Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
  • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
• • 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
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
  • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
• • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

• the present invention relates to treatments for cancer, especially multi-drug resistant cancers. More specifically, the present invention relates to combination treatments of taxoids and immunotherapy.
• PD-1 and PD-L1 inhibitors are immune checkpoint inhibitors that are used to treat various forms of cancer.
• PD-L1 expression by tumor cells which may be one of the important parameters that correlates with and may even be required for efficacy of PD-1/ PD-L1 inhibitors, varies by tumor type and among individual patients (see, e.g., Taube et al., Clin Cancer Res; 20(19): 5064-74 (2014) and Sunshine and Taube, Current Opinion in Pharmacology, 23:32-38 (2015)).
• CTLA-4 inhibitors are also checkpoint inhibitors that are being developed to treat various forms of cancer. CTLA-4 expression has also been shown to correlate with efficacy of CTLA-4 inhibitors.
• Pancreatic ductal adenocarcinoma is a lethal and aggressive disease with the lowest 5-year patient survival rate of any tumor type routinely tracked (6%). The incidence of PDAC is rising, and it is projected to become the second leading cause of cancer-related death in the United States by
• PDAC is distinguished by a dense desmoplastic stroma, rich in fibroblasts, extracellular matrix, and inflammatory leukocytes (but few infiltrating effector T cells). Although certain combination chemotherapies are increasingly effective for PDAC, tumor response rates remain low and durability is short. The presence of cancer initiating or stem cells (CSCs) as a subpopulation in human pancreatic tumor has been confirmed and the CSCs have been attributed to increased tumor growth, invasion and metastasis as well as resistance to chemo and radiation therapy.
• CSCs cancer initiating or stem cells
• Vascular dysfunction is another hallmark property of PDAC that limits the ability to penetrate into and deliver drugs to the deeply seated cancer cells within the tumor mass.
• Vascular dysfunction is further related to sub-optimal oxygen availability within the tumor, giving rise to a hypoxic microenvironment that has been implicated in imparting resistance to chemo and radiotherapy as well as increasing the invasiveness and metastatic potential of the cancer cells.
• a hypoxic environment harbors highly drug-resistant, quiescent cells that show CSC-like traits and, therefore, this sub population of stem cells are important targets for therapy to effectively treat the disease and to address clinical recurrences. All of these factors working in tandem present an insurmountable obstacle in designing a safe and effective therapy against PDAC.
• FOLFIRINOX regimen is associated with significant toxicities, which includes neutropenia, diarrhea, and sensory neuropathy, limiting its use to only patients with good performance status.
• Another new combination gemcitabine regimen with nanoparticle albumin bound (nab)-paclitaxel (ABRAXANE ® ) was introduced in the Metastatic Pancreatic Adenocarcinoma Trial (MPACT) Phase III clinical trial.
• Gemcitabine plus ABRAXANE ® increased the median overall survival to 8.7 months as compared to 6.6 months with gemcitabine monotherapy.
• gemcitabine plus (nab)-paclitaxel has less toxicity as compared to FOLFIRINOX, making it the most widely used regimen in the community setting for patients with newly diagnosed metastatic pancreatic cancer (PC) in the United States.
• PC metastatic pancreatic cancer
• the progression free survival in both regimens remains dismal. Many patients treated with either of the two regimens have ultimately relapsed and require second line therapy.
• new immuno-oncology "10" agents such as anti- PD-1 or anti CTLA-4 antibody therapies, have not shown efficacy in PDAC due to a highly immunosuppressive microenvironment and dense stroma that inhibits T-cell infiltration in the tumor mass.
• An ideal drug to provide in a combination treatment regimen would be one that both de-bulks the tumor as well as targets the cancer stem cell population.
• One of the advantages of drugs like paclitaxel is that they target tubulin/microtubule, a basic component of the cell that is absolutely necessary for cell survival; however, resistance can be achieved by up-regulation of multi-drug resistance (MDR) mechanisms such as efflux pumps or by tubulin mutations. Accordingly, a continuing challenge in cancer chemotherapy is to develop new cytotoxic agents with greater selectivity for the tumor, overcoming MDR, improved pharmacology and a reduction in toxicity.
