Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/66—Phosphorus compounds
  • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
  • A61K31/409—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having four such rings, e.g. porphine derivatives, bilirubin, biliverdine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
  • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
  • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
  • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7135—Compounds containing heavy metals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
  • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
  • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
  • A61K41/0076—PDT with expanded (metallo)porphyrins, i.e. having more than 20 ring atoms, e.g. texaphyrins, sapphyrins, hexaphyrins, pentaphyrins, porphocyanines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

• the present invention relates to treatment of cancer and to combinational therapies therefor.
• Bchl bacteriochlorophyll: Bchl-D: bacteriochlorophyll derivative
• BLI bioluminescence imaging
• Bpheid bacteriopheophorbide a (the C-17 -free carboxylic acid derived from Bphe without the central metal atom)
• CTX CY: cyclophosphamide
• DC dendritic cells
• DDW double distilled water
• GEM Gemzar, gemcitabine: GFP, Green Fluorescent Protein
• IVIS In Vivo Optical Imaging System
• Luc luciferin, luciferase
• IVIS In Vivo Optical Imaging System
• luc luciferase
• Ly6G lymphocyte antigen 6 complex is a 21-25kD glycosylphosphatidylinositol (GPI)-linked differentiation antigen that is expressed by myeloid-derived cell
• Ly6G lymphocyte antigen 6 complex is a 21-25kD glycosylphosphatidy
• Combination of therapeutic modalities that target the cancer and the immune suppressing cells constitute a promising avenue for controlling primary tumors growth and for preventing metastatic growth.
• MDSCs Myeloid-derived suppressor cells
• G-MDSC granulocytic
• M-MDSC monocytic
• MDSCs exhibit potent immunosuppressive activities in cancer. MDSCs infiltrate tumors and strongly inhibit cancer-specific cytotoxic T cells (Gabrilovich, 2017). Tumors have evolved to 'harness' these properties of MDSCs to restrain antitumor immunity and to promote tumor expansion in the surrounding environment and at distant sites, through effects on angiogenesis and metastasis. New therapies to restrain MDSC activity are crucial for the efficient control of tumor cells by immune responses (Condamine et al., 2015; Diaz-Montero et al., 2009; Gabrilovich, 2017; Marvel and Gabrilovich, 2015; Najjar and Finke, 2013; Quail, 2013 #18).
• Myeloid-derived suppressor cells of the mono or polymorphonuclear type promote immune suppression and thereby cancer cells evasion from anti-tumor immunity by neutralizing cytotoxic T cells through nitric oxide and peroxynitrite generation (Condamine et al., 2015; Gabrilovich and Nagaraj, 2009).
• the early differentiation of myeloid progenitor cells into MDSCs comes on the account of dendritic cells maturation (Gabrilovich, 2017) needed for professional presentation of tumor-associated antigens (TAAs), innate immunity and initiation of adaptive immunity response.
• TAAs tumor-associated antigens
• the MDSCs elevation augments immune suppression by blocking CD4 and CD8 T cell activation (Gabrilovich, 2017).
• This elevation has been primarily correlated with the secretion of cytokines such as G-CSF, GM-CSF, IL-6 and others from the cancer cells followed by autocrine stimulation through IL-6 and others from the MDSCs (Gabrilovich and Nagaraj, 2009).
• cytokines such as G-CSF, GM-CSF, IL-6 and others from the cancer cells followed by autocrine stimulation through IL-6 and others from the MDSCs (Gabrilovich and Nagaraj, 2009).
• the MDSCs provide protection to the primary tumors and circulating cancer cells from immune surveillance and toxicity and help establishing the tumor niche.
• MDSCs Multiple methods of inhibiting MDSCs are currently under investigation. These can broadly be categorized into methods that (a) promote differentiation of MDSC into mature, non-suppressive cells (all-trans retinoic acid (ATRA), vitamin D) (Najjar and Finke, 2013), (b) decrease MDSC levels (sunitinib, gemcitabine (GEM), flurouracil (f- 5U), bardoxolone methyl (CDDO-Me) (Najjar and Finke, 2013), or (c) functionally inhibit MDSC ( phosphodiesterase type 5 (PDE-5) inhibitors, cyclooxygenase 2 (COX-2) inhibitors) (Najjar and Finke, 2013).
• ATRA all-trans retinoic acid
• GEM gemcitabine
• f- 5U gemcitabine
• CDDO-Me bardoxolone methyl
• PDE-5 phosphodiesterase type 5
• COX-2 cyclooxygenase 2
• Gemcitabine (GEM, Gemzar), fluorouracil (f-5U) and other FDA-approved chemotherapeutic agents that are used for the treatment of a variety of cancers in the clinical arena, were found to selectively reduce the content of MDSC in both tumor- bearing mice and humans (Le et al., 2009; Suzuki et al., 2005; Vincent et al., 2010). However, despite a delay in cancer progression, none of these drugs was found to significantly increase time to metastases and patients survival (e.g. in treatment of pancreatic cancer).
• Photodynamic therapy (PDT) in general and vascular-targeted PDT (VTP) in particular using novel bacteriochlorophyll derivatives (hereinafter Bchl-D) were shown to selectively ablate localized solid tumors in different targets (WO 00/33833; WO 2004/045492).
• VTP with the Bchl-D designated WST11 after successful clinical trials (Azzouzi et al., 2013, 2017; Eymerit-Morin et al., 2013), has been recently approved for use in early stage prostate cancer treatment.
• Metronomic chemotherapy which was originally designed to inhibit angiogenesis, involves low-dose chemotherapeutic agents administered in a frequent regular schedule with no prolonged breaks and minimizes severe toxicities (Ge et al., 2012; Ghiringhelli et al., 2007). Such treatments were found to particularly impact pro tumor immune cells proliferation in a specific cell population manner.
• the present invention provides an anti-myeloid-derived suppressor cells agent (hereinafter “anti-MDSCs agent”) and a bacteriochlorophyll derivative (hereinafter “Bchl-D) for use in combination therapy for cancer, wherein the anti-MDSC agent and the Bchl-D are administered sequentially and the administration of the Bchl-D is followed by photodynamic therapy (PDT).
• anti-MDSCs agent an anti-myeloid-derived suppressor cells agent
• Bchl-D bacteriochlorophyll derivative
• the present invention provides a method for treatment of cancer by combination therapy comprising administering to a patient in need thereof: (i) a therapeutically effective amount of an anti-myeloid-derived suppressor cells (MDSCs) agent (hereinafter “anti-MDSCs agent”); and (ii) a therapeutically effective amount of a bacteriochlorophyll derivative (Bchl-D) followed by photodynamic therapy (PDT) (hereinafter “Bchl-D PDT").
• MDSCs anti-myeloid-derived suppressor cells
• Bchl-D PDT photodynamic therapy
• Fig. 1A shows in- vivo accumulation and clearance of STL-6014 (upper row) and STL-7012 (lower row) in mice bearing 4T1 breast tumor.
