ALPHA-2 ADRENERGIC RECEPTOR AGONISTS FOR THE TREATMENT OF CANCER

Abstract

The present invention relates to the treatment of cancer. In particular, the invention relates to the therapeutic use of alpha-2 adrenergic receptor agonists for the treatment of cancer. More particularly, apraclonidine, clonidine, guanfacine and guanabenz, which are alpha-2 adrenergic receptor agonists, are all capable of efficiently reducing the growth of solid tumors. The effect is immune-mediated and is abolished in the presence of an alpha-2 antagonist or in mice that are knockout for the alpha-2 adrenergic receptor.

Claims

1-15. (canceled)

16. A method for the prevention and/or the treatment of cancer in an individual in need thereof comprising the administration of a therapeutic effective amount of an alpha-2 adrenergic receptor agonist, as an active agent.

17. The method according to claim 16, wherein said agonist is selected from the group consisting of amitraz, apraclonidine, bethanidine, brimonidine, bromocriptine, cirazoline, clonidine, detomidine, dexmedetomidine, dipivefrin, droxidopa, epinephrine, ergotamine, etilefrine, etomidate, fadolmidine, guanabenz, guanfacine, guanoxabenz, guanethidine, indanidine, lofexidine, medetomidine, mephentermine, metamfetamine, metaraminol, methoxamine, dl-methylephedrine, methyldopa, mivazerol, moxonidine, naphazoline, norepinephrine, norfenefrine, octopamine, oxymetazoline, pergolide, phenylpropanolamine, propylhexedrine, pseudoephedrine, racepinephrine, rilmenidine, romifidine, (R)-3-nitrobiphenyline, synephrine, talipexole, tizanidine, xylazine, xylometazoline, and a functional derivative thereof.

18. The method according to claim 16, wherein said agonist is selected from the group consisting of apraclonidine, clonidine, guanfacine, romifidine, and a functional derivative thereof.

19. The method according to claim 16, wherein said agonist is selected from the group consisting of an antibody, an antibody fragment, an afucosylated antibody, a diabody, a triabody, a tetrabody, a nanobody, and an analog thereof.

20. The method according to claim 16, wherein said agonist does not cross the blood/brain barrier.

21. The method according to claim 16, wherein said agonist is to be administered at a dose ranging from about 0.0001 mg/kg body weight to about 100 mg/kg body weight.

22. The method according to claim 16, wherein said agonist is to be administered systemically.

23. The method according to claim 16, wherein said agonist is to be administered with an additional treatment selected from the group consisting of chemotherapy, immunotherapy, radiation, and the like.

24. The method according to claim 16, wherein said cancer is selected from the group consisting of myelofibrosis, acute lymphoblastic leukemia, acute myeloblastic leukemia adrenal gland carcinoma, bile duct cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, gastrointestinal stromal tumors, glioblastoma, head and neck cancer, hepatocellular carcinoma, Hodgkin's lymphoma, kidney cancer, lung cancer, melanoma, Merkel cell skin cancer, mesothelioma, multiple myeloma, myeloproliferative disorders, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, salivary gland cancer, sarcoma, squamous cell carcinoma, testicular cancer, thyroid cancer, urothelial carcinoma, and uveal melanoma.

25. The method according to claim 16, wherein the cancer is a solid cancer.

26. The method according to claim 25, wherein the solid cancer is selected from the group consisting of melanoma, breast carcinoma, colon carcinoma, renal carcinoma, adrenocortical carcinoma, testicular teratoma, skin sarcoma, fibrosarcoma, lung carcinoma, adenocarcinoma, liver carcinoma, fibrosarcoma, glioblastoma, prostate carcinoma, ovarian cancer and pancreatic carcinoma.

27. The method according to claim 25, wherein the solid cancer is selected in the group consisting of colon carcinoma, ovarian cancer, melanoma, breast carcinoma, liver carcinoma, lung carcinoma, renal carcinoma, prostate carcinoma and fibrosarcoma.

28. A method for reducing the volume and/or the weight of a solid tumor in an individual in need thereof comprising the administration of a therapeutic effective amount of an alpha-2 adrenergic receptor agonist, as an active agent.

29. The method according to claim 28, wherein said agonist is selected from the group consisting of apraclonidine, clonidine, guanfacine, romifidine, and a functional derivative thereof.

30. The method according to claim 28, wherein said agonist is selected from the group consisting of an antibody, an antibody fragment, an afucosylated antibody, a diabody, a triabody, a tetrabody, a nanobody, and an analog thereof.

31. The method according to claim 28, wherein said agonist does not cross the blood/brain barrier.

32. A kit for preventing and/or treating cancer comprising: an alpha-2 adrenergic receptor agonist or a pharmaceutical composition comprising the same, and a means to administer the alpha-2 adrenergic receptor agonist or the pharmaceutical composition.

