DISCONNECTION AGENTS

Abstract

The invention relates to the use of disconnection agents before, or together with, toxic agents for the treatment of solid tumours.

Claims

1. A method to identify a disconnection agent useful for the treatment of tumours, comprising the following steps: i. obtaining donor and acceptor cells, where the donor cells are distinguishable from the acceptor cells; ii. loading the donor cells with an intracellular marker substance that can be transferred from the donor cells to the acceptor cells via connections; iii. mixing the donor cells and the acceptor cells and culturing in the presence of a candidate disconnection agent, and optionally in the absence of a candidate disconnection agent; and iv. detecting the degree of transfer of the intracellular marker substance into the acceptor cells.

2. The method of claim 1, where the donor and acceptor cells are the same cell type.

3. The method of claim 2, where the donor and acceptor cells are non-tumour human cells.

4. The method of claim 3, where the cells are HepG2 cells.

5. The method of claim 2, where the cells are human tumour cells from the tumour type to be treated.

6. The method claim 5 where, the cells are glioblastoma-derived cells.

7. The method of claim 6, where the cells are U737 cells.

8. The method of any preceding claim, where the intracellular marker substance is Cell Tracker Green.

9. The method of any preceding claim, comprising labelling donor and/or acceptor cells, to obtain the donor cells and the acceptor cells which are distinguishable.

10. The method of any preceding claim, where the candidate disconnection agents is contacted with the donor and acceptor cells 1 to 6 hours after the donor and acceptor cells are mixed.

11. A method of treating a mammal with a tumour in need of such treatment comprising administration of a disconnection agent prior to, or concomitant with, administration of a toxic agent toxic to cells of the tumour.

12. The method of claim 11, where the disconnection agent is a modulator of Protein Kinase C activity.

13. The method of claim 12, where the disconnection agent is a stimulator of Protein Kinase C activity.

14. The method of claim 13, where the stimulator of Protein Kinase C activity is selected from: esters of phorbol (including PMA), bryostatin 1, bryostatin 2 and TPPB.

15. The method of any of claims 11-14, where the tumour type is not glioblastoma.

16. The method of any of claims 11-14, where the tumour type is glioblastoma.

17. The method of any of claims 11-14, where the tumour type is breast cancer, pancreatic cancer, lung cancer, liver cancer, stomach cancer or ovarian cancer.

18. The method of any of claims 11-17, where the disconnection agent is administered systemically.

19. The method of any of claims 11-17, where the disconnection agent is administered locally to the tumour.

20. The method of any of claims 11-19, where the disconnection agent is administered between 7 days and 1 hour prior to the administration of the toxic agent.

21. The method of any of claims 11-20, where the toxic agent is radiotherapy.

22. The method of any of claims 12-20, where the toxic agent is a chemotherapeutic agent.

23. The method of any of claims 11-20, where the toxic agent is an immunotherapeutic agent.

24. The method of claim 16, or any claim dependent on claim 16, where the disconnection agent is a stimulator of Protein Kinase C activity and the disconnection agent is administered between 7 days and 1 hour prior to the toxic agent.

25. The method of claim 24, where the stimulator of Protein Kinase C activity is selected from among the following list: esters of phorbol (including PMA), bryostatin 1, bryostatin 2 and TPPB.

26. A disconnection agent and a toxic agent for use in a method of treating a mammal with a tumour in need of such treatment, wherein the disconnection agent is administered prior to, or concomitant with, the toxic agent.

27. Use of a disconnection agent and a toxic agent in the manufacture of a medicament for a method of treating a mammal with a tumour in need of such treatment, wherein the disconnection agent is administered prior to, or concomitant with, the toxic agent.

28. A composition comprising a disconnection agent useful for the treatment of tumours, optionally wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, excipient or diluent.

29. A composition according to claim 28, comprising a toxic agent.

30. A combined preparation comprising a disconnection agent and a toxic agent.

31. A composition or combined preparation according to any of claims 28-30 where the disconnection agent is selected from among the following list: esters of phorbol (including PMA), bryostatin 1, bryostatin 2 and TPPB.