• Fig. IB shows the accumulation and/or retention of STL-6014 in tumor and different organs at 16 h post administration.
• Fig. 2 shows: (2A) The treatment Scheme 1 of mice bearing 4T1 tumors in their mammary pad using a combination of STL-6014 PDT and Gemzar administration starting either at two days prior PDT (blue arrows) or one day after PDT (orange arrows); (2B) Illuminated tumors at 6 h post-bolus injection of STL-6014; (2C) Luciferin bioluminescence of mice with primary and metastatic tumor (left), primary tumor only (middle) and cured mice (right).
• Fig. 3 shows the percentage of animals free of orthotopic 4T1 tumors at day 7 (blank) and day 30 (black) post one of five treatment regimens.
• STL-6014 PDT treatment was applied at day 7 post 4T1 grafting in the mammary pad and Gemzar (GEM) administration followed treatment Scheme 1 depicted in Fig. 2.
• GEM Gemzar
• Fig. 4 presents Kaplan-Meier survival curves for Balb/C mice bearing orthotopic 4T1 tumors and treated by the different therapeutic regimens described in Fig. 3.
• Fig. 5 depicts pie charts showing the percentage of animals found with 4T1 lung metastasis at the day of animals sacrifice following two different regimens of STL-6014 PDT combined with Gemzar.
• Fig. 6 illustrates 4T1 tumor progression in Balb/C mice that have been re- challenged by second orthotropic grafting of 2xl0 5 4T1 cells after being cured of 4T1 tumors by STL-6014 PDT combined with Gemzar given 2 days prior PDT. Re-challenge was performed at day 120 post first cure (240 days post first tumor grafting).
• Fig. 7 depicts treatment Scheme 2 for combined WST11 VTP and Gemzar administration for mice bearing MB49 (bladder) tumors.
• MB49 cancer cells (10 6 ) were grafted on the hind leg of Balb/C mice at day -15.
• First Gemzar administration (50mg/kg) was performed at day -3, then at day 4 and continued in cycles of 2 times/week, 3 weeks/month to a total of 12 treatments/90days.
• WST11 VTP was performed at day 15 post tumor grafting.
• WST11 (9mg/kg) was infused i.v. for 5 minutes followed by 10 minutes illumination at 753 nm using Modulight laser at 120mW/cm .
• Fig. 8 shows the response of mice grafted with MB49 tumors to different treatment regimens based on treatment Scheme 2 (Fig. 7).
• (8A) Mean tumor growth (error bars- SEM). Black-control (0/17 cured, p ⁇ 0.0001 vs VTP+ Gemzar); Blue-Gemzar alone (0/19 cured, p ⁇ 0.0001 vs VTP+Gemzar); Red- VTP alone (3/19 cured, p ⁇ 0.0005 vs VTP +Gemzar); Green- VTP+Gemzar (11/17 cured). P value calculations are based on two way Anova test.
• (8B) Tumor growth in the hind leg of individual mice following the above WST11 VTP treatment combinations.
• Fig. 9 shows the evolution of systemic anti-tumor response following different treatment regimens.
• MB 49 cancer cells were grafted on the right hind leg of Balb/C mice at day -15.
• (9A) First Gemzar administration (50mg/kg) was performed at day -3, then at day 4 and continued in cycles of 2 times/week, for 3 weeks. Then one week off and again a three weeks cycle to a total of 12 treatments/90days.
• WSTl l VTP was performed at day 15 post grafting with WSTl l at 9mg/kg infused i.v. for 5 minutes followed by 10 minutes illumination at 753 nm using Modulight laser at 120mW/cm .
• Second tumor was grafted at the hind left leg at day 1 post VTP.
• Fig. 10 shows that lack of T-cell and B-cell populations reduces the therapeutic effect of VTP, gemcitabine and combination thereof in MB49 tumors.
• Fig. 11 provides representative diagrams of immune cells populations in the spleen of 4Tl-bearing mice in non-treated (control) animals: (HA) Granulocytic/monocytic myeloid-derived suppressor cells (G/M-MDSCs) and neutrophils; (11B) Dendritic cells (DC); (11C) Tumor associated macrophages (TAMs); (11D) CD4 & CD8 T cells; (HE) Gating of T regulatory cells (Tregs).
• Fig. 12 illustrates the treatment impact of STL-6014-PDT combined with Gemzar administration as described by treatment Scheme 1 (depicted in Fig. 2) on the splenic immune cells populations in 4T1 -bearing mice at 1-3 weeks post treatment. (12A) G/M- MDSCs (blank), TAMs (black). (12B) DC (blank), CD8 (black); (12C) Tregs.
• Fig. 13_ shows changes in the spleen weight of animals orthotopically grafted with 4T1 cancer cells following at 1 and 3 weeks post no treatment of animal bearing tumors at 7 days post grafting treatment by STL-6014 PDT alone, treatment by Gemzar alone and combination treatment. Treatment scheme is illustrated in Fig 2.
• Fig. 14 depicts the innate immune cell population in the spleen of animals grafted with MB49 tumors in the hind leg and underwent no treatment (control) and animals that underwent WST11 VTP in combination with one of two Gemzar treatment regimens.
• the animals selected for FACS analysis were dosed by Gemzar at day -3, 1, 4, 7 at either low dose (60mg/kg, red) or high dose (120mg/kg, green) (** p ⁇ 0.01, * p ⁇ 0.05).
• Fig. 15 depicts the adaptive immune response of mice bearing MB49 tumors to different treatment regimens.
• Fig. 16 depicts the WST11 VTP treatment scheme given at day 7 after tumor grafting with and without immune modulation by cyclophosphamide (CTX) given at day 3 before VTP, in mice bearing 4T1 tumors in the hind leg.
• CX cyclophosphamide
• Fig. 17 illustrates the local response of individual Balb/C mice bearing 4T1 tumors in the hind leg, to different therapeutic regimens with a fixed dose 9.0 mg/kg WSTl l under different light intensities (mW/cm ) and CTX dosages.
• Fig. 18 presents Kaplan-Meier survival curves for Balb/c mice bearing 4Tl-luc breast tumors grafted s.c. at the hind leg and subjected to WST11 VTP alone using low light intensity (lOOmW/cm 2 ) (18A, 18B) or high light intensity (150 mW/cm 2 ) (18C, 18D) or in combination with single dose CTX (CY) administration (150 or 50 mg/kg) at three days prior VTP.
• Treatment results are illustrated for all treated mice (18A, 18C,18D) or for animals that underwent non-reversible full ablation of the primary tumor and survived with no metastases at day 90 (18D).
• Fig. 19 shows the impact of different treatment regimens on the percentage of animals with lung metastases in Balb/c mice bearing s.c. 4Tl-luc breast tumors in their hind leg.
• (19A) a Petri dish showing 4Tl-luc colonies evolved from cells isolated from the lungs of Balb/c mouse at day 7 post grafting of 4Tl-luc breast cancer cells at the hind leg (day of WST11 VTP).
• Fig. 20 shows Foxp3 positive cells (Treg cells) infiltration to 4Tl-luc tumors.
• 4T1 tumors grafted at the hind leg of Balb/C mice were excised when reaching 30-60 mm at indicated times post CTX or saline administration, formalin-fixed and paraffin embedded. Sections were prepared and stained for Foxp3 expression. Number of positive cells was measured using Fiji software.
• Fig 20A depicts representative pictures at day 7 post tumor grafting and quantification of untreated control
• Fig. 20B depicts representative picture of animals treated by CTX.
• Fig. 20C depicts the percentage of Foxp3 (Treg cells) in untreated animals, animals treated by CTX at 24 hours post treatment and of animals treated by CTX at 72 hours post treatment.
• Fig. 21 shows the effect of low dose CTX administration at 3 days prior WST11 VTP on T cell populations in draining lymph nodes and spleens of mice bearing 4Tl-luc tumors.
• Fig. 21A Total cell counts in draining lymph nodes (LN, left) and spleen (right). LN and spleens were harvested at indicated times after WST11 VTP with (black) and without (blank) CTX administration following treatment Scheme 4 as depicted in Fig. 16. Cells were isolated and stained for flow cytometry with anti-CD4, CD8 and Foxp3 antibodies. Total cells in spleens were counted after erythrocytes depletion.
• Fig. 21B The effect of low dose CTX administration at 3 days prior WST11 VTP on T cell populations in draining lymph nodes and spleens of mice bearing 4Tl-luc tumors.
• Fig. 21A Total cell counts in draining lymph nodes (LN, left) and spleen (right). L
• Fig. 21C Percentage of CD8+ (left), CD4+ (middle) and Foxp3 (Treg, right) in draining LN at different times after WSTl l VTP with (Black) or without CTX (blank) treatment at 3 days before VTP (*p ⁇ 0.001, **p ⁇ 0.05); Fig. 21C. Percentage of CD8+ (left), CD4+ (middle) and Foxp3 (Treg, right) in the spleen at different times after WSTl l VTP with (Black) or without CTX (blank) treatment at 3 days before VTP (*p ⁇ 0.001, **p ⁇ 0.001, ***p ⁇ 0.05)
• Fig. 22 depicts the effect of low dose CTX administration on myeloid cell populations in tumors and spleens of mice bearing s.c. 4Tl-luc tumors at the hind leg on 7 days post grafting (VTP day). Left-tumors; Right-spleen.
• Fig. 23 A shows treatment Scheme 5 for the combined treatment of mice bearing 4T1 tumors in the mammary pad using WSTl l VTP alone (at 7 days post grafting, when tumors reach diameters of 5-7 mm); WSTl l VTP combined with metronomic administration of CTX starting at 3 days prior VTP and given three more times (black arrows) (CTX 2); WSTl l VTP combined with one dose administration of CTX at 1 days prior VTP WSTl l (CTX 1) and three more times (red arrows); VTP combined with metronomic administration of Gemzar starting at two days (blue) or one day (yellow) prior VTP and continues at 5 days intervals for 3 more times.
• Fig. 23B presents Kaplan-Meier curves that illustrate survival of 4Tl-luc tumors bearing animals in the mammary after treatment by different protocols of WSTl l VTP combinations with CTX or Gemzar.
• Fig. 23C presents percentage of mice bearing 4T1 tumors in their mammary pad that were completely cured (blank), cured of primary tumor but developed metastases (dashed) and animals presented both recurrence of primary tumors and development of metastases (black) at day 90 after different treatment regimens.
• the invention relates to combinational therapies using bacteriochlorophyll-based photodynamic therapy (PDT) or vascular-targeted PDT (VTP) and immune cells, particularly myeloid-derived suppressor cells (MDSCs), modulating agents for the elimination of primary tumors and prevention of cancer dissemination.
• PDT bacteriochlorophyll-based photodynamic therapy
• VTP vascular-targeted PDT
• MDSCs myeloid-derived suppressor cells
• the present invention is directed to the non-thermal ablation of localized solid tumors and elimination of their remote micrometastases.
• the present invention provides an anti-myeloid-derived suppressor cells agent (hereinafter “anti-MDSCs agent”) and a bacteriochlorophyll derivative (hereinafter “Bchl-D) for use in combination therapy for cancer, wherein the anti-MDSC agent and the Bchl-D are administered sequentially and the administration of the Bchl-D is followed by photodynamic therapy (PDT).
• anti-MDSCs agent an anti-myeloid-derived suppressor cells agent
• Bchl-D bacteriochlorophyll derivative
• the combination therapy of the invention has enhanced therapeutic effect compared to the effect of the anti-MDSCs agent or of the Bchl-D followed by PDT, when each is administered alone.
• this enhanced therapeutic effect is a synergistic therapeutic effect, namely, much stronger than the additive therapeutic effects of the individual treatment modalities.
• the anti-MDSCs agent for use according to the invention may be, without being limited to, gemcitabine, 5-fluorouracyl (5-FU), cisplatin, paclitaxel, cyclophosphamide (CTX, CY), sunitinib, a cyclooxygenase 2 (COX-2) inhibitor such as SC-58236 and SC-58125, a bisphosphonate such as zoledronic acid or an aminobisphosphonate such as alendronate, all-trans retinoic acid (ATRA), vitamin D3, vitamin A, a KIT-specific antibody, a nitroaspirin derivative, a synthetic triterpenoid derivative such as bardoxolone methyl (known as CDDO-Me), and a phosphodiesterase-5 (PDE-5) inhibitor such as sildenafil and taladafil.
• 5-FU 5-fluorouracyl
• CX cyclophosphamide
• COX-2 cyclooxygenase
• the anti-MDSCs agent is gemcitabine or cyclopho sphamide .
• the Bchl-D for use according to the present invention is preferably a water-soluble anionic bacteriochlorophyll derivative optionally conjugated with an RGD-containing peptide or RGD-peptidomimetic residue.
• the Bchl-D is an anionic Bchl-D of the formula I:
• M represents 2H or Pd
• Ri is 0-R 4 or -NHR 5 , wherein R 4 is H, H + , an ammonium group or a monovalent metal cation such as Na + or K + and R5 is an RGD-containing peptide or RGD peptidomimetic residue;
• R 2 is -0-Ci-C 6 alkyl, preferably methyl
• R3 IS -NH-(CH 2 ) n -S0 3 ⁇ R6 + , wherein n is 2 or 3 and R 6 + is a monovalent metal cation such as Na + or K + ;
• the anionic Bchl-D for use in the invention has the formula I wherein Ri at position 17 is OR 4 , namely, it is not conjugated to an RGD- containing peptide or RGD peptidomimetic residue (hereinafter "non-conjugated Bchl- D") such as, but not limited to, the Bchl-Ds herein designated WST11 and STL-7012.
• WSTl l the rhodobacteriochlorin derivative Palladium B ⁇ oxo-lS- methoxycarbonylmethyl-rhodobacteriochlorin 13 1 -(2-sulfoethyl) amide dipotassium salt, was synthesized in the laboratory of Prof. Avigdor Scherz, a co-inventor in the present application, at the Weizmann Institute of Sciences (Rehovot, Israel) and disclosed in WO 2004/045492. After successful clinical trials, WSTl l has been recently approved for use in early stage prostate cancer treatment.
• STL-7012 is the non-metalated Bchl-D B ⁇ oxo-lS-methoxycarbonylmethyl- Pvhodobacteriochlorin lS ⁇ -sulfoethyl) amide dipotassium salt (disclosed in WO 2008/023378, formula in Appendix hereinafter).
• Bchl-Ds that can be used according to the invention include the following compounds disclosed in WO 2004/045492: Palladium S ⁇ oxo-lS- methoxycarbonylmethyl-rhodobacteriochlorin ⁇ -(S-sulfopropyl) amide dipotassium salt; B ⁇ oxo-lS-methoxycarbonylmethyl-rhodobacteriochlorin 13 1 -(2-sulfoethyl) amide dipotassium salt; B ⁇ oxo-lS-methoxycarbonylmethyl-rhodobacteriochlorin 13 x -(3- sulfopropyl) amide dipotassium salt; and Palladium B ⁇ oxo-lS-methoxycarbonylmethyl- rhodobacteriochlorin 13 1 -(2-sulfoethyl) amide potassium salt.
• the PDT is vascular- targeted PDT (VTP) and an area of the local to be treated is illuminated at a short period after the administration of the non-conjugated Bchl-D is completed. This period is usually of 0-30 min, for example, 0, 10, 15, 20, 25 or 30 min.
• the Bchl-D for use in the invention is a Bchl-D formula I conjugated to an RGD-containing peptide or RGD peptidomimetic residue, has the formula I wherein Ri at position 17 is NH-R5, namely, it is conjugated to an C(hereinafter "conjugated Bchl-D").
• the RGD-containing peptide or RGD peptidomimetic residue may be a non-cyclic or cyclic peptide.
• the conjugated Bchl-D for use in the invention is conjugated to a cyclic RGD-containing peptide or RGD peptidomimetic residue.
• Bchl-Ds conjugated with cyclic RGD-containing peptide or RGD peptidomimetic residues for use in the present invention include, but are not limited to, those disclosed in the publications WO 2008/023378 and WO 2010/046900 such as the Bchl-Ds herein designated STL-6014, STL-6033, STL-6038, and STL-6068 (structures presented in Appendix hereinafter).
• the conjugated Bch-D is STL-6014, the 3 l -oxo-
• conjugated Bchl-Ds disclosed in WO 2008/023378 that can be used according to the invention include, but are not limited to: Palladium B ⁇ oxo-lS-methoxycarbonylmethyl-Rhodobacteriochlorin 13 1 -(2- sulfoethyl)amide-17 -(cycloRGDfK) amide potassium salt
• the PDT is tissue- targeted and an area of the local for treatment is illuminated after some time, to allow accumulation and optimal concentration of the conjugated Bchl-D in the targeted tissue.
• This time may be of at least 4h, preferably 6 h, after the administration of the conjugated Bchl-D is completeed.
• the anti-MDSC agent and the Bchl-D for use according to the invention are administered sequentially according to several different regimens.
• the PDT or VTP treatment comprises a sole administration of the Bchl-D followed by illumination of an area of the local to be treated
• the anti-MDSC treatment comprises several administrations of the anti-MDSC agent at various determined time intervals. If necessary, the session may be repeated one or more times if and when a new metastasis is found later on.
• the anti-MDSC agent is administered once before the PDT or VTP treatment and several times thereafter at determined time intervals.
• the anti-MDSC agent is administered several times at determined time intervals after the PDT or VTP treatment.
• the anti-MDSC treatment may comprise from 4 to 12 administrations or more of the anti-MDSC agent at determined time intervals of 5 to 12 days or more.
• the anti-MDSC treatment may comprise 4, 5, 6, 7, 8, 9, 10, 11 or 12 or more administrations of the anti-MDSC agent at determined time intervals of 5, 6, 7, 8, 9, 10, 11 or 12 days or more.
• the treatment protocol may be modified by the physician by changing the number of anti-MDSCs administrations and/or changing the number of days of the determined time interval between the anti-MDSCs administrations.
• the physician may determine to make a pause in the treatment and provide during the treatment a longer interval than the determined interval and then reassume the original protocol after this break.
• the physician may decide to make a break of 10 to 20 days or more after the 2 nd or 3 rd administration, and then go back to the determined interval of 5 days for the following administrations. This break interval is always longer than the determined protocol interval.
• the anti-MDSC agent is gemcitabine and PDT is performed with the Bchl-D STL-6014. 18. In other embodiments, the anti-MDSC agent is gemcitabine and VTP is performed with the Bchl-D WST11.
• the anti-MDSC agent is cyclophosphamide and VTP is performed with the Bchl-D WST11.
• the anti-MDSC agent is administered in a low dose such as a dose 3 or 4 times lower than the conventional dose of the agent in conventional monochemotherapy.
• a low dose such as a dose 3 or 4 times lower than the conventional dose of the agent in conventional monochemotherapy.
• cancer solid tumors and metastases including but not limited to, melanoma, renal cell carcinoma, colon, breast, lung, prostate, bladder, brain, adenocarcinoma of the pancreas or head and neck tumors.
• the invention further relates to an anti-MDSC agent for use in combination therapy with a Bchl-D for the treatment of cancer, wherein the anti-MDSC agent and the Bchl-D are administered sequentially and administration of the Bchl-D is followed by photodynamic therapy (PDT).
• PDT photodynamic therapy
• the invention additionally relates to a Bchl-D for use in combination therapy with an anti-MDSC agent for the treatment of cancer, wherein the anti-MDSC agent and the Bchl-D are administered sequentially and administration of the Bchl-D is followed by photodynamic therapy (PDT).
• PDT photodynamic therapy
• the combination therapy has enhanced therapeutic effect compared to the effect of the anti-MDSCs agent or the Bchl-D followed by PDT, when each of them is administered alone. This enhanced therapeutic effect may be a synergistic therapeutic effect.
• the present invention provides a method for treatment of cancer by combination therapy comprising administering to a patient in need thereof: (i) a therapeutically effective amount of an anti-myeloid-derived suppressor cells (MDSCs) agent (hereinafter “anti-MDSCs agent”); and (ii) a therapeutically effective amount of a bacteriochlorophyll derivative (Bchl-D) followed by photodynamic therapy (PDT) (hereinafter “Bchl-D PDT").
• MDSCs anti-myeloid-derived suppressor cells
• Bchl-D PDT photodynamic therapy
• the combination therapy of the method of the invention provides an enhanced therapeutic effect compared to the effect of the anti-MDSCs agent or the Bchl-D PDT, each administered alone.
• This enhanced effect may be a synergistic therapeutic effect
• the present invention synchronizes administration of chemotherapeutic agents that function as anti-MDSCs with cell/tissue-directed photodynamic therapy (PDT) or vascular-targeted photodynamic therapy (VTP) using different chemical derivatives of bacteriochlorophyll (Bchl-D).
• PDT cell/tissue-directed photodynamic therapy
• VTP vascular-targeted photodynamic therapy
• Bchl-D different chemical derivatives of bacteriochlorophyll
• Low dose administration of the chemotherapeutic agent to the treated subject several times at determined time intervals, achieves reduction in the MDSCs load.
• PDT or VTP using Bchl-D is applied to ablate the primary tumors or observed metastases.
• metronomic application weekly application of low doses continues for several weeks thereafter.
• Treatment efficacy is monitored by, but not limited to, follow up of primary tumor ablation using different imaging techniques (e.g. MRI, ultra-sound), histology (e.g. biopsies), reduction of MDSC counts in the circulation, delay or elimination of metastases growth and prolonged subjects survival.
• imaging techniques e.g. MRI, ultra-sound
• histology e.g. biopsies
• the treatment can be repeated later on in cases where new metastases are found in the patient.
