effect of PDT Using a Rose Bengal-C(KLAKLAK).SUB.2 .(SEQ ID NO. 2) Conjugate or a Rose Bengal-C(KLAKLAK) (SEQ ID NO. 6) Conjugate on MCF-7 Breast Cancer Cells

(37) Human MCF-7 invasive breast ductal carcinoma cells were maintained Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% foetal bovine serum (FBS) and 1% penicillin streptomycin. The cells were incubated at 37° C. in a humidified 5% (v/v) CO.sub.2 atmosphere. Cells were seeded at a concentration of 5×10.sup.3 cells per well in 96-well tissue culture plate for 24 hours. The medium was removed from each well and the cells incubated for 3 h with drug (100 μL) at concentrations of 0.05 μM, 0.1 μM, 0.5 μM and 1 μM in media. The drug solution was then removed and the cells washed twice with PBS, fresh media was added and selected wells exposed to white light for 1 min (22.8 J/cm.sup.2). Fresh media was then added to each of the wells and cell viability determined 24 h later using a MTT assay. Each experiment was repeated on 3 separate occasions with an n=6 on each occasion.

(38) The RB-C(KLAKLAK).sub.2 (SEQ ID NO. 2) conjugate (which is a conjugate of Rose Bengal and a peptide having the amino acid sequence defined by SEQ ID NO. 2) and the RB-C(KLAKLAK) (SEQ ID NO. 6) conjugate (which is a conjugate of Rose Bengal and a peptide having the amino acid sequence defined by SEQ ID NO. 6) effectively killed MCF-7 invasive breast ductal carcinoma cells as part of PDT.

(39) These results are presented in FIG. 8.

Example 8

The Effect of PDT Using a Rose Bengal-C(KLAKLAK).SUB.2 .(SEQ ID NO. 2) Conjugate or a Rose Bengal-C(KLAKLAK) (SEQ ID NO. 6) Conjugate on PANC-1 Pancreatic Cancer Cells

(40) Human PANC-1 epithelioid carcinoma cells were maintained Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% foetal bovine serum (FBS) and 1% penicillin streptomycin. The cells were incubated at 37° C. in a humidified 5% (v/v) CO.sub.2 atmosphere. Cells were seeded at a concentration of 5×10.sup.3 cells per well in 96-well tissue culture plate for 24 hours. The medium was removed from each well and the cells incubated for 3 h with drug (100 μL) at concentrations of 0.05 μM, 0.1 μM, 0.5 μM and 1 μM in media. The drug solution was then removed and the cells washed twice with PBS, fresh media was added and selected wells exposed to white light for 1 min (22.8 J/cm.sup.2). Fresh media was then added to each of the wells and cell viability determined 24 h later using a MTT assay. Each experiment was repeated on 3 separate occasions with an n=6 on each occasion.

(41) The RB-C(KLAKLAK).sub.2 (SEQ ID NO. 2) conjugate (which is a conjugate of Rose Bengal and a peptide having the amino acid sequence defined by SEQ ID NO. 2) and the RB-C(KLAKLAK) (SEQ ID NO. 6) conjugate (which is a conjugate of Rose Bengal and a peptide having the amino acid sequence defined by SEQ ID NO. 6) effectively killed PANC-1 epithelioid carcinoma cells as part of PDT.

(42) These results are presented in FIG. 9.

REFERENCES

(43) Costley, D., Nesbitt, H., Ternan, N., Dooley, J., Huang, Y. Y., Hamblin, M. R., McHale A. P., & Callan, J. F. (2017). Sonodynamic inactivation of Gram-positive and Gram-negative bacteria using a Rose Bengal-antimicrobial peptide conjugate. International Journal of Antimicrobial Agents.

(44) Fowley C, Nomikou N, McHale A P, McCarron P A, McCaughan B, Callan J F. Water soluble quantum dots as hydrophilic carriers and two-photon excited energy donors in photodynamic therapy. J Mater Chem 2012; 22,6456-62.