• MDR multi-drug resistance
• DHA-SBT-1214 One of the next-generation taxoids, such as DHA-SBT-1214, addresses these issues. It has several unique properties that make it a potentially useful therapy in the clinic either as a stand-alone treatment or in combination with other therapeutic modalities. DHA-SBT-1214 is active against many drug resistant tumor types and is not a substrate for several MDR mechanisms, such as over-expression of the P-glycoprotein (P-gp) transporter and the treatment results in complete tumor regression in pancreatic, colon and prostate cancer xenograft models. DHA-SBT-1214, exhibited two-to-three orders of magnitude higher potency than those of paclitaxel and docetaxel against drug-resistant cell lines expressing MDR phenotypes.
• P-gp P-glycoprotein
• DHA-SBT-1214 has been shown to down-regulate stem related genes in CSCs purified from three human colon cancer cell lines, DLD-1, HTC-116, and HT-29.
• DHA-SBT- 1214 was effective in a patient-derived prostate cancer stem cell xenograft model, where paclitaxel was completely ineffective and ABRAXANE ® only marginally effective in delaying tumor growth and improving survival.
• Paclitaxel and docetaxel are effective initially against breast, ovary, and lung cancers, and show limited efficacy against pancreatic cancer; however human albumin formulated paclitaxel has shown some benefit.
• PDAC is inherently refractory due to the over expression of Pgp, an effective ATP- binding cassette (ABC transporter), which effluxes hydrophobic anticancer agents including paclitaxel and docetaxel.
• AAC transporter an effective ATP- binding cassette
• DHA-SBT-1214 shows remarkable activity against drug- resistant cancer cells, expressing MDR phenotypes including PDAC cells and tumor xenografts. Accordingly, there is every indication that DHA-SBT-1214 a powerful tumor-targeting chemotherapeutic agent, overcoming the weaknesses of paclitaxel, docetaxel and ABRAXANE ® and substantially improve the quality of life of PDAC patients.
• DHA-SBT-1214 and other taxoids are very hydrophobic, a safe formulation that can solubilize the molecule and afford systemic delivery potential is needed.
• the DHA molecule linked through an ester bond is susceptible to cleavage in the aqueous environment and especially in the presence of esterases.
• Nanoemulsions are heterogenous systems composed of oil in water where the oil droplets are reduced to nanometer size using either ultrasound or high-pressure homogenization methods.
• the surface of the oil droplets is decorated with amphiphilic molecules to lower the interfacial tension and afford stability in the presence of aqueous medium.
• DHA-SBT-1214 can be encapsulated in the oil droplet of the nanoemulsion and is protected from hydrolysis by esterases.
• Surface modification of the oil droplet with polyethylene glycol) (PEG) prolongs the circulation half-life upon systemic administration and passive targeting to solid tumors due to the leaky vasculature by the enhanced permeability and retention (EPR) effect.
• EPR enhanced permeability and retention
• Taxane treatment has been shown to, stimulate tumor-associated macrophage cytotoxicity, induce the activation of dendritic cells, natural killer cells, tumor specific cytotoxic T-cells as well as downregulate regulatory T cells (“T re gs”) ⁇ It has also been shown to inhibit myeloid-derived suppressor cell function. Combining such approaches with anti-PD-Ll/PD-1 therapies will broaden the clinical benefit to include a greater proportion of patients.
• NE-DHA-SBT-1214 provides a unique opportunity to both de-bulk the tumor and kill cancer stem cells thus creating an unprecedented path forward to combine with 10 agents.
• the temporal sequencing of the combination of NE-DHA-SBT-1214 and 10 therapy will be important in uncovering the potential synergistic effects of this combination therapy.
• de-bulking the tumor and affecting stromal permeability we are potentially releasing antigen into the system and by killing cancer stem cells we are both releasing cancer stem cell antigens, as well as, decreasing the re-population effect of these cells.
• