COMPOUND FOR PREVENTION OR TREATMENT OF A SKIN CANCER OR SKIN PRECANCER
20230218589 · 2023-07-13
Inventors
- James WELLS (St Lucia, Queensland, AU)
- Brian William DYMOCK (St Lucia, Queensland, AU)
- Andrew HARVEY (St Lucia, Queensland, AU)
- Terrie-Anne COCK (St Lucia, Queensland, AU)
- Rebecca POUWER (St Lucia, Queensland, AU)
- Kimberley BEAUMONT (St Lucia, Queensland, AU)
Cpc classification
A61K31/436
HUMAN NECESSITIES
A61K31/436
HUMAN NECESSITIES
A61K9/0021
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K9/0014
HUMAN NECESSITIES
International classification
A61K31/436
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
Abstract
The present invention relates, inter alia, to a method of preventing or treating skin cancer or skin precancer, the method comprising locally administering to the skin of a subject in need thereof an effective amount of a compound of Formula (I) or a pro-drug thereof, Formula (I) wherein the subject is being administered an immunosuppressant agent that binds to FKBP12. The agent that binds to FKBP12 may be tacrolimus. The invention also relates to methods of preventing or treating a skin condition, disorder or disease associated with administration of an immunosuppressant agent that binds to FKBP12, and to uses of the compound of Formula (I) in treating skin cancer or skin precancer, or a skin condition, disorder or disease associated with administration of an immunosuppressant agent that binds to FKBP12.
##STR00001##
Claims
1. A method of preventing or treating skin cancer or skin precancer, the method comprising locally administering to the skin of a subject in need thereof an effective amount of a compound of Formula (I) or a prodrug thereof, ##STR00012## wherein the subject is being administered an immunosuppressant agent that binds to FKBP12.
2. The method of claim 1, wherein the compound of Formula (I) is Compound 1: ##STR00013##
3. The method of claim 1, wherein substantially no compound of Formula (I), or prodrug thereof enters the blood stream after local administration to the skin of the subject.
4. The method of claim 1, wherein the subject is a human.
5. The method of claim 1, wherein the immunosuppressant agent that binds to FKBP12 forms a complex with FKBP12 and a further molecule.
6. The method of claim 1, wherein the immunosuppressant agent that binds to FKBP12 is selected from the group consisting of tacrolimus, sirolimus and everolimus.
7. The method of claim 6, wherein the immunosuppressant agent that binds to FKBP12 is tacrolimus.
8. The method of claim 1, wherein the subject is an organ transplant recipient.
9. The method of claim 8, wherein local administration of the compound of Formula (I), or prodrug thereof to the skin of the subject does not result in organ transplant rejection.
10. The method of claim 8, wherein the organ transplant recipient is being administered a combination of immunosuppressive agents which includes tacrolimus.
11. The method of claim 8, wherein the transplanted organ is selected from the group consisting of: liver, kidney, pancreas, heart, trachea, lung, face, intestine, eye, limb, cornea, bone and bone marrow.
12. The method of claim 1, wherein the method results in a reduction in the size or volume of a skin cancer or skin precancer, or results in eradication or elimination of a skin cancer or skin precancer.
13. The method of claim 1, wherein the skin cancer is a cutaneous squamous cell carcinoma (cSCC).
14. The method of claim 1, wherein the skin precancer is an actinic keratosis (AK), an intraepidermal carcinoma or Kaposi's sarcoma.
15. (canceled)
16. The method of claim 1, wherein the compound of Formula (I) or prodrug thereof is administered to the epidermis.
17. The method of claim 16, wherein the compound of Formula (I) or prodrug thereof is administered topically by rubbing or massaging a cream, ointment or salve containing the compound of Formula (I) or prodrug thereof onto the skin.
18. The method of claim 1, wherein the compound of Formula (I) or prodrug thereof is administered locally to the skin by intradermal injection.
19. (canceled)
20. A method of preventing or treating a skin condition, disorder or disease associated with administration of an immunosuppressant agent that binds to FKBP12, the method comprising locally administering to the skin of a subject in need thereof an effective amount of a compound of Formula (I) or a prodrug thereof, ##STR00014## wherein the subject is being administered an immunosuppressant agent that binds to FKBP12.
21. The method of claim 20, wherein the skin condition, disorder or disease is selected from the group consisting of: skin cancer, skin precancer, a fungal infection, a parasitic infection, a yeast infection, a viral infection, a bacterial infection, an inflammatory skin condition, a vascular skin condition and a benign skin lesion.
22. The method of claim 20, wherein the skin condition, disorder or disease is selected from the group consisting of: pruritis, folliculitis, onychopathy, a lesion or sore, poor wound healing, a rash, oedema, stomatitis, hair loss and hypertrichosis.
Description
BRIEF DESCRIPTION OF DRAWINGS
[0095] Examples of the invention will now be described by way of example with reference to the accompanying Figures, in which:
[0096]
[0097]
[0098]
[0099]
[0100]
[0101]
[0102]
[0103]
[0104] Preferred features, embodiments and variations of the invention may be discerned from the following Examples which provides sufficient information for those skilled in the art to perform the invention. The following Examples are not to be regarded as limiting the scope of the preceding Summary of the Invention in any way.
EXAMPLES
[0105] Examples of the present invention will now be described with reference to
Example 1
Synthesis of 17-Ethyl-1,14,20-trihydroxy-12-[2′-(4″-hydroxy-3″-methoxycyclohexyl)-1′-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-[22.3.1.0.SUP.4,9.]octacos-18-ene-2,3,10,16-tetraone (Compound I)
[0106] ##STR00011##
[0107] Reaction was performed as 5 parallel batches and combined for purification. To a stirred solution of 17-ethyl-1,14-dihydroxy-12-[2′-(4″-hydroxy-3″-methoxycyclohexyl)-1′-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-[22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tetraone (2.0 g, 2.5 mmol) (ascomycin, purchased from Angene Chemical) in a mixture of acetic acid (32 mL) and water (32 mL) was added selenium dioxide (0.42 g, 3.79 mmol) at ambient temperature. The reaction mixture was stirred for 16 h, then a further portion of selenium dioxide (0.42 g, 3.79 mmol) was added, and stirring was continued for 24 h. The reaction mixture was neutralised by the addition of saturated sodium hydrogen carbonate solution, and then extracted with ethyl acetate. The combined organic phase was dried over sodium sulfate and concentrated in vacuo. The residue was purified by silica gel column chromatography (dichloromethane/methanol) then reverse-phase column chromatography (C18 80 g column, PrepChrom C-700 Purification system, 0.1% formic acid in water/acetonitrile) to give 17-ethyl-1,14,20-trihydroxy-12-[2′-(4″-hydroxy-3″-methoxycyclohexyl)-1′-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-[22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tetraone (3.1 g, 30.4% yield for 5 combined parallel reactions) as a white solid. A portion of the material was further purified by prep-HPLC using the following conditions: Shimadzu UFLC XR, Column: Xterra Prep MS C18 OBD, 19×150 mm, 10 micron, Column temperature: ambient temperature, Mobile Phase A : H2O+0.05% Formic Acid, Mobile Phase B: acetonitrile, Flow rate: 15 mL/min, Mobile phase gradient details: T=0 min (95% A, 5% B); T=1 min (95% A, 5% B); T=9 min (50% A, 50% B); T=13.5 min (50% A, 50% B); T=13.6 min (5% A, 95% B), analysis time 15.5 min.
[0108] .sup.1H NMR (400 MHz, Chloroform-d) (selected signals) δ 5.64 (br d, J=9.0 Hz, 1H), 4.11 (br d, J=3.9 Hz, 1H).
[0109] LCMS: Rt=2.76 min, [M+Na]+=830, [M+NH.sub.4]+=825, [M+H]+=808. The sample was analysed using the following conditions: Shimadzu LCMS-2020 Nexera UHPLC, Column: Xterra MS-C18, 2.1×50 mm, 3.5 micron, Column temperature: 40° C., Mobile Phase A: H2O+0.05% Formic Acid, Mobile Phase B: acetonitrile, Mobile phase gradient details: T=0 min (95% A, 5% B); T=0.3 min (95% A, 5% B); gradient to T=3 min (5% A, 95% B); end of run at T=4 min (5% A, 95% B), Flow rate: 0.5 mL/min, analysis time 5.5 min.
Example 2
TR-FRET Assay (Carried Out by Selcia Discovery, UK)
[0110] A 384 well plate time resolved fluorescence resonance energy transfer (TR-FRET) assay was used to determine the effect of inhibitors to compete for tacrolimus (FK506) binding to the FKBP12 enzyme. The FKBP12 enzyme used in the assay is tagged with a polyhistidine sequence. An anti-6xHis antibody, labelled with a fluorescent donor, F(d), binds the tagged enzyme. The enzyme ligand, FK506 is tagged with a fluorescent acceptor, F(a) and binds the enzyme. When the components of the antibody/enzyme/ligand complex are in close proximity, excitation of the F(d) labelled antibody at a particular wavelength A results in F(a) emission at wavelength B due to non-radiative energy transfer. In the presence of a test inhibitor which competes for FK506, the complex is disrupt& and is no longer in close proximity, resulting in emission at wavelength A due to the F(d).