• the present invention provides a method of treating cancer, said method comprising administering to a patient in need thereof: (i) a therapeutically effective amount of an anti-myeloid-derived suppressor cells (MDSCs) agent (hereinafter “anti-MDSCs agent”); and (ii) a therapeutically effective amount of a bacteriochlorophyll derivative (Bchl-D) followed by photodynamic therapy (PDT) (hereinafter “Bchl-D PDT”), or vascular-targeted PDT (hereinafter “Bchl-D VTP”) to provide a combination therapy having an enhanced therapeutic effect compared to the effect of the anti-MDSCs agent or the Bchl-D PDT each administered alone.
• MDSCs anti-myeloid-derived suppressor cells
• Bchl-D PDT bacteriochlorophyll derivative
• Bchl-D VTP vascular-targeted PDT
• treating and “treatment” or the phrase “to treat” as used herein refers to any type of treatment that imparts a benefit to a patient afflicted with a cancer, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the condition, prolongation of survival time, etc.
• therapeutically effective amount refers to an amount that is therapeutically effective in the treatment of cancer as defined hereinabove when used in the combination therapy and administered in repeated doses according to the invention.
• therapeutically effective amount refers to its capability of causing ablation of the tumor or of the metastasis after performance of the PDT or VTP.
• the immune modulated Bchl-D PDT or VTP can be repeated since new metastases may indicate the occurrence of new micrometastases of different clone compared with the primary tumor.
• Working solution was prepared by dissolving STL-6014 K + salt in 5% mannitol solution (lmg/ml), the pH of the solution was adjusted to 8 by addition of tris buffer (0.15%) in 5% mannitol, the solution was aliquoted to several tubes based on the experiment demands, frozen and lyophilized to give formulated STL-6014 K + salt. Concentration was verified by spectrophotometric measurement. STL-7012 was provided as dipotassium salt.
• Working solution was prepared by dissolving STL-7012 K + salt in 5% mannitol solution (lmg/ml). Adjustment of pH, preparation of aliquots and verification of concentration were performed as for STL-6014.
• WST11 was supplied as a powder and kept in the dark at - 20°C. Stock was prepared by dissolving WST11 in DDW containing 5% glucose and 0.67% mannitol, aliquoted and lyophilized. Working solution was reconstituted with DDW and the concentration of solution was confirmed spectrophotometrically.
• Clinical WST11 (batch: P00611, and 10-130611) was supplied as lyophilized formulation. Material was dissolved in 5% dextrose, aliquoted and stored at - 20°C. At the day of treatment the aliquot was thawed, filtered and the concentration of solution was confirmed spectrophotometrically.
• Chemotherapeutic drugs preparation and storage- Gemcitabine (GEM, Gemzar ® , Lilly) was purchased from a local pharmacy as a powder, dissolved in saline to a concentration of 38 mg/ml, aliquoted and stored at -20 °C. Aliquots were thawed, diluted with saline to a desired concentration and used on the same day at a dose of 75mg/kg.
• Cyclophosphamide (CTX, Endoxan ® , Baxter Oncology Gmbh, Germany) was purchased from a local pharmacy. A stock solution of 20 mg/ml was prepared by dissolving in 0.9% NaCl, aliquoted and stored at -20°C. Aliquots were thawed, diluted with saline if needed to a desired concentration and used on the same day prepared at indicated doses.
• Murine cell lines - Mammary luciferase (luc)-labeled 4T1 cell line (4T1-Luc) was a kind gift from Dr. Zvi Granot (Faculty of Medicine, The Hebrew University of Jerusalem, Israel); melanoma B 16-F10 cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA); bladder MB49 cell line was a kind gift from James Allison (MSKCC, New York, USA), and luc-labeled MB49-cell line was established using the murine stem cell virus (MSCV)-puromycin-luc-GFP construct (kindly provided by Dr. Emily Cheng, Human Oncology and Pathogenesis Program, MSKCC).
• MSCV murine stem cell virus
• 4T1 and MB49 cells were maintained in DMEM medium and B 16-F10 cells in RPMI medium supplemented with 1 mmol/L sodium pyruvate, 10% fetal calf serum (FCS), 250 ⁇ g/ml hygromycin, 0.06 mg/ml penicillin and 0.1 mg/ml streptomycin.
• FCS fetal calf serum
• the cells were grown as monolayer at 37°C in a humidified atmosphere (5% C0 2 , 95% air).
• Murine tumor models Female, Balb/c mice (6-8 week old) were grafted either subcutaneously (s.c.) into the right hind leg or orthotopically with lxlO 6 4T1 cells suspended in 50 or ⁇ PBS. For the rechallenge study, 2xl0 5 4T1 cells were orthotopically grafted. For bladder cancer model, 5xl0 4 of MB49 or MB49-luciferase cell line were s.c. injected into the right flank of C57B/6 male mice (7-8 weeks old, Taconic, Hudson, NY) 15 days prior to VTP treatment.
• mice 4T1-Luc cells were grafted in the mammary pad of Balb/c mice.
• mice When primary tumors reached ⁇ 50mm , mice were i.v. injected with 7.5 mg/kg STL-6014 and left under dim light conditions for 6h, after which an area of 1 cm including the tumor was illuminated with a 753 nm laser, applied through frontal face optical fibers (Medlight SA, Switzerland) at 200 mW/cm .
• the mice received analgesia (2.5 mg/kg Flunexin, once daily).
• Ablation of primary tumors was assessed every 7 days by imaging the tumor's luciferase-mediated bioluminescence signal, using IVIS. Mice were sacrificed (according to the guidelines of the Weizmann Institute of Science) when tumors reached the diameter of 15 mm, or when the mouse developed metastases, determined by the IVIS whole-body fluorescence.
• VTP treatment protocol For s.c. tumor treatment Tumor-bearing mice were anesthetized by i.p. injection of a mixture of Ketamine/Xylazine solution 100 mg/10 mg per kg body weight, respectively. WSTl l was then administered at a dose of 9 mg/kg by 5 min constant rate i.v. infusion into the tail vein. Illumination at fluency rate of 100 or 150 mW/cm" for 10 min was initiated immediately after infusion completion using a 4W, 755 nm diode laser (Ceramoptec, Germany) equipped with frontal optical diffuser. Mouse was placed on the left side to expose the tumor and covered with non-transparent material to protect normal tissues.
• mice were anesthetized as before.
• WSTl l dose was 9 mg/kg given by 5min infusion.
• Mouse was placed on the back, the tumor was lifted with a clip to separate it from the body and the animal was shielded with black material to prevent vital organ damage.
• Illumination at fluency rate of 200 mW/cm for 15 min was initiated immediately after infusion completion using a 4W, 755 nm diode laser (Modulight, Finland) equipped with frontal optical diffuser.
• CTX cyclophosphamide
• Ly6G staining (PDT studies): Mice bearing orthotopic 4T1-Luc tumors at two sites (right and left mammary fat pad) were subjected to PDT only on one side, combined with the second GEM treatment protocol. Tumors were excised 24 h, 3 days and 3 weeks post-PDT, and fixed and stained for Ly6G. Images were acquired for offline analysis using a slide scanner.