32. A composition or combined preparation according to any of claims 28-31, for use as a medicament.

33. A composition or combined preparation according to any of claims 28 to 31, for use in treating tumours.

34. The method of any of claims 1-10 further comprising the following steps: v. Identifying a disconnection agent which reduces or prevents transfer of the intracellular marker substance into the acceptor cells; vi. Producing a medicament consisting of or comprising the identified disconnection agent.

35. A disconnection agent produced by the method of claim 34.

Description

FIGURES

[0095] FIG. 1 shows an example of an SMS assay according to the invention. In the figure, “A” denotes an unstained cell, “B” denotes a cell dye-stained cell, “C” denotes a cell tracker-stained cell, and “D” denotes a cell dye- and cell tracker-stained cell. In step 1, stain cells with a cell dye and plate. In step 2, stain cells with a cell tracker dye. In step 3, harvest cell dye-stained cells and pre-treat each set of stained cells separately with compounds of interest. In step 4, combine sets of stained cells and plate with compounds of interest. In step 5, harvest cells, stain with viability dye and assess cell dye transfer by flow cytometry. In the graph, the x-axis is CellTracker Fluorescence, the y-axis is Calcein AM Fluorescence, and the relative position of cell dye positive cells, cell tracker positive cells and cell dye plus cell tracker double positive cells depicted. Graphic shows dye can be transferred from a cell dye-stained cell to a cell tracker-stained cell but cell tracker cannot be transferred from a cell tracker-stained cell to a cell dye-stained cell;

[0096] FIGS. 2A-2C show examples of PKCm-mediated inhibition of cell dye-sharing in the SMS assay. FIG. 2A left graph shows SMS in TPPB-treated Hs578T cells and right graph shows SMS in bryostatin-treated Hs578T cells. FIG. 2B left graph shows SMS in PMA-treated Hs578T cells and right graph shows SMS in indolactam V-treated Hs578T cells. FIG. 2C left graph shows SMS in bryostatin 1-treated HepG2 cells and right graph shows SMS in bryostatin 2-treated U373 cells. In each graph, x-axis is concentration (μM) and y-axis is percentage of acceptor cells (%). In FIGS. 2A and B, upper horizontal dotted line is vehicle, lower horizontal dotted line is PMA (5 nM). In FIG. 2C, upper horizontal line is vehicle, lower horizontal dotted line is PMA (10 nM);

[0097] FIGS. 2D-2E show examples of PKCm not interfering with cell dye-sharing in the SMS assay. FIG. 2D left graph shows SMS in FR 236924-treated Hs578T cells and right graph shows SMS in 6-(N-decylamino)-4-hydroxymethylindole-treated Hs578T cells. FIG. 2E shows SMS in SC-9-treated Hs578T cells. In each graph, x-axis is concentration (i.t.M) and y-axis is percentage of acceptor cells (%); while the upper horizontal dotted line is vehicle and the lower horizontal dotted line is PMA (5 nM);

[0098] FIG. 3 shows in vivo MPLSM images of tumours treated with TPPB and radiotherapy. Top row is control, with images (left to right) at D0, D7, D14, D21 (D7 after RTx), D28 (D14 after RTx), D35 (D21 after RTx) and D42 (D28 after RTx). Middle row is TPPB continuous dosing day 0-28, with columns as top row (except no D42 image). Bottom row is TPPB 3 doses days 13, 14, 15, with columns as top row (except no D21 image);

[0099] FIG. 4 is a graph indicating differences between continuous and intermittent TPPB treatment. Depicted are: (i) vehicle; (ii) TMZ 25; (iii), TMZ 50, (iv) TPPB 0.7 2× per week+TMZ 25; and (v) TPPB 0.2 QD+TMZ 25. X-axis is days after tumour implantation and y-axis is relative tumour volume; and

[0100] FIG. 5 shows in vivo MPLSM images of tumours treated with TPPB and radiotherapy (column D) versus control treatment with radiotherapy alone (columns A, B and C) at various times (days 0, 7, 21, 28, 35, 42, 70 and 91 after initiation of radiotherapy in rows 1-8, respectively). Block E highlights treatment leading to absence of tumours. Two randomly selected fields of view are shown from each p animal, but the same field of view is depicted in each row (at each timepoint).

EXAMPLES

Example 1: A Test for Disconnection Agents (SMS Assay)

[0101] The SMS assay was developed to assess the transfer of cell dye (for example the acetoxymethyl [AM]-based Calcein AM), between cancer cells. The SMS assay can be applied to any cell lines forming tumour networks such as tumour microtubes (TM) or tunneling nanotubes (TNT), which enable the transfer of molecules across cells. The SMS assay can be used to assess networking capabilities of any cancer cell lines including HepG2 hepatocellular carcinoma cells, Hs578T cell line for human breast cancer, and U373 cell line for human glioblastoma.

[0102] The SMS assay is illustrated in FIG. 1. For the SMS assay, two sets of cells are stained with the cell dye of choice and a customary cell tracker dye (for example cell tracker blue) using established staining techniques (steps 1 and 2). Each set of cells are then co-incubated with the compound of interest to assess its capabilities to modulate the cells small molecule sharing characteristics (step 3). Then the two sets of cells are combined on a plate, for example at a ratio of 1:1, and cultured in the presence of the compound of interest (Step 4).

[0103] Finally, live cells are evaluated for viability and analyzed by flow cytometry for the uptake of cell dye in the flow tracker-stained cells (Step 5). The comparison of cell tracker uptake against positive and negative control enables the assessment of the compound's ability to modulate the tumour networks including those formed by TM and TNTs

Example 2: Demonstration of Disconnection of Tumour Cells in Vitro Using PKC Modulators

[0104] The SMS assay has been applied to assess the modulation of network sharing capabilities of Protein Kinase C Modulators (PKCm) at various concentrations in the Hs578 cell line for human breast cancer, U373 cell line of human glioblastoma, and HepG2 hepatocellular carcinoma cells. PBS vehicle was used as negative control and the PKCm PHORBOL 12-MYRISTATE 13-ACETATE (PMA) at a concentration of 5 nM was used as the positive control. Surprisingly, the application of the PKCm TPPB, PMA, Bryostatin 1, Bryostatin 2, and Indolactam V in the SMS assay showed a marked inhibition of cell dye-sharing at various doses that are not cytotoxic in one or more cell lines. However, the PKCm FR236924, SC-9, 6-(N-Decylamino)-4-Hydroxymethylindole, and Ingenol-3-angelate did not have any effect on cell dye-sharing at a concentration range of 0.1 to 30 μM.

[0105] FIG. 2A and FIG. 2B show the percentage of cell tracker cells that were accepting the cell dye transfer from separately stained donor cells according to the SMS assay of Example. The percentage of acceptor cells detected is shown in comparison to vehicle (upper horizontal line) and PMA at 5-10 nM concentration (lower horizontal line). Active PKCm in the SMS assay (FIG. 2A) show a decrease in acceptor cells relative to vehicle at certain concentration. Non-active PKCm in the SMS assay (FIG. 2B) do not show a decrease of acceptor cells relative to vehicle. The upper limit of the tested concentrations was determined as the maximal non-cytotoxic dose.

[0106] The present invention thus identified TPPB, PMA, Bryostatin 1, Bryostatin 2, and Indolactam V as PKCm to disrupt tumour networks formed for example by TM and TNT. The invention extends to analogs of these compounds.

Example 3: Specificity of PKC Modulators Against PKC Families and Isozymes

[0107]

TABLE-US-00001 Family Enzyme Gene TPPB PMA Bryostatin 1 Indolactam V Ingenol cPKCs PKCα PRKCA Alpha ++ + + + + PKCβI/PKCβII PRKCB Beta + + (βI) ++ + PKCγ PRKCG Gamma + + + nPKCs PKCδ PRKCD Delta + + + ++ + PKCε PRKCE Epsilon + + + + + PKCη PRKCH Eta + + PKCθ PRKCQ Theta + aPKCs PKCζ PRKCZ Zeta PKC.Math./λ PRKCI Iota/Lamda +—activation, ++—strong activation, blanks—no data Protein Kinase C is a multifunctional protein kinase consisting of 9 isozymes that are clustered in 3 families of isozymes as depicted in the table above. PKCm are differentiated by their specificity to these PKC families and their isozymes. The differentiation of active and inactive PKCm in the SMS assay can be explained by differences in the activation of PKC families and individual isozymes. Such activated PKC families can be cPKC and/or nPKC. Such activated isozymes can be PKC alpha, beta, gamma, delta, epsilon, eta, and/or theta. The activity of a PKCm in the SMS assay depends on their potency against these targets, for example as noted below. Alexander et. al. (2012) suggest TPPB and Bryostatin 1 activate PKC-alpha, delta and epsilon. Other PKCs not investigated. Sharkey et al. (1984) demonstrated that DAG and PMA share a common binding site on PKC. This binding site (C1 domains; Giorgione et al. (2003)) on cPKCs and nPKCs is activated by PMA/DAG, however aPKC family isoforms have an atypical C1 domain that is activated by IP3 rather than DAG. Wender et. al (2011) demonstrate activation and translocation of PKCβI following Bryostatin 1 treatment in transfected CHO cells. No literature on the specificity of Bryostatin 2. Masuda et. al. (2002) show binding of Indolactam V to synthetic peptide analogues of the stated PKC isozyme C1 domains. Kedai et. al. (2004) provide inhibitor constants (K.sub.i) values for the binding of ingenol 3-angelate to the stated isozymes. Parker et. al. (2008) performed KD of PKC-alpha with siRNA in U87MG cells, found no consistent impact on cell death in complete growth media, but did see an impact on cell cycle progression (stuck in S1 phase). Shi et. Al. (2019) were able to develop a PD-L1.sup.KO, EGFRVIII.sup.+ U373 cell line by CRISPR/Cas9.

Example 4: Demonstration of Disconnection of Tumour Cells in Vivo Using TPPB and Radiotherapy in an Orthoptically Implanted Solid Brain Tumour

[0108] Tumour Implantation

[0109] 8-10 weeks old male NMRI nude mice were used for all studies with human primary brain tumour cells. Cranial window implantation in mice was done in a modification of what has been previously described (see References), including a custom-made titanium ring for painless head fixation during imaging. 2-3 weeks after cranial window implantation, 30,000 tumour cells of the patient-derived S24 glioblastoma model were stereotactically injected into the mouse brain at a depth of 500 μm. Tumours were injected on day minus 35, i.e., 35 days before treatment start. Treatment was started on day 0.

[0110] Treatment

[0111] Control animals (top row of FIG. 3) were untreated. TPPB continuous treatment (middle row) was administered intraperitoneally once per day on days 0 to 28 at variable daily doses ranging from 10 to 700 μg/kg. TPPB 3 doses (bottom row of FIG. 3) were administered intraperitoneally once per day on days 13, 14 and 15 at 200 μg/kg.

[0112] Radiation Treatment (RTx)

[0113] Tumours were irradiated with 7 Gy on three consecutive days (days 14, 15, 16, total dose 21 Gy) in regions matching in tumour cell density using a 6 MV linear accelerator with a 6 mm collimator (adjusted to the window size) at a dose rate of 3 Gy min.sup.−1 (Faxitron MultiRad225). The used radiation schedule is in the range of the commonly prescribed 60 Gy in 2 Gy. fractions for malignant glioma patients, assuming an α/β of ˜10 in the linear quadratic model and taking into account the radiation time of 3 days.