Assay Protocol for Determination of Binding of Compound 1 to the FKBP12 Enzyme
[0111] An 8-point dilution series was performed for Compound 1 and unlabelled tacrolimus (FK506—used as a control) over a concentration range of 0.001 nM to 10 μM. The inhibitors were added to the master mix in the assay plate containing the enzyme/antibody/ligand complex, with a final detergent concentration of 0.005%. The reaction was incubated for 30 min at room temperature and then read on a SpectraMax M5 (Molecular Devices) at wavelengths A (615 nm) and B (665 nm). The ratio between light emission (wavelength B/A) is calculated and the blank subtracted values were plotted against the inhibitor concentration in Logio molar and fitted using one site K.sub.i non-linear regression to determine the Kd of the bound test inhibitor.
[0112] Results: The Kd of the reference compound was determined to be 1.078 nM and the Kd of Compound 1 was 2.801 nM.
Example 3
Mouse T Cell Proliferation Assay
[0113] Compound preparation: Tacrolimus 5 mg/mL stock (FK506—LC Labs) was dissolved in 80% ethanol and Compound 1 10 mM stock was dissolved in DMSO.
Procedure:
[0114] Spleens, inguinal and brachial lymph nodes were harvested from two C57BL/6 mice in sterile conditions and placed in sterile Phosphate Buffered Saline (PBS), then cells were dissociated into a single cell suspension by gently crushing all organs through a 70 μm cell strainer. Cells were washed through the strainer into a 50 mL falcon tube with 5 mL sterile ACK lysis buffer (0.15 M NH.sub.4Cl, 1 mM KHCO.sub.3 pH 7.3). Cells were incubated for 3 min at room temperature to lyse red blood cells. 5 mL sterile PBS was added to the cell suspension and cells were centrifuged at 350 g for 10 min at room temperature. Supernatant was removed and the cell pellet resuspended in 10 mL sterile PBS. Cells were centrifuged again at 350 g for 10 min at room temperature. The supernatant was removed and the cells were resuspended in 1 mL of sterile PBS.
[0115] 1 μμL of 5 mM CellTrace Violet (Thermo Fisher) was added to the cell suspension and the mixture was swirled to ensure even dispersion of the dye. Cells were incubated for 20 min in the dark at room temperature. 5 mL of pre-warmed complete RPMI medium [(RPMI 1640 (Gibco), 10% heat inactivated FBS (Gibco), 1× Penicillin/Streptomycin/L-Glutamine, 100 μM 2-Mercaptoethanol] was added to the cell suspension and incubated in the dark at room temperature for 5 min. Cells were centrifuged at 350 g for 5 min at room temperature. Supernatant was removed without disrupting the cell pellet. Cells were resuspended in 1 mL complete RPMI medium.
[0116] Cells were counted using a haemocytometer and cells were adjusted to 2×10.sup.6 cells/mL using complete RPMI medium. 500 μL of cell suspension was added to wells in a 48-well plate (Nunc) so the final number of cells per well was 1×10.sup.6.
[0117] A vial of Dynabeads Mouse T Activator CD3/CD28 beads (4×10.sup.7 beads/mL) was vortexed for 30 sec and 625 μL of beads was aliquoted into a 5 mL polypropylene FACS tube (Sarstedt). 1 mL of complete RPMI medium was added to the beads and mixed by pipetting. The FACS tube was placed in a StemCell Technologies EasySep magnet for 1 minute and supernatant was discarded. The tube was removed from the magnet and washed beads were resuspended in 625 μL of complete RPMI medium. 12.5 μL of bead suspension (5×10.sup.5 beads) was added into appropriate wells of the 48-well plate with cell suspensions to induce T cell activation.
[0118] Tacrolimus or vehicle control was diluted in complete RPMI medium and added to appropriate wells with cell suspension for a final concentration of 0.6 ng/mL. Cells were incubated in a humidified incubator at 37° C. with 5% CO.sub.2 for 1 hour before Compound 1 (or vehicle only) was diluted in complete RPMI medium and added to the appropriate wells at the concentration required. The final volume in all wells was 1 mL. Cells were incubated for 3 days in a humidified incubator at 37° C. with 5% CO.sub.2.
[0119] Cells were harvested from each well by resuspending cells with a pipette and transferring to polypropylene FACS tubes. Any remaining cells were washed off wells using an additional 1 mL FACS buffer and transferred to appropriate FACS tubes. Cells were pipetted up and down to remove attached cells from CD3/CD28 beads. FACS tubes with cells were placed in a StemCell Technologies EasySep magnet for 1-2 min to separate magnetic CD3/CD28 beads from solution. Supernatant containing cells were transferred to appropriate polystyrene FACS tubes. 5 mL FACS buffer was added to FACS tubes and cells were centrifuged at 350 g for 5 min at 4° C. Supernatant was removed and cells were resuspended in 5 mL of FACS buffer (2% FBS in PBS) and centrifuged again at 350 g for 5 min at 4° C. Supernatant was removed leaving the cell pellet.
[0120] Mouse FC block (Biolegend) was diluted 1:200 in FACS buffer. 50 μL of diluted mouse FC block was added to each tube with cells. Tubes were incubated on ice in the dark for 15 min. A mix of fluorescent antibodies including CD45.2-PE-Cy7, TCRb-FITC and CD8b-APC (all Biolegend) was prepared at a 1:200 dilution in FACS buffer. After the 15 min incubation in FC block 50 μL of diluted antibody solution was added to each tube. Tubes were incubated on ice in the dark for 30 min. 5 mL of FACS buffer was added to each tube and tubes were centrifuged at 350 g for 5 min at 4° C. Supernatant was removed and cell pellet was resuspended in 500 μL of FACS buffer. 5 μL of 7AAD live/dead discrimination dye (Thermo Fisher) was added to each sample 15 min before acquiring samples on an LSR Fortessa X20. Data was analysed using FlowJo software.
[0121] Compound I (Cmpd 1) rescues mouse CD8+ T cell proliferation in vitro in the presence of tacrolimus: Lymphocytes from mouse spleen and lymph nodes were stained with Cell Trace Violet, then pre-incubated with 0.6 ng/mL tacrolimus (Tac) or vehicle control (No tacrolimus used in
[0122] Results depicted in
Example 4: Human T Cell Proliferation Assay
[0123] Compound preparation: Tacrolimus 5 mg/mL stock (FK506—LC Labs) was dissolved in 80% ethanol and Compound 1 (Cmpd 1) 10 mM stock was dissolved in DMSO. Cyclosporine A 1 mg/mL stock (CsA—Sigma) was dissolved in DMSO, Rapamycin 5 mg/mL stock (LC Labs) was dissolved in ethanol.
Procedure:
[0124] Under sterile conditions, a 96 well flat bottom plate with white opaque walls (Perkin Elmer) was coated with 10 μg per well of purified human anti-CD3 antibody (clone OKT-3; Biolegend) in a volume of 50 μL per well in sterile PBS. The plate was covered in parafilm and incubated overnight at 4° C. Just prior to addition of Peripheral Blood Mononuclear Cells (PBMCs), the antibody solution was removed and discarded using a multichannel pipette. Wells were washed by adding 200 μL sterile PBS to each well and incubating for 2 min. PBS wash solution was removed and discarded following incubation. The wash step was repeated once, for a total of two PBS washes.
[0125] 20 mL of blood from a consenting healthy volunteer was collected into lithium heparin vacuette containers (Greiner Bio-One). Under sterile conditions, blood from each container was pooled into a single tube and mixed thoroughly with 20 mL FACS buffer (2% FBS in 1×PBS). 15 mL Ficoll-Paque (GE Healthcare) was added to two 50 mL Falcon tubes, before 20 mL of whole blood/FACS buffer mixture was carefully and slowly dispensed on top of the Ficoll layer. Tubes were centrifuged at 800 g for 20 min at room temperature with the brake off. After centrifugation, the resulting PBMC layer between the plasma and Ficoll layers was carefully removed and transferred to a new 50 mL Falcon tube. Tubes were topped up to 45 mL with FACS buffer, then centrifuged at 500 g for 15 min. Supernatant was carefully removed without disturbing cell pellet, then PBMCs from both tubes were pooled. 30 mL of pre-warmed complete RPMI media [RPMI 1640 (Gibco), 10% heat inactivated FBS (Gibco), 1× Penicillin/Streptomycin/L-Glutamine (Gibco), 100 μM 2-Mercaptoethanol (Sigma)] was added to PBMCs, before centrifugation at 500 g for 15 min at room temperature. Supernatant was removed carefully without disturbing cell pellet. PBMCs were resuspended in 1 mL complete RPMI media.
[0126] PBMCs were counted manually using a haemocytometer and cell concentration was adjusted to 1.5×10.sup.6 cells/mL with complete RPMI media. 50 μL of cell suspension was added to appropriate wells of the 96 well flat bottom plate with white opaque walls pre-coated in human anti-CD3. The final number of cells per well was 7.5×10.sup.4.
[0127] Tacrolimus, Cyclosporine A, Rapamycin (sirolimus) or vehicle control was diluted in complete RPMI medium and added to appropriate wells with PBMC suspension at the concentration required. Cells were incubated in a humidified incubator at 37° C. with 5% CO.sub.2 for 1 hour before Compound 1 (or vehicle only) was diluted in complete RPMI medium and added to the appropriate wells at the concentration required. The final volume in all wells was 100 μL. Cells were incubated for 5 days in a humidified incubator at 37° C. with 5% CO.sub.2.