• Flow cytometry FACS analysis: Spleens were excised from humanly sacrificed animals and strained through 70 ⁇ mesh. Red blood cells were lysed with ammonium chloride lysis solution (ACK) and cells counted and adjusted for staining. Lymph nodes were minced in PBS, washed and adjusted for staining. Tumors were cut to small pieces and incubated for 30min at 37°C with shaking in the mixture of 2.5mg Collagenase II, 2.5mg CoUagenase IV and 0.5mg DNAase per 1ml 1% BSA containing PBS.
• ACK ammonium chloride lysis solution
• cells were subsequently incubated for 30min at RT with fluorescent antibodies including percp-cy5.5/APC-Cy7-CDl lb, PE/PerCP-Cy5-Ly6G, PE-cy7-Ly6C, APC-F4/80, Alexa488/PE-CDl lc, eVolve655/v450-MHCII, percp-cy5.5- CD3, APC/v450-CD4, Alexa488-CD8, PE-cy7-Foxp3, CD4-APC, Alexa488/PerCP- Cy5/PE-Texas Red-CD8 and PE/APC-Foxp3, Alexa 700/FITC-CD45, FITC-Ki67, APC- Cy7-CD25, PE Texas Red-Granzyme B, PE-CD62L, PE-Cy7-CD44, and APC-CD86 (the antibodies were purchased from eBioScience (San Diego, CA and from BioLe
• FIG. 1A lower panel
• Fig. 1A lower panel
• WST11 Data not shown
• Fig. IB Upper panel-bioluminescence of different tissues in the tumor- bearing mice; lower panel-fluorescence of STL-6014 in these organs.
• mice were inoculated with lxlO 6 4T1-Luc cells in the mammary fat pad.
• mice bearing 4T1-Luc could be subjected to one of several treatment options as described by treatment scheme 1 (Fig.2A) as follows:
• Balb/c bearing 4T1 tumor were divided in 5 groups: (1) non-treated (control); (2) treatment with STL-6014 PDT; (3) treatment with metronomic Gemzar: (4) treatment with STL-6014 PDT and metronomic Gemzar starting 1 day post PDT; and (5) treatment with STL-6014 PDT and metronomic Gemzar starting 2 days prior to PDT.
• Fig. 3 shows primary tumor responses at day 7 (white) and 30 (black) post STL-6014 PDT alone or in combination with Gemzar provided in one of the two treatment modalities described in treatment Scheme 1. Absence of primary tumor was observed in 70-80% of mice treated by STL-6014 PDT alone or in combination with Gemzar at day 7 post treatment.
• Fig. 4 shows the Kaplan-Meier survival curves over 120 days follow up of 4T1-Luc bearing mice subjected to different treatment regimens and controls.
• Fig. 6B shows the survival of individual animals re-challenged with a second grafting of 2xl0 5 4T1 cells at 120 days after they underwent complete cure from first grafted tumor as evident by the lack of 4T1 cell's bioluminescence at day 120 post first grafting.
• Fig. 7 depicts a second treatment scheme applied herein to MB49 MB49 (mice bladder cancer) bearing Balb/C mice when tumor reached about 4-7 mm in diameter at day 15 of grafting 2xl0 5 MB49 cells in the right hind leg.
• the treatment scheme comprises application of WSTl l VTP alone, or ip administration of 50mg/kg Gemzar at day 12 post grafting followed by 3 weeks cycles of Gemzar administration at day 1 and 7 of each week, followed by no administration at week 4 of each month. All together, animals received 12 Gemzar administrations.
• 9.0 mg/kg WSTl l was I.V. infused for 5 minutes followed by 10 minutes illumination at 753 nm using Modulight laser at 120m W/cm 2 .
• Example 8 Response of mice grafted with MB49 tumors to different regimens of treatment with WSTll VTP and Gemzar
• Figs. 8A-D show the response of Balb/C mice grafted with MB49 tumors to the different treatment regimens described in Fig. 7.
• Figs. 8A-8B show the average and individual MB49 tumor growth, respectively, in the mouse hind leg following the treatment with the above WSTl l VTP/Gemzar combination (error bars, SEM control vs combination therapy p ⁇ 0.0001, Gemzar vs combination therapy p ⁇ 0.0001, VTP vs combination therapy p ⁇ 0.0005 (Two way Anova test)). All control animals had to be sacrificed because of tumor burden at 20-25 days post grafting.
• FIG. 8C depicts the prevention of lung distant metastases (imaging, A103+A112) at day 25 in response to non-treatment (control) and treatment by the different treatment regimens (Gemzar, VTP or VTP+Gemzar), monitored weekly by IVIS bioluminescence imaging.
• Example 9 Evolution of adaptive anti-tumor immunity as reflected in the metastatic evolution and re-challenge success in animals treated by WSTll VTP and Gemzar
• Fig. 9A depicts the treatment Scheme 3.
• VTP was performed when at day 15 post grafting, tumors reached 4-7 mm in diameter (day 0). Mice were challenged the following day after VTP ablation with a second injection of MB49 on the left hind leg.
• Gemzar was administered weekly (3 week cycle, 3 week off) at 50 mg/kg to the cohorts of Gemzar and combination up to 90 days starting 3 days prior to VTP.
• Fig. 9B relates to second tumor growth in individual animals of a bilateral model.
• Fig. 9C presents Kaplan-Meier surviving mice that at day 125 post VTP treatment (the last Gemzar dose was given at day 90) were re- challenged via tail vein injection of MB49 cell line. All of untreated naive mice succumbed to death by 35 days after injection of MB49 cells while 70% of mice survived after WST11 VTP or WST11 VTP/Gemzar rejected the tumor cells.
• Example 10 The impact of T and B cells deficiency on the response of tumor bearing mice to treatment with WST11 VTP and Gemzar
• Examples 11-18 below provide insight to the innate and immune response of tumor-bearing animals to the different treatment protocols described in the present application.
• Example 11 Immune cells population in the spleen of non-treated Balb/C mice
• Fig. 11 FACS analyses of immune cells populations in the spleen of control (no treatment) 4T1 -bearing mice was carried out as described in Material and Methods hereinabove. The results are shown in Fig. 11: (A) Granulocytic/monocytic myeloid- derived suppressor cells (G/M-MDSCs) and neutrophils; (B) Dendritic cells (DC); (C) Tumor associated macrophages (TAMs); (D) CD4 & CD8 T cells; (E) T regulatory cells (Tregs).
• G/M-MDSCs Granulocytic/monocytic myeloid- derived suppressor cells
• DC Dendritic cells
• TAMs Tumor associated macrophages
• TAMs Tumor associated macrophages
• D CD4 & CD8 T cells
• Regs T regulatory cells
• Example 12 The treatment impact of STL-6014-PDT combined with Gemzar administration on the immune cell profile in spleen of 4T1 -bearing mice.
• Fig. 12 presents the treatment impact of STL-6014-PDT combined with Gemzar administration on the immune cell profile in spleen of 4T1 -bearing mice.
• the profile of splenic cell populations was assessed following 3 treatment regimens: STL-6014 PDT, STL-6014 PDT with Gemzar (starting 2 days before PDT), and Gemzar alone.
• Treated groups were compared to tumor-bearing controls. Animals with good treatment responses (as judged by primary tumor ablation at 1, 2, and 3 weeks post-treatment) were taken for analysis. Splenocytic cells were stained for innate and adaptive immune markers and analyzed by LSRII flow cytometer (BD Bioscience, San Jose, CA) and analyzed using FlowJo software (Tree Star, Ashland, OR).
• Example 13 Impact of WST11 VTP and Gemzar on the spleen of treated animals.
• Fig. 13 compares changes in the spleen weight of animals orthotopically grafted with 4T1 cancer cells and not treated (control) with tumor bearing animals that underwent Gemzar treatment, WST11 VTP treatment or their combination.