[0114] In Vivo Multiphoton Laser Scanning Microscopy (MPLSM) and Image Processing

[0115] MPLSM imaging was done with a Zeiss 7MP microscope (Zeiss) equipped with a Coherent Chameleon Ultrall laser (Coherent). MPLSM images were acquired by the ZEISS ZEN Software.

[0116] White to light gray areas of images in FIG. 3 depict S24 glioblastoma cells growing in the mouse brain. Black or dark gray areas depict vessels or background.

[0117] Results

[0118] Control treatment (top row) showed progressing S24 glioblastoma tumours in the mouse brain between days 0 and d7. Control animals had stable tumour burden between days 7 and 14, i.e., before radiotherapy (RTx), and stable tumour burden from days 28 to 42, i.e., after RTx on day 14. Tumour networks were identified as from day 7 and persisted through day 42, noticeable by the filament-like connections between the tumour cells.

[0119] TPPB continuous dosing (middle row of FIG. 3) showed no effect against control as described above. The tumour burden and connectivity between days 0 and 35 was similar to control. No images were taken on day 42.

[0120] Mice treated with 3 doses of TPPB on days 13, 14, and 15 had a similar tumour growth trajectory as untreated control animals on the pre-treatment days 0 to 14. Following treatment and RTx, mice treated with 3 doses of TPPB showed marked tumour regression on day 28 or 14 days after start of RTx. The tumour connectivity was markedly reduced on day 28. On days 35 and 42, i.e., 21 and 28 days after RTx, the S24 glioblastoma cells were barely noticeable, rounded, and completely disconnected amounting to complete remission from the tumour burden. No images were taken on day 21.

[0121] References:

[0122] Osswald, M.; Jung, E.; Sahm, F.; Solecki, G.; Venkataramani, V.; Blaes, J.; Weil, S.; Horstmann, H.; Wiestler, B.; Syed, M.; et al. Brain tumour cells interconnect to a functional and resistant network. Nature 2015, 528, 93-98.

Example 5: Demonstration of the Impact of Disconnection Using TPPB Followed by Chemotoxic Insult on a Solid Tumour in Vivo

[0123] Tumour Implantation

[0124] NMRI nude mice were subcutaneously implanted with patient-derived glioblastoma cells from the Glio10535 model (Orthmann et al.) on day 0.

[0125] Treatment

[0126] Animals were stratified into the treatment groups on day 6 after tumour implantation to achieve similar average tumour volumes per group. Minimal tumour sizes were 44 mm.sup.3 and maximal tumour sizes were 247 mm.sup.3. Vehicle animals (n=9) were treated with a mix of DMSO, Tween20 and PBS. Each 5 animals were orally dosed with temozolomide chemotherapy at 25 mg/kg (TMZ 25) and at 50 mg/kg (TMZ 50) on consecutive days per week. Each week represents one chemotherapy cycle of 5 treatment days and 2 off-treatment days. TPPB was dosed intraperitoneally in combination with 25 mg/kg oral temozolomide either continuously at 0.2 mg/kg (n=10) or intermittently (n=10) at 0.1, 0.4, and 0.7 mg/kg starting with the last dose of every TMZ 25 cycle and ending the day before the next treatment cycle. Before the start of chemotherapy, TPPB was titrated to its target dose.

[0127] Results

[0128] FIG. 4 shows the evolution of tumour volumes relative to treatment start on day 10 (relative tumour volume, RTV). By 38, when the majority of vehicle-treated animals reached critical tumour sizes, the anti-tumour effect achieved with intermittent TPPB dosing (TPPB 0.7 2× per week+TMZ 25 group) was additive to the effect of temozolomide 25 mg/kg monotherapy (TMZ 25), superior to continuous treatment with TPPB (TPPB 0.2 QD+TMZ 25 group), and similar to temozolomide 50 mg/kg monotherapy (TMZ 50).

[0129] The study confirmed that continuous dosing with TPPB has no effect over control, while intermittent dosing results in tumour growth inhibition.

[0130] References:

[0131] A. Orthmann , A. Hoffmann , R. Zeisig , J. Hayback, A. Jödicke, S. Kuhn, M. Linnebacher, J. Hoffmann, I. Fichtner, Therapeutic response to chemotherapeutic drugs of glioma-PDX and correlation to common mutations identified by panel sequencing, poster presentation (see https://www.epo-berlin.com/dokumente/2016_Poster_PAMM_Orthmann.pdf)

Example 6: Demonstration of Complete Cure of an Orthoptically Implanted Solid Brain Tumour in Mice Using a Disconnection Agent Followed by Radiotherapy in Accordance with the Method of the Invention

[0132] Method

[0133] Orthotopic brain tumours were induced in NMRI mice using human patient-derived S24 glioblastoma cells exactly as described in Example 4. Once significant tumours were established in vivo (approximately 35 days after implantation), as shown by intravital microscopy exactly as described in Example 4, mice received 4 intraperitoneal injections on day -1, day 6 and day 13 and day 20, with Control mice receiving vehicle only and Treated mice receiving TPPB at 200 μg/kg. All mice then received radiotherapy on days 0, 1 and 2 exactly as described in Example 4, except that the radiation dose was 6 Gy as opposed to 7 Gy on each occasion (18 Gy total radiation dose as opposed to 21 Gy total radiation dose). The impact of treatment on the tumour was then monitored by intravital microscopy at intervals after the initiation of radiotherapy on day 0.

[0134] Results

[0135] The results are shown in FIG. 5.

[0136] Mice that received control (vehicle) injections (columns A, B and C in FIG. 5), and were therefore not treated with a disconnection agent prior to radiotherapy in accordance with on aspect of the present invention, showed tumour progression throughout the study. Although there was an initial reduction in tumour cell number (white areas in each field of view in FIG. 5) immediately following radiotherapy, consistent with the toxic effect of the radiation, tumour burden subsequently increased throughout the observation period such that by day 91 after initiation of radiotherapy (row 8 in FIG. 5), the number of tumour cells had increased significantly in all fields of view from all animals. The progression observed in this study is consistent with reported studies using the same model (see references under Example 4), where progression is always seen following radiotherapy.

[0137] In marked contrast, mice that received active treatment with a disconnection agent (TPPB) initiated prior to radiotherapy, in accordance with one aspect of the present invention (column D in FIG. 5) showed a complete cure of the orthotopic solid brain tumour by day 91 after initiation of radiotherapy (row 8 in FIG. 5). By this time point, there were no detectable tumour cells (white areas in each field of view in FIG. 5) in any field of view. The only difference in treatment between the two groups was the use of a disconnection agent initiated prior to radiotherapy in the treated group (column D) compared to the control group (columns A,B and C) exactly in accordance with the method of the invention. A similar cure was observed among animals treated according to the method of the invention in Example 4. However, no other treatments (unrelated to the present invention) described in the literature have ever effected a cure in this model.

[0138] The impact of initiating treatment with a disconnection agent prior to radiotherapy was evident from day 35 after initiation of radiotherapy (row 5 in FIG. 5), when the tumour cells exhibited a marked change in morphology (in addition to the disconnection we observed, exactly as observed in Example 4), with a bright, punctate staining pattern visible at high magnification. It is likely that this represents the induction of apoptosis within the tumour cell population, which result in the complete absence of tumour cells observed at later timepoints (rows 6-8 in FIG. 5; highlighted as block “E”).

[0139] Treatment with a disconnection agent (TPPB) followed by radiotherapy, in accordance with the method of the invention, was well tolerated in all mice, with no behavioural or clinical signs associated with the treatment, and the mice remained well throughout the period of the study, in marked contrast to control mice that did not receive prior treatment with the disconnection agent, which increasingly demonstrated neurological symptoms consistent with the expanding tumour.