33. The kit according to claim 32, further comprising an anti-tumor compound.

34. The kit according to claim 32, wherein the anti-tumor compound is selected from the group consisting of a chemotherapy agent and an immunotherapy agent.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0170] FIG. 1 is set of curves showing that clonidine promotes a decrease of the tumor volume in a model of colon carcinoma. BALB/c mice bearing CT26 colon carcinomas received daily injections of vehicle control (black circles), clonidine (5 mg/kg, i.p.) (inverted grey triangles), clonidine (5 mg/kg, i.p.) plus phentolamine (5 mg/kg, i.p.) (black squares), or clonidine (5 mg/kg, i.p.) plus propranolol (5 mg/kg, i.p.) (grey triangles), when the tumor size was around 50 mm.sup.3, until the day of sacrifice. Tumor volume is expressed in mm.sup.3. *** p<0.001.

[0171] FIG. 2 is set of curves showing that guanabenz promotes a decrease of the tumor volume in a model of colon carcinoma. BALB/c mice bearing CT26 colon carcinomas received daily injections of vehicle control (black circles), guanabenz (5 mg/kg, i.p.) (black squares), or guanabenz (5 mg/kg, i.p.) plus phentolamine (5 mg/kg, i.p.) (grey circles), when the tumor size was around 50 mm.sup.3, until the day of sacrifice. Tumor volume is expressed in mm.sup.3. * p<0.05.

[0172] FIG. 3 is set of curves showing that the effect of clonidine in promoting a decrease of the tumor volume in a model of colon carcinoma is strongly attenuated by yohimbine. BALB/c mice bearing CT26 colon carcinomas received daily injections of vehicle control (black circles), clonidine (5 mg/kg, i.p.) (black squares), clonidine (5 mg/kg, i.p.) plus yohimbine (10 mg/kg, i.p.) (grey triangles), or yohimbine (10 mg/kg, i.p.) (black inverted triangles), when the tumor size was around 50 mm.sup.3, until the day of sacrifice. Tumor volume is expressed in mm.sup.3. ** p<0.01.

[0173] FIG. 4 is set of curves showing that the effect of guanabenz in promoting a decrease of the tumor volume in a model of colon carcinoma is strongly attenuated by yohimbine. BALB/c mice bearing CT26 colon carcinomas received daily injections of vehicle control (black circles), guanabenz (5 mg/kg, i.p.) (black diamonds), guanabenz (5 mg/kg, i.p.) plus yohimbine (10 mg/kg, i.p.) (grey circles), or yohimbine (10 mg/kg, i.p.) (black inverted triangles), when the tumor size was around 50 mm.sup.3, until the day of sacrifice. Tumor volume is expressed in mm.sup.3. *** p<0.001.

[0174] FIGS. 5A-B is a set of histograms showing the increase of tumor infiltration of CD8+T cells upon administration of romifidine or clonidine. TiRP-tumor bearing mice received adoptive cell transfer (ACT) of 10 million of P1A-specific activated CD8+T cells. Daily injections of romifidine (5 mg/kg i.p., FIG. 5A) or clonidine (5 mg/kg i.p., FIG. 5B) were administered from the day of the ACT, when the tumor size was around 500 mm.sup.3, until the day of sacrifice. Tumor infiltration of CD8+T cells is expressed as the %CD8+T cells among total CD45+T cells. * p<0.05.

[0175] FIGS. 6A-B is a set of curves and histograms showing the tumor growth (FIG. 6A) in TiRP+/+ mice that have received daily injections of clonidine (5 mg/kg, i.p., curve 2) or vehicle (PBS, i.p., curve 1), after ACT with 10 million P1A-specific activated CD8+T cells, which was performed when the tumor size was around 400 mm.sup.3. Tumor infiltration of CD8+cells evaluated at the day of sacrifice by FACS (FIG. 6B).

[0176] FIGS. 7A-B is a set of curves and histogram showing that clonidine strongly increased the anti-tumor efficacy of human PBMC against ovarian carcinoma cells SKOV3. SKOV3 human ovarian tumor cells were injected subcutaneously in immune deficient NSG (NOD Scid-gamma) mice. When tumors were palpable (50 mm.sup.3), tumor-bearing mice received adoptive cell transfer of 3 million of allogeneic human PBMC (i.v.) and were injected daily with clonidine (5 mg/kg, i.p.; grey squares; curve 2) or vehicle (PBS, i.p.; black circles; curve 1) until sacrifice. (FIG. 7A) depicts the evolution of the tumor volume (in mm.sup.3) during the time course upon treatment (in days). (FIG. 7B) depicts the comparison of the clonidine treatment combined with ACT (grey bar) as compared to ACT only (black bar), with respect to the tumor weight (in g). * p<0.05; **** p<0.0001.