• the six fold increase in the spleen weight found in non-treated animals is completely prevented by VTP and VTP+Gemzar and almost completely avoided by Gemzar alone.
• Combining with data presented in Figs 11 and 12 the spleen weight increase is mainly due to increased population of granulocytes and monocytes which is greatly reduced by the VTP+Gemzar treatment.
• Example 14 The impact of WSTll VTP combined with Gemzar administration on the evolving of anti-tumor innate immunity in 49MB tumor bearing mice
• Fig. 14 The Innate immune response to different therapeutic regimens of mice bearing MB49 tumors is depicted in Fig. 14.
• IHC Imunohistochemical staining of CDl lb+ in the spleen shows that both VTP and gemcitabine alone depletes CDl lb+ cells in the spleen but the combined treatment has much stronger effect.
• B FACS analysis on splenocytic cells of the treated animals shows that at day 9 post treatment neutrophils/G- MDSC and monocytes/M-MDSC are depleted systemically by WSTl l VTP alone or gemcitabine treatment. Here, the combination didn't lower myeloid population suggesting that VTP and Gemzar might target the same cell population.
• Dosing schedule of Gemzar for flow was days -3, 1, 4, 7 at either low dose (60mg/kg) or high dose (120mg/kg). Importantly, in the 4T1 mice models the combinational treatment had more profound effect that each of the individual treatment modalities.
• Example 15 The impact of WSTll VTP combined with Gemzar administration on the evolving of anti-tumor adaptive immunity
• VTP/Gem combination decreased Treg population and significantly increased the Teff population in the tumor. Enhancement of Teff/Treg value was maintained at day 9 post treatment. The level of central memory T cell (T CM ) was increased by combination in tumors at day 6 and 9 post VTP. This population has been reported to confer superior protection against cancer in several different model systems compared with T EM cells.
• Example 16 Treatment scheme for WSTll VTP combined with low dose application of cyclophosphamide (CTX).
• CX cyclophosphamide
• Fig. 16 illustrates the treatment scheme for WST11 VTP combined with low dose application of cyclophosphamide (CTX) as immune modulator for treatment of 4T1 tumor bearing mice.
• Cyclophosphamide e.g. 50/150 mg/kg
• CX cyclophosphamide
• Example 17 Impact of low and high dose cyclophosphamide (CTX) administration on WSTll VTP-mediated ablation of subcutaneous (s.c.) 4Tl-luc breast tumors grafted in the mouse hind leg
• CX cyclophosphamide
• Figs. 17 and 18 depict the impact of low and high dose cyclophosphamide (CTX) administration on WSTl l VTP-mediated ablation of subcutaneous (s.c.) 4Tl-luc breast tumors grafted in the mouse hind leg.
• Mice bearing 4Tl-luc breast tumors at the hind leg were untreated or subjected to a single CTX administration alone (upper panel); treated by WSTl l (VTP at low or high light intensity (middle panel); or treated by WSTl l (9.0 mg WSTl l/kg) VTP combined with single dose CTX (50 or 150 mg/kg) given three days before VTP (lower panel).
• CTX cyclophosphamide
• Example 18 Impact of different treatment regimens on the percentage animals with lung metastases in Balb/c mice bearing s.c. 4Tl-luc breast tumors.
• Example 19 The impact of CTX on the population of regulatory T cells (Tregs) in grafted tumors.
• CTX was previously shown to attenuate Treg population when provided to patients at low doses under metronomic regimen. Hence, its synergistic effect on the outcome of WST11 VTP could be due to enhancement of Cytotoxic T cells activity by reducing the relative percentage of the protumoric Tregs.
• Foxp3 positive cells infiltration to 4Tl-luc tumors grafted in the hind leg of Balb/C mice is depicted in Fig. 20.
• 4T1 tumors were excised when reaching 30-60 mm at indicated times post CTX or saline administration, formalin-fixed and paraffin embedded. Sections were prepared and stained for Foxp3 expression. Number of positive cells was measured using Fiji software. Representative pictures at VTP day and quantification are shown (Figs. 19A,B).
• CTX administration significantly reduces tumor infiltration by regulatory Foxp3 + T cells three days later (Fig. 19C). This relieves immunosuppressive microenvironment in the tumor allowing anti-tumor immune responses to evolve following VTP.
• Example 20 Effect of low dose CTX administration at 3 days prior WSTll VTP on T cell populations in draining lymph nodes and spleens of mice bearing 4Tl-luc tumors.
• Fig, 20 shows that low dose CTX administered three days prior WSTl l VTP reduced the number of cells in the draining lymph nodes and spleens of tumor bearing mice on the VTP day.
• a significant increase in total cell numbers was observed in CTX treated animals compared to those received only VTP that show a decrease in cell counts.
• This finding suggested a CTX-mediated depletion of immune cells followed by repopulation with naive immune cells.
• the CTX administration reduced the number of regulatory Foxp3+ T cells in both lymph nodes and spleens following VTP compared to animals subjected to VTP alone.
• the spleens there was also small but significant increase in CD8+ cytotoxic T cells demonstrating overall shift towards immune active status beneficial for anti-tumor immunity development.
• Example 21 Effect of low dose CTX administration on myeloid cell populations in tumors and spleens of mice bearing s.c. 4Tl-luc tumors at the hind flank.
• Tumors and spleens were harvested three days post CTX administration (VTP day), cells isolated and stained for flow cytometry with anti- CD l ib, Ly6G and Ly6C antibodies.
• Fig. 21 shows that Low dose CTX reduced G-MDCS/neutrophils known to suppress anti-tumor immune response in the tumor microenvironment allowing for more efficient immune response to evolve following VTP.
• Example 22 Survival of Balb/C mice bearing bearing s.c. 4Tl-luc tumors at the Mammary pad following different treatment regimens Animal survival following WST11 VTP combined with CTX administration (Fig. 23 A, black, 50mg/kg) was compared to those of animals treated by WST11 VTP combined with Gemzar (Fig. 23 A, blue, 75mg/kg).
• VTP alone with 9mg/kg WST11 VTP at 200mW/cm 2 illumination for 15min resulted in very limited survival with most of the mice developing local recurrence and lung metastases (Fig. 23B-Green).
• VTP combined with Gemzar according to protocol No. 2 starting on day -2 followed by three additional doses every 5 days
• increased the survival rate to above 30% Fig. 23B-blue).
• CpG oligodeoxynucleotide as immune adjuvant enhances photodynamic therapy response in murine metastatic breast cancer.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Animal Behavior & Ethology (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• General Health & Medical Sciences (AREA)
• Pharmacology & Pharmacy (AREA)
• Medicinal Chemistry (AREA)
• Epidemiology (AREA)
• Molecular Biology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Organic Chemistry (AREA)
• Gastroenterology & Hepatology (AREA)
• Biochemistry (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Priority Applications (18)