[0128] CellTiter-Glo Luminescent Cell Viability Assay (Promega) was performed to determine the level of metabolically active cells per well based on ATP quantification. CellTiter-Glo Buffer and lyophilised CellTiter-Glo Substrate were equilibrated to room temperature. CellTiter-Glo Substrate was reconstituted with 10 mL CellTiter-Glo Buffer and gently vortexed for 1 minute to create the CellTiter-Glo Reagent. The 96 well plate containing PBMCs was equilibrated to room temperature for 30 min. Once at room temperature, 100 μL of CellTiter-Glo Reagent was dispensed using a multichannel pipette into each well containing 100 μL cells and media. The plate was mixed for 2 min at room temperature on an orbital shaker to induce cell lysis. The plate was then incubated for 10 min at room temperature in the dark to stabilize the luminescence signal. Luminescence signal was recorded using a CLARIOstar Plus plate reader (BMG Labtech).
[0129] Compound I (Cmpd 1) rescues human T cell proliferation in vitro in the presence of tacrolimus: PBMCs isolated from human blood were pre-incubated with 0.6 ng/mL tacrolimus (Tac) or vehicle control (no tacrolimus used in
[0130] Results depicted in
Compound 1 (Cmpd 1) Rescues Human T Cell Proliferation In Vitro in the Presence of FKBP12-binding Rapamycin, but Not Cyclophilin-Binding Cyclosporine A:
[0131] PBMCs isolated from human blood were pre-incubated with 1 ng/mL rapamycin (Rapa), 50 ng/mL cyclosporine A (CsA) or vehicle control for 1 h before Compound 1 (Cmpd 1) was added at concentrations 0, 0.3 μM, 1 μM or 3 μM as indicated. PBMCs were stimulated with human anti-CD3 antibody and T cells were allowed to proliferate at 37° C. with 5% CO.sub.2 in a humidified incubator for 5 days. Proliferation/viability was assessed via a CellTiter-Glo Luminescent Cell Viability Assay and luminescence was read using a CLARIOstar Plus plate reader.
[0132] Results depicted in
Example 5
Mouse Tumour Model
[0133] Mice: All animal procedures were approved by the University of Queensland Animal Ethics Committee (approval no UQDI/512/17). K14HPV38E6/E7 mice, which express the E6 and E7 genes of Human Papillomavirus (HPV) type 38 under the control of the Keratin 14 promoter (Viarisio et al., “E6 and E7 from Beta HPV38 Cooperate with Ultraviolet Light in the Development of Actinic Keratosis-like Lesions and Squamous Cell Carcinoma in Mice.” PLoS Pathog. 2011 e1002125) were bred and maintained locally at the Translational Research Institute Biological Research Facility (Brisbane, Australia). All mice used were between 12 and 20 weeks and were housed under specific pathogen-free conditions.
[0134] Tacrolimus diet: All customised mice diet was manufactured by Specialty Feeds (Perth, Wash.). Briefly, tacrolimus (MedChemExpress) was mixed with caster sugar and then incorporated into standard mouse diet. 1.5 g of tacrolimus was mixed with 100 g of caster sugar and 9.9 kg of standard mouse diet to result in tacrolimus-diet (150 ppm). Food colouring was added to distinguish the drug. During the manufacturing process the pellets were air-dried overnight rather than dried in an oven in order to minimise the amount of heat applied. The final product was sealed in airtight bags and stored at 4° C. protected from light to ensure minimal degradation. Pellet volumes in feed hoppers were kept to a minimum and restocked every 3-4 days for the duration of the experiments.
[0135] Compound 1 preparation: Compound 1 (Cmpd 1) was prepared at the beginning of each dosing week as a 1 or 2 mg/mL solution as required in 4% ethanol/0.2% Tween-80/PBS. Compound 1 solution was stored at 4° C. for up to one week.
[0136] Mouse back cSCC tumour model: K14-HPV38-E6/E7 mice were randomised into groups based on body weight and age and were fed tacrolimus (150 ppm in the diet) for 7 days prior to tumour cell injection and throughout the study. HPV38-E6/E7 cells (cSCC cell line derived from UV-induced tumours from the mouse strain above) were cultured and passaged for 1 week prior to injection in complete F-12 media [3:1 v/v F-12 (Gibco) and DMEM high glucose (Gibco) medias supplemented with 5% heat inactivated FBS (Gibco), 0.4 μg/mL Hydrocortisone (Sigma), 5 μg/mL Insulin (Sigma), 8.4 ng/mL Cholera Toxin (Sigma), 10 ng/mL Human rEGF (Invitrogen), 24 μg/mL Adenine (Sigma), 1× Penicillin/Streptomycin/Glutamine (Gibco)]. Cells were washed twice in PBS prior to injection. Mice were injected with 1×10.sup.6 HPV38-E6/E7 SCC cells in PBS subcutaneously into the center of the shaved lower back in a 100 μL volume using a 30G syringe. Tumour size was monitored 3× weekly throughout the study using digital callipers. Once tumours reached approximately 0.05-0.1 cm.sup.3 mice were treated twice daily (as required) with intratumoural injections (40 μL) with Compound 1 or vehicle for up to 4 weeks or until euthanasia criteria were met. Mice were euthanased once tumours reached 1 cm.sup.3.
[0137] Mouse back 5117-RE tumour model: Method as per the above section (Mouse back cSCC tumour model), with the following changes; BALBc mice were used, and 5117-RE cells were cultured and passaged for 1 week prior to tumour cell injection in RPMI media (Gibco) supplemented with 10% heat inactivated FBS (Gibco) and 1× Penicillin/Streptomycin/Glutamine (Gibco).
[0138] CD8 T cell depletion: CD8b depleting antibody (BioXCell; clone 53-5.8) or isotype control antibody (BioXCell; clone HRPN) were administered by intraperitoneal injection on days 8, 15, and 22 post SCC challenge. 250 μg per mouse in 200 μL PBS was administered on days 8 and 15. 100 μg per mouse in 200 μL PBS was administered on day 22. Mice were bled on day 10 post cSCC challenge to check depletion efficiency via FACS.
[0139] Dissociation of tumour cells for FACS analysis: To release cells from tumours, harvested tissue was cut into small fragments and digested for 60 min at 37° C. in RPMI media containing 2% FBS, 3 mg/mL collagenase D and 5 ug/mL DNase I. Tissues were then gently pressed through a 70 μm cell strainer to create a single-cell suspension. Cells from each tumour were resuspended into 300 μL of RPMI media with 10% FCS, 1× Penicillin-Streptomycin-Glutamine (Gibco), 100 μM 2-Mercaptoethanol (Sigma), before staining with appropriate antibodies as described below.
[0140] Ex vivo stimulation and staining protocol Ji cytokine detection: 100 μL of each suspension of tumour dissociated cells was incubated in 96-well cell culture plates coated with CD3 antibody (clone 145-2C11—Biolegend) along with soluble CD28 antibody (2.5 μg/ml—clone 37.51, Biolegend) at 37° C. for 30 min. As a control (no stimulation), 100 μL of each suspension of tumour dissociated cells was also incubated an uncoated 96-well cell culture plate without soluble CD28 antibody and incubated at 37° C. for 30 min. After 30 min of incubation, 5 μg/mL Brefeldin A was added to all wells, and cells were incubated at 37° C. for a further 3.5 h. After a 4 h total incubation, cells were resuspended in FACS buffer with 5 μg/mL Brefeldin A (eBioscience)+Fc block (Purified Rat Anti-Mouse CD16/CD32: isotype Rat IgG2a, clone: 93, Biolegend) for 20 min on ice to block non-specific antibody staining. Monoclonal antibodies for surface staining (CD45.1-PE-Dazzle, TCRh-FITC, CD8a-PE-Cy7, CD4-Ax700) were subsequently added and incubated on ice for 30-40 min in concert with Live/Dead Aqua. Stain (Biolegend) to elucidate live cell populations. Cells were then resuspended in fixation buffer (eBioscience), and incubated in the dark at room temperature for 20 min. Cells were washed and resuspended in 1× Permeabilisation buffer (eBioscience)+intracellular antibodies including interferon gamma (IFNg-APC clone XMG1.2—eBioscience) and TNF alpha (TNFa-BV785 clone MP6-XT22—Biolegend). Cells were incubated in the dark at room temperature for 20 min.
[0141] CD59 staining protocol: 100 μL of tumour dissociated cells was resuspended in FACS buffer and incubated with Fe-block (Purified Rat Anti-Mouse CD16/CD32: isotype Rat IgG2a, clone: 93, Biolegend) for 20 min on ice to block non-specific antibody staining. Monoclonal antibodies for surface staining (CD45.1-PE-Dazzle. TCRb-FITC, CD8a-PE-Cy7, CD4-Ax700, CD69-APC; Biolegend) were subsequently added and incubated on ice for 30 min in concert with Live/Dead Aqua Stain (Biolegend) to elucidate live cell populations. Cells were then resuspended in fixation buffer and incubated at morn temperature for 20 min.
[0142] FACS analysis: Stained tumour dissociated cells were then washed twice, resuspended in FACS buffer and Flow cytometric analysis was performed using LSR Fortessa X20 (BD Biosciences) flow cytometers with FACSDiva. software (Becton Dickinson, Sparks, Md., USA). Data were exported and analyzed using FlowJo software (I'reestar Inc., Ashland, Oreg., USA).