[0177] FIGS. 8A-C is a set of histograms showing that clonidine cooperates with the therapeutic efficacy of human T cells in a human xenograft model. SKOV3 human ovarian tumor cells were injected subcutaneously in immune deficient NSG mice. When tumors were palpable, tumor-bearing mice received adoptive cell transfer of 3 million of allogeneic human PBMC (i.v.) and were injected daily with clonidine (5 mg/kg, i.p.; grey squares) or vehicle (PBS, i.p.; black circles) until sacrifice. Following clonidine administration (5 mg/kg, i.p.), infiltration of both CD8+(FIG. 8A) and CD4+(FIG. 8C) human T cells into human SKOV3 tumors was monitored, as well as the expression of CD44 on tumor-infiltrated CD8+T cells (FIG. 8B). * p<0.05.

[0178] FIG. 9 is a set of curves showing that clonidine strongly sensitized immune-resistant autochthonous melanoma tumors (TiRP) to adoptive cell transfer (ACT). TiRP-tumor bearing mice received adoptive cell transfer (ACT) of 10 million P1A-specific activated CD8+T cells and daily injections of vehicle control (black circles), clonidine (5 mg/kg, i.p.) (black squares), or clonidine (5 mg/kg, i.p.) plus phentolamine (5 mg/kg, i.p.) (grey triangles), when the tumor size was around 500 mm.sup.3, until the day of sacrifice. Tumor volume is expressed in mm.sup.3. ** p<0.01.

[0179] FIGS. 10A-B is a set of curves showing the tumor growth of B16F1 melanoma in C57BL/6J (FIG. 10A) or immunodeficient NSG mice (FIG. 10B) treated with PBS (Control, curve 1) or clonidine (5 mg/kg, i.p., curve 2).

[0180] FIGS. 11A-B is a set of curves showing the tumor growth of B16F1 melanoma in C57BL/6J (FIG. 11A) or NSG mice (FIG. 11B) treated with PBS (Control, curve 1) or guanfacine (5 mg/kg, i.p., curve 2).

[0181] FIGS. 12A-B is a set of curves and histograms showing the tumor growth (FIG. 12A) in TiRP+/+ mice that have received daily injection of guanfacine (5 mg/kg, i.p., curve 2) or vehicle (PBS, i.p., curve 1), after ACT with 10 million P1A-specific activated CD8+T cells, which was performed when the tumor size was around 400 mm.sup.3. Tumor infiltration of CD8+cells evaluated at the day of sacrifice by FACS (FIG. 12B).

[0182] FIGS. 13A-E is a set of curves and histograms showing that clonidine or guanabenz treatment benefits myelofibrosis (blood cancer) induced by JAK2V617F mutant bone marrow transplantation. FIGS. 13A-C: Trends of JAK2 V617F mutant allele burden variation (as reflected by CD45.2 donor chimerism in the blood of primary recipients) in mice treated with PBS (panel A), clonidine (5 mg/kg i.p.; panel B) or guanabenz (5 mg/kg i.p.; panel C) over 7 days. FIG. 13D is a histogram showing that clonidine or guanabenz treatment significantly reduces the platelet (PLT) counts (m/mm.sup.3) in primary myelofibrosis, induced by JAK2V617F mutant bone marrow transplantation. ** P<0.01. FIG. 13E is a histogram showing that clonidine treatment significantly reduces the platelet (PLT) counts (m/mm.sup.3) in myelofibrosis induced by romiplostim. * P<0.05; ns: not significant.

[0183] FIGS. 14A-B is a set of curves showing the tumor growth (melanoma) in TiRP+/+ mice that have received daily injections of guanfacine (5 mg/kg, i.p. triangles; FIG. 14A), clonidine (5 mg/kg, i.p., circles; FIG. 14B), or control (PBS, i.p., squares), when the tumor size was around 250 mm.sup.3 until sacrifice.

[0184] FIGS. 15A-C is a set of curves and histograms showing the tumor growth (FIG. 15A) of CT26 colon cancer in C57BL/6J mice treated with PBS (Control, circles), clonidine (squares), clonidine mixed with yohimbine (dark grey inverted triangles), or yohimbine alone (light grey inverted triangles). Tumor infiltration of CD8+cells evaluated at the day of sacrifice by FACS (FIG. 15B). Tumor growth of CT26 colon cancer in NSG mice treated with PBS (Control), clonidine, clonidine mixed with yohimbine, or yohimbine alone (FIG. 15C).