Applications Claiming Priority (2)

Related Child Applications (2)

Publications (1)

Family

ID=58794130

Family Applications (1)

Country Status (14)

Families Citing this family (4)

Citations (8)

• 2017
  • 2017-04-10 KR KR1020187032029A patent/KR20190008206A/ko not_active Abandoned
  • 2017-04-10 BR BR112018070847A patent/BR112018070847A2/pt not_active Application Discontinuation
  • 2017-04-10 CN CN202410235521.9A patent/CN118304400A/zh active Pending
  • 2017-04-10 MX MX2018012392A patent/MX390751B/es unknown
  • 2017-04-10 US US16/092,742 patent/US11278555B2/en active Active
  • 2017-04-10 WO PCT/IL2017/050440 patent/WO2017179053A1/en not_active Ceased
  • 2017-04-10 JP JP2018553149A patent/JP2019510807A/ja active Pending
  • 2017-04-10 SG SG11201808849TA patent/SG11201808849TA/en unknown
  • 2017-04-10 CN CN201780035366.4A patent/CN109862918A/zh active Pending
  • 2017-04-10 KR KR1020227034306A patent/KR20220139445A/ko not_active Abandoned
  • 2017-04-10 RU RU2021110230A patent/RU2021110230A/ru unknown
  • 2017-04-10 RU RU2018139523A patent/RU2747258C2/ru active
  • 2017-04-10 CA CA3020067A patent/CA3020067A1/en active Pending
  • 2017-04-10 IL IL262191A patent/IL262191B2/en unknown
  • 2017-04-10 AU AU2017250732A patent/AU2017250732B2/en active Active
  • 2017-04-10 EP EP17726380.3A patent/EP3442582A1/en not_active Withdrawn
• 2018
  • 2018-10-10 MX MX2022003190A patent/MX2022003190A/es unknown
  • 2018-10-26 ZA ZA2018/07187A patent/ZA201807187B/en unknown
• 2021
  • 2021-11-29 JP JP2021193514A patent/JP2022031823A/ja active Pending
• 2022
  • 2022-03-21 US US17/700,063 patent/US20220202838A1/en not_active Abandoned
• 2023
  • 2023-02-06 AU AU2023200591A patent/AU2023200591A1/en not_active Abandoned

Patent Citations (8)

Non-Patent Citations (32)

Also Published As

Similar Documents

Legal Events