[0143] Intra-tumoural injection of Compound 1 (Cmpd 1) significantly reduced tacrolimus-dependent cutaneous Squamous Cell Carcinoma (cSCC) tumour growth: K14-HPV38-E6/E7 mice were fed tacrolimus (150 ppm in the diet) throughout the experiment, beginning 7 days prior to tumour cell injection. Mice were injected sub-cutaneously with 1×10.sup.6 HPV38-E6/E7 SCC cells into the lower back and tumour size was monitored 3 times a week throughout the experiment. When tumours reached approximately 0.06 cm.sup.3 (Day 8), BID intra-tumoural (TT) injections were performed with 40 μL of 2 mg/mL Compound 1 or vehicle control for 12 days. Results depicted in
[0144] Intra-tumoural injection of Compound 1 (Cmpd 1) significantly increased cSCC tumour-infiltrating CD8 T cell activation and intracellular interferon gamma and TNF alpha: After 12 days BID IT injections of 2 mg/mL Compound 1 or vehicle control, 10 mice per treatment group were euthanised, tumours harvested, dissociated into single cells and stained for fluorescence activated cell sorting (FACS) analysis. Results depicted in
[0145] Depletion of CD8 T cells prevents Compound I mediated regression of tacrolimus-dependant cSCC tumours: K14-HPV38-E6/E7 mice were fed tacrolimus (150 ppm in the diet) throughout the experiment, beginning 7 days prior to tumour cell injection. Mice were injected sub-cutaneously with 1×10.sup.6 HPV38-E6/E7 SCC cells into the lower back and tumour size was monitored 3 times a week throughout the experiment. CD8 T cells were depleted on day 8 by the intraperitoneal injection of CD8b-depleting antibody (CD8b), and again on day 15 and 22 post SCC challenge. When tumours reached approximately 0.1 cm.sup.3 (Day 11), BID intra-tumoural (IT) injections were performed with 40 μL of 2 mg/mL Compound 1 or vehicle control for 3 weeks or until euthanasia criteria (tumour size 1 cm.sup.3—ethical limit) was reached. Results depicted in
[0146] Intra-tumoural injection of Compound I (Cmpd 1) significantly reduced tacrolimus-dependent spindle cell sarcoma (5117-RE) tumour growth: BALBc mice were fed tacrolimus (150 ppm in the diet) throughout the experiment, beginning 7 days prior to tumour cell injection. Mice were injected sub-cutaneously with 1×10.sup.6 5117-RE (spindle cell sarcoma) cells into the lower back and tumour size was monitored 3 times a week throughout the experiment. This cell line can be considered a model of Kaposi's sarcoma (KS), as KS is generally regarded to be a tumour of spindle cell lineage origin (Duman, Nephrology Dialysis Transplantation, 2002 https://doi.org/10.1093/ndt/17.5.892). When tumours reached approximately 0.1 cm.sup.3 (Day 11), BID intra-tumoural (IT) injections were performed with 40 μL of 2 mg/mL Compound 1 or vehicle control for 13 days. Results depicted in
[0147] Intra-tumoural injection of Compound 1 (Cmpd 1) significantly increased 5117-RE tumour-infiltrating CD8 T cells producing cytokines TNF alpha and interferon gamma: After 13 days BID IT injections with Compound 1 or vehicle control, 10 mice per treatment group were euthanised, 5117-RE tumours harvested, dissociated into single cells and stained for fluorescence activated cell sorting (FACS) analysis. Results depicted in
Intra-Tumoural Injection of Compound 1 (Cmpd 1) and cSCC Tumour Pharmacokinetics Indicates High Levels of Compound 1 in Tumours:
[0148] In another study of the mouse back tumour model, the concentration of Compound 1 and tacrolimus in SCC tumours was assessed at 1, 6, and 18 h post final dose. Table 1 below tabulates data demonstrating that average tumour concentrations of Compound 1 were more than 2000-fold that of tacrolimus over 18 h.
[0149] Tumour processing: The whole tumour was removed from the mouse. The weight of the tumour was recorded and tumour was placed into an appropriate cryovial on dry ice then transferred to a −80° C. freezer. 3 tumours per time point were assessed.
[0150] Sample processing: A LC/MS/MS based bioanalytical method was developed for the simultaneous detection and quantification of Compound 1 and tacrolimus in mouse tumours. Calibration standards and quality control samples were prepared by adding 2.5 μL of stock solutions of test compound of different concentrations into 25 μL of naive mouse blood or skin homogenates. Control samples were prepared by spiking 2.5 μL of water or acetonitrile into 25 μL of naïve mouse blood or skin homogenates. Tumour samples were homogenised in 1 mL of PBS then transferred into polypropylene Eppendorf tubes. 100 μL of 0.1 M zinc sulfate was added into the tubes, vortexed for 10 sec, and 250 μL of HPLC-grade acetonitrile containing internal standard (pimecrolimus) was added, vortexed for 2 min, and centrifuged for 3 min at 800×g. 20-40 μL of the supernatant was analysed by LCMS/MS. Instrument: Acquity UPLC, Waters. Column: Acquity BEH C18 100×2.1 mm, 1.7 micron; Mobile phase A: methanol; Mobile phase B: 5 mM ammonium acetate with 0.1% formic acid; Mobile phase gradient details: T=0 min (10% A, 90% B); T=0.01 min (10% A, 90% B); gradient to T=1.5 min (95% A, 5% B); T=3.2 min (95% A, 5% B). Flow rate: 0.3 mL/min, run time: 4.5 min; Ionisation mode: Electrospray ionisation (positive).
TABLE-US-00001 TABLE 1 Concentration of Compound 1 and Tacrolimus in Tumour Time Average Concentration Average Concentration (h) Compound 1 in tumour (ng/g) tacrolimus in tumour (ng/g) 1 164606 33 6 148276 73 18 168496 52
Example 6: Topical Application of Compound
[0151] The single dose pharmacokinetics of Compound 1 (Cmpd 1) was assessed by topical administration of the compound as a formulation in propylene glycol (3% concentration) applied once per day (QD) to one ear only of C57BL/6 female mice, 8 weeks of age (N=2 or 3 per timepoint; samples taken 1, 6 and 24 h post administration of Compound 1).
[0152] After a single topical dose of Compound 1 to the ear (10 μL of 3% solution in propylene glycol), measured concentrations of Compound 1 in mouse ears are between 22.8 to 30.7 μg/g over 24 hours. Blood concentrations of Compound 1 were all below the limit of quantitation (LOQ) for all samples (<3.5 ng/mL).
[0153] Table 2 below provides tabulated data showing the average concentration of Compound 1 quantified in mouse ears following a single topical administration of a 3% solution of Compound 1 in propylene glycol. The average concentration of Compound 1 was between 22.8 to 30.7 μg/mL over 24 h, with no compound detected in mouse blood down to the limit of quantification (LOQ) (3.5 ng/mL).
TABLE-US-00002 TABLE 2 Compound 1 quantified in mouse ears Time Concentration Average concentration (h) in blood (ng/mL) in ear (ng/g) 1 BLQ 28961 6 BLQ 30714 24 BLQ 22847 Limit of Quantitation (LOQ): 3.5 ng/mL; BLQ: Below Limit of Quantitation in blood
Methods:
[0154] Compound Application: Compound 1 (10 μL of a 3% solution in propylene glycol) was applied to mouse ears using a silicone brush. The brush was cleaned with distilled water and ethanol between applications. The vehicle solution was 100% propylene glycol.
[0155] Blood processing: Cryovials were prepared containing 10 μL 0.5 M EDTA and labelled appropriately. Cardiac bleed was performed on mice and blood transferred to an Eppendorf tube. Blood (110 μL) was transferred immediately to a cryovial containing EDTA and well mixed to prevent clotting. The cryovial was placed on dry ice and transferred to a −80° C. freezer.
[0156] Ear processing: Before taking ears for pharmacokinetics the skin was cleaned with distilled water and dried with cotton balls/swab before doing 2 tape strips (one piece of tape per strip). The whole ear was removed from the mouse. The weight of the whole ear was recorded and placed into an appropriate cryovial which was placed on dry ice then transferred to a −80° C. freezer.
[0157] Sample processing: A LC/MS/MS based bioanalytical method was developed for the detection and quantification of Compound 1 in mouse blood and ears. Calibration standards and quality control samples were prepared by adding 2.5 μL of stock solutions of test compound of different concentrations into 25 μL of naïve mouse blood or ear homogenates. Control samples were prepared by spiking 2.5 μL, of water or acetonitrile into 25 μL of naïve mouse blood or ear homogenates. The blood or ear samples were transferred into polypropylene Eppendorf tubes. 100 μL of 0.1 M zinc sulfate was added into the tubes, vortexed for 10 sec, and 250 μL, of HPLC-grade acetonitrile containing internal standard (pimecrolimus) was added, vortexed for 2 min, and centrifuged for 3 min at 800×g. 20-40 μL of the supernatant was analysed by LCMS/MS, using the same instrument, column, Mobile phase A, Mobile phase B, gradient and other parameters as outlined above for the tumour sample processing.
[0158] In compliance with the statute, the invention has been described in language more or less specific to structural or methodical features. It is to be understood that the invention is not limited to specific features shown or described since the means herein described comprises preferred forms of putting the invention into effect. The invention is, therefore, claimed in any of its forms or modifications within the proper scope of the appended claims appropriately interpreted by those skilled in the art.
CITATIONS
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