[0185] FIGS. 16A-C is a set of curves and histograms showing the tumor growth of CT26 colon cancer in C57BL/6J mice treated with PBS (Control, circles), guanabenz (diamonds), guanabenz mixed with yohimbine (light grey inverted triangles), or yohimbine alone (dark grey inverted triangles) (FIG. 16A); the tumor infiltration of CD8+ cells evaluated at the day of sacrifice by FACS (FIG. 16B); and the tumor growth of CT26 colon cancer in NSG mice treated with PBS (Control), guanabenz, guanabenz mixed with yohimbine, or yohimbine alone (FIG. 16C).

[0186] FIGS. 17A-B is a set of curves showing the tumor growth of LS411N ovarian cancer in mice treated with PBS (Control, curve 1), clonidine (curve 2), clonidine mixed with yohimbine (curve 3), or yohimbine alone (curve 4) (FIG. 17A); with PBS (Control), guanabenz, guanabenz mixed with yohimbine, or yohimbine alone (FIG. 17B); upon adoptive transfer of allogeneic human PBMC. Adoptive transfer of human PBMC was performed when tumor size reaches 100 mm.sup.3.

[0187] FIGS. 18A-B is a set of curves showing the tumor growth of MC38 colon cancer in Wild type (FIG. 18A) or Adra2a-KO mice (FIG. 18B) treated with PBS (Control; curves 1) or clonidine (curves 2).

[0188] FIGS. 19A-B is a set of curves showing the tumor growth of MC38 colon cancer in Wild type (FIG. 19A) or Adra2a-KO mice (FIG. 19B) treated with PBS (Control; curves 1) or guanabenz (curves 2).

[0189] FIGS. 20A-B is a set of curves showing the tumor growth of B16F10-OVA melanoma in Wild type (FIG. 20A) or Adra2a-KO mice (FIG. 20B) treated with PBS (Control; curves 1) or clonidine (curves 2).

[0190] FIGS. 21A-B is a set of curves showing the tumor growth of B16F10-OVA melanoma in Wild type (FIG. 21A) or Adra2a-KO mice (FIG. 21B) treated with PBS (Control; curves 1) or guanabenz (GBZ, curves 2).

[0191] FIGS. 22A-H is a set of curves showing the tumor growth of mice treated with PBS (Control, circles) or clonidine (squares) in different tumor models: such as mammary gland tumor (FIG. 22A), hepatocellular carcinoma (FIG. 22B), T cell lymphoma (FIG. 22C), lung cancer (FIG. 22D), myeloma (FIG. 22E), mammary adenocarcinoma (FIG. 22F), renal cancer (FIG. 22G) and fibrosarcoma (FIG. 22H).

[0192] FIGS. 23A-H is a set of curves showing the tumor growth of mice treated with PBS (Control, circles) or guanabenz (GBZ, triangles) in different tumor models: such as mammary gland tumor (FIG. 23A), hepatocellular carcinoma (FIG. 23B), T cell lymphoma (FIG. 23C), lung cancer (FIG. 23D), myeloma (FIG. 23E), mammary adenocarcinoma (FIG. 23F), renal cancer (FIG. 23G) and fibrosarcoma (FIG. 23H).

[0193] FIGS. 24A-B is a set of curves showing the tumor growth of NSG mice that received allogeneic human PBMC and a prostate cancer as xenograft, and further treated with PBS (Control, circles) or clonidine (squares) (FIG. 24A) or with PBS (Control, circles) or guanabenz (GBZ, triangles) (FIG. 24B)

[0194] FIGS. 25A-B is a set of curves showing the tumor growth of MC38 (FIG. 25A) or CT26 (FIG. 25B) colon cancer in mice treated with PBS (Control; closed circles), clonidine (5 mg/kg, i.p; closed squares) or apraclonidine (5 mg/kg, i.p; closed triangles).

EXAMPLES

[0195] The present invention is further illustrated by the following examples.

Example 1

Materials and Methods

[0196] 1—Mice

[0197] TiRP mice have been created by crossing Ink4a/Are.sup.flox/flox mice with mice carrying a transgenic construct controlled by the tyrosinase promoter and driving the expression of H-Ras .sup.12V and Trapl a which encodes a MAGE-type tumor antigen P1A; the promoter is separated from the coding region by a stop cassette made of a floxed self-deleting CreER (Huijbers et al., 2006, Cancer Res 66, 3278-3286). Those mice were backcrossed to a B10.D2 background and bred to homozygosity. TCRP1A mice heterozygous for the H-2Ld/P1A35-43-specific TCR transgene were kept on the B10.D2;Rag1.sup.−/− background (Shanker et al., 2004, J Immunol 172, 5069-5077). BALB/c mice were used in tumor transplantation experiments. TiRP-derived T429.11 transplanted melanoma model: T429.11 clone was derived from an induced Amela TiRP tumor referred to as T429. It was cloned from the T429 induced melanoma primary tumor line. Two million of T429.11 tumor cells were injected subcutaneously into recipient mice for tumor establishment (Zhu et al., 2017, Nat Commun. 10; 8(1):1404). Adra2a tm1Bkk mice (Strain Name: B6.129-Adra2a tm1Bkk /J, # 004367) were obtained from Jackson Laboratory (Bar Harbor, Me., USA).

[0198] All mice used in this study were produced under specific pathogen free (SPF) conditions at the animal facility of the de Duve Institute. All the rules concerning animal welfare have been respected according to the 2010/63/EU Directive. All procedures were performed with the approval of the local Animal Ethical Committee, with reference 2015/UCL/MD/15.

[0199] 2—Tumor Induction with 40H-Tamoxifen

[0200] A fresh solution of 40H-Tamoxifen was prepared by dissolving 40H-Tamoxifen (Imaginechem®) in 100% ethanol and mineral oil (ratio 1:9) followed by 30 min sonication, and injected subcutaneously (2 mg/200 uL per mouse) in the neck area of gender-matched 7 weeks old TiRP mice. Tumor appearance was monitored daily and tumors were measured three times/week. Tumor volume (in mm.sup.3) was calculated by the following formula: Volume=width.sup.2×length/2.

[0201] 3—Cell Cultures

[0202] SKOV3 human ovarian cancer cells and CT26 murine colon carcinoma cells were cultured in IMDM medium supplemented with 10% Fetal bovine serum (FBS).

[0203] 4—Drug Administration

[0204] Mice received a daily intra-peritoneal injection of guanabenz (5 mg/kg) or vehicle (PBS) from the day of randomization (and the day of ACT when applicable) until the day of sacrifice. For the co-administration of adrenergic receptor agonist and antagonist, BALB/c mice bearing CT26 colon carcinomas received daily injections of vehicle control, guanabenz (5 mg/kg, i.p.), or guanabenz (5 mg/kg, i.p.) plus yohimbine (10 mg/kg, i.p.), when the tumor size was around 50 mm.sup.3, until the day of sacrifice.

[0205] 5—Adoptive Cell Transfer with TCRP1A CD8+T Cells

[0206] For the adoptive cell transfer (ACT), P1A-specific (TCRP1A) CD8+T cells were isolated from spleens and lymph nodes of TCRP1A mice as described hereinabove, and stimulated in vitro by co-culture with irradiated (10,000 rads) L1210.P1A.B7-1 cells (Gajewski et al., 1995, J Immunol 154, 5637-5648) at 1:2 ratio (0.5×10.sup.5 CD8+T cells and 10.sup.5 L1210.P1A.B7-1 cells per well in 48-well plates) in IMDM (GIBCO®) containing 10% fetal bovine serum supplemented with L-arginine (0.55 mM, Merck®), L-asparagine (0.24 mM, Merck®), glutamine (1.5 mM, Merck®), beta-mercaptoethanol (50 μM, Sigma Aldrich®), 50 U mL.sup.−1 penicillin and 50 mg mL.sup.−1 streptomycin (Life Technologies®). Four days later, TCRP1A CD8+T cells were purified on a Lymphoprep gradient (StemCell®) and 10.sup.7 living cells were injected intravenously in 200 μL PBS in TiRP-tumor bearing mice on the day of randomization.

[0207] 6—Evaluation of Alpha-2 Adrenaline Receptor Agonists/Antagonists on Tumor Growth

[0208] a) TiRP Tumor Model

[0209] TiRP-tumor bearing mice received adoptive cell transfer (ACT) of 10.sup.7 P1A-specific activated CD8+T cells and daily injections of vehicle control, clonidine (5 mg/kg, i.p.), or clonidine (5 mg/kg, i.p.) combined with phentolamine (5 mg/kg, i.p.), when the tumor size was around 500 mm.sup.3, until the day of sacrifice. Tumor size was monitored every 2 days. Mice were sacrificed when the tumor in control group reached around 2,000 mm.sup.3.

[0210] b) CT26 Tumor Model

[0211] C57BL/6 mice were injected subcutaneously with 2×10.sup.6 CT26 colon tumor cells at an age of 6 to 8 weeks. For evaluation of phentolamine on the effect of clonidine and guanabenz, the mice were randomized into different groups when tumors size arrived around 50 mm.sup.3 and received daily injections of guanabenz (5 mg/kg, i.p.), a mix of guanabenz (5 mg/kg, i.p.) and phentolamine (5 mg/kg, i.p.), clonidine (5 mg/kg, i.p.), a mix of clonidine (5 mg/kg, i.p.) and phentolamine (5 mg/kg, i.p.), a mix of clonidine (5 mg/kg, i.p.) and propranolol (5 mg/kg, i.p.) or vehicle control. For evaluation of yohimbine on the effect of clonidine and guanabenz, the mice were randomized into different groups when tumors size arrived around 100 mm.sup.3 and received daily injections of guanabenz (5 mg/kg, i.p.), yohimbine (10 mg/kg), a mix of guanabenz (5 mg/kg, i.p.) and yohimbine (10 mg/kg, i.p.), clonidine (5 mg/kg, i.p.), a mix of clonidine (5 mg/kg, i.p.) and yohimbine (10 mg/kg, i.p.) or vehicle control. Tumor size was measured every 2 days and mice were sacrificed when the tumor in control group reached 1,000 mm.sup.3.

Example 2

Colon Carcinoma Model

[0212] BALB/c mice bearing CT26 colon carcinomas received daily injections of vehicle control, clonidine (5 mg/kg, i.p.), clonidine (5 mg/kg, i.p.) plus phentolamine (5 mg/kg, i.p.), or clonidine (5 mg/kg, i.p.) plus propranolol (5 mg/kg, i.p.), when the tumor size was around 50 mm.sup.3, until the day of sacrifice. As shown on FIG. 1 and FIG. 2, respectively, clonidine and guanabenz strongly inhibited CT26 tumor growth. Phentolamine, an alpha-2 adrenergic receptor antagonist, attenuated the inhibitory effect of clonidine and guanabenz on tumor growth, indicating that inhibition of tumor growth is mediated via the agonistic effect on alpha-2 adrenergic receptor. In contrast, propranolol, a beta-adrenergic receptor antagonist, did not revert the effect of clonidine on tumor growth (FIG. 2). The anti-tumor effect of clonidine and guanabenz through the agonistic effect on alpha-2 adrenergic receptor was further confirmed by the fact that yohimbine, an alpha-2 adrenergic receptor antagonist, also reduced the inhibition of tumor growth (see FIG. 3 and FIG. 4).

Example 3

Romifidine and Clonidine Administration Promote an Increase of Tumor Infiltration of CD8+T Cells

[0213] TiRP-tumor bearing mice received adoptive cell transfer (ACT) of 10.sup.7 of P1A-specific activated CD8+T cells. Daily injections of 5 mg/kg i.p. of romifidine or clonidine were thus administered from the day of the ACT, when the tumor size was around 500 mm.sup.3, until the day of sacrifice. As shown on FIGS. 5A-B, following romifidine or clonidine administration, tumor infiltration of CD8+T cells was increased. These results demonstrate that alpha-2 adrenergic receptor agonists, when combined with adoptive T cell transfer, efficiently promote an anticancer response. As shown on FIGS. 6A-B, clonidine, when combined with ACT, efficiently control the growth of immune-resistant autochthonous melanoma tumors (TiRP).

Example 4

Effect of the Combined Treatment of Clonidine and ACT Towards Human Ovarian Tumor

[0214] SKOV3 human ovarian tumor cells were injected subcutaneously in immune deficient NSG mice. When tumors were palpable, tumor-bearing mice received adoptive cell transfer of 3×10.sup.6 of allogeneic human PBMC (i.v.) and were injected daily with clonidine (5 mg/kg, i.p.) or vehicle (PBS, i.p.) until sacrifice. As shown on FIG. 7, clonidine strongly increased the anti-tumor efficacy of human PBMC against human ovarian carcinoma cells SKOV3. Both tumor growth (FIG. 7A) and tumor weight on the day of sacrifice (FIG. 7B) were significantly decreased with clonidine used with an ACT, as compared to ACT alone. These results demonstrate that clonidine improves the therapeutic efficacy of human T cells in a human xenograft model, and that the therapeutic effect of clonidine could be translated to human tumors.

Example 5

Clonidine Improves the Therapeutic Efficacy of Human T Cells in a Human Xenograft Model

[0215] SKOV3 human ovarian tumor cells were injected subcutaneously in immune deficient NSG mice. When tumors were palpable, tumor-bearing mice received adoptive cell transfer of 3×10.sup.6 of allogeneic human PBMC (i.v.) and were injected daily with clonidine (5 mg/kg, i.p.) or vehicle (PBS, i.p.) until sacrifice. Following clonidine administration (5 mg/kg, i.p.), infiltration of both CD8+(FIG. 8A) and CD4+(FIG. 8C) human T cells into human ovarian tumors SKOV3 was increased, and the tumor-infiltrated CD8+T cells were also more active in the mice that received clonidine, as shown with the increased expression of CD44 on tumor-infiltrated CD8+T cells (FIG. 8B).

Example 6

Clonidine Strongly Sensitized Immune-Resistant Autochthonous Melanoma Tumors (TiRP) to Adoptive Cell Transfer (ACT)

[0216] TiRP-tumor bearing mice received adoptive cell transfer (ACT) of 10.sup.7 P1A-specific activated CD8+T cells and daily injections of vehicle control, clonidine (5 mg/kg, i.p.), or clonidine (5 mg/kg, i.p.) plus phentolamine (5 mg/kg, i.p.), when the tumor size was around 500 mm.sup.3, until the day of sacrifice. As shown on FIG. 9, clonidine strongly sensitized immune-resistant autochthonous melanoma tumors (TiRP) to adoptive cell transfer (ACT). Phentolamine attenuated the inhibitory effect of clonidine on TiRP tumor growth.

Example 7

Both Clonidine and Guanfacine Treatment as a Monotherapy Result in an Immune-Mediated Tumor Inhibition of Melanoma

[0217] Treatments of B16F1 melanoma with either clonidine (FIGS. 10A-B) or guanfacine (FIGS. 11A-B) inhibited tumor growth when B16F1 was implanted in immune competent C57BL/6 mice. No tumor inhibition was found when B16F1 was implanted in immune deficient NSG Mice. Both clonidine and guanfacine inhibited the tumor growth in the induced TiRP model (FIGS. 6A-B and FIGS. 12A-B) despite the fact that this model is extremely immune suppressive.

Example 8

Anti-Tumor Efficiency of Clonidine or Guanabenz as Monotherapy in the Myelofibrosis (Blood Cancer)

[0218] Myelofibrosis (MF) is a clonal malignant disease resulting from acquisition of JAK-STAT activating driver mutations in bone marrow cells, of which the JAK2V617F mutation is the most prevalent.

[0219] Bone marrow from primary CD45.2 JAK2V617F mice was mixed 1:1 with CD45.1 C57BL/6 marrow and transplanted into lethally irradiated CD45.2 C57BL/6 recipients. 1 month after bone marrow transplantation, mice were randomized into three groups and received daily i.p injection of 5 mg/kg of clonidine, guanabenz or PBS. Blood was taken on Day 0 (Just before the treatment) and on Day 7 (7 days after the treatment). The allele burden was assessed as a fraction of CD45.2 cells along total CD45 cells in the blood of mice.

[0220] Decreased JAK2V617F allele burden is a golden standard method to evaluate the disease progression of MF. As shown in FIG. 13A, in mice that did not receive treatment (PBS group), the JAK2V617F allele burden continues to increase as expected. In mice treated with clonidine (FIG. 13B) or guanabenz (FIG. 13C), 1 week after treatment, there is a decrease of JAK2V617F allele burden in a number of mice.

[0221] Murine femurs and tibias from JAK2V617F mutant mice were first harvested and cleaned thoroughly. Marrow cells were flushed into PBS with 2% fetal bovine serum using a 25G needle and syringe. Resulting cell suspensions were then filtered through a 40 uM cell strainer. Recipient mice were irradiated with two doses of 500 rad 4 h apart. 1 million of donor cells were injected into wild-type recipients by standard intravenous injection using a 27G insulin syringe. One week after bone marrow transplantation, mice were treated with guanabenz or clonidine at a dose of 5 mg/kg daily for three weeks. After three weeks of treatment, mice were dissected and peripheral blood was drawn by cardiac puncture, the platelet concentration was measured by Cell Counter Analyzer MS9-5V. Wildtype mice that did not receive bone marrow transplantation or clonidine/guanabenz treatment were used as controls. As shown in FIG. 13D, both clonidine and guanabenz treatments significantly reduced the high platelet counts induced by JAK2V617F mutated bone marrow.

[0222] Romiplostim is a ligand binding to the Thrombopoietin Receptor (TPO) Ligand. It has been shown that thrombopoietin (TPO)/myeloproliferative leukemia protein (MPL; TPO receptor) signaling pathway plays a certain role in the development of MF. Myeloproliferative leukemia protein activation directly induces fibrocyte differentiation to cause myelofibrosis. Administration of TPO ligand induced MF.

[0223] 8-week female C57BL/6 J wild-type and Adra2a KO mice were administered saline or 1 mg/kg of romiplostim by a subcutaneous injection into the neck skin on days 1, 8 and 15. Clonidine or guanabenz were administrated i.p. at a dose of 5 mg/kg daily for two weeks starting from day 8. Mice were dissected on day 22 and peripheral blood was drawn by cardiac puncture, the platelet concentration was measured by Cell Counter Analyzer MS9-5V.

[0224] As shown in FIG. 13E, TPO activation increases the platelet counts in the blood, which is another phenotype of MF. Clonidine treatment reduces the platelet count in the wildtype mice but not in the KO mice, indicating that alpha-2 adrenergic receptor activation inhibits the increase of platelet related to the disease progression.

Example 9

Anti-Tumor Efficiency of Guanfacine or Clonidine as Monotherapy in the TiRP Melanoma Model

[0225] FIGS. 14A-B shows that both guanfacine (FIG. 14A) and clonidine (FIG. 14B) are efficient in decreasing the tumor volume in a TiRP melanoma model, when administered as a monotherapy.

Example 10

Anti-Tumor Efficiency of Clonidine or Guanabenz Treatment Towards Colon Cancer is Blocked by Yohimbine, an Alpha-2 Adrenergic Receptor Antagonist

[0226] Clonidine treatment (FIGS. 15A-C) or guanabenz treatment (FIGS. 16A-C) inhibited CT26 colon cancer tumor growth, and these effects were strongly attenuated when combined with yohimbine, an alpha-2 adrenergic receptor antagonist, indicating both clonidine and guanabenz inhibit CT26 tumor growth via their agonistic effect on alpha 2 adrenergic receptor.

Example 11

Anti-Tumor Efficiency of Clonidine or Guanabenz Treatment Towards Human Ovarian Cancer is Blocked by Yohimbine, an Alpha-2 Adrenergic Receptor Antagonist

[0227] Clonidine treatment (FIG. 17A) or guanabenz treatment (FIG. 17B) resulted in a significant tumor inhibition in human LS411N ovarian cancer xenograft tumor model upon adoptive transfer of human allogenic PBMC, therefore indicating the therapeutic effect observed in the murine tumor models could be translated to human tumors. This effect was strongly attenuated when combined with yohimbine, an alpha-2 adrenergic receptor antagonist, indicating that also for human tumors, the mechanism of action for both clonidine and guanabenz is via their agonistic effect on alpha-2 adrenergic receptor.

Example 12

Anti-Tumor Efficiency of Clonidine or Guanabenz Treatment Towards Colon Carcinoma is Abolished in Adra2a KO Mice

[0228] C57BL/6 Wildtype or C57BL/6 Adra2a KO mice bearing MC38 colon carcinomas received daily injections of vehicle control (PBS), clonidine (5 mg/kg, i.p.) or guanabenz (5 mg/kg, i.p.), when the tumor size was around 50 mm.sup.3, until the day of sacrifice. In the wildtype mice, in consistence with previous observations, a significant tumor inhibition was observed both with clonidine treatment (FIG. 18A) and guanabenz treatment (FIG. 19A). However, this tumor inhibition was almost completely attenuated in Adra2a KO mice (FIG. 18B and FIG. 19B). These results provided evidences that alpha-2 adrenergic receptor is the valid immunotherapeutic target which inhibition can promote anti-tumor immune response.

[0229] Similar observation was made for C57BL/6 Wildtype (FIG. 20A and FIG. 21A) or C57BL/6 Adra2a KO (FIG. 20B and FIG. 21B) mice bearing B16F10 OVA melanoma.

Example 13

Anti-Tumor Efficiency of a Clonidine and Guanabenz Treatments Towards Various Cancer Types

[0230] Clonidine treatment led to a strong tumor inhibition in various tumor models, such as mammary gland tumor (FIG. 22A), hepatocellular carcinoma (FIG. 22B), T cell lymphoma (FIG. 22C), lung cancer (FIG. 22D), myeloma (FIG. 22E), mammary adenocarcinoma (FIG. 22F), renal cancer (FIG. 22G) and fibrosarcoma (FIG. 22H). The same observations were made for guanabenz treatment (FIGS. 23A-H). Finally, clonidine or guanabenz treatment led to a strong tumor inhibition in a xenograft model of prostate cancer in NSG mice treated with PBMC (see FIGS. 24A-B).

[0231] Altogether, these results are suggesting that adrenergic receptor alpha-2 agonists can be used to treat a broad range of cancer types.

Example 14

Anti-Tumor of Apraclonidine Treatment Towards Colon Cancer is Mediated by Peripheral Rather than Nervous Central Effects

[0232] Apraclonidine does not cross the blood-brain barrier as compared to clonidine. FIGS. 25A-B show that tumor growth of MC38 colon cancer (FIG. 25A) or CT26 colon cancer (FIG. 25B) treated with PBS, clonidine or apraclonidine. The anti-tumor effect of apraclonidine suggests that the pharmacological profile of alpha-2 adrenergic receptor agonists may be explained by peripheral effects rather than nervous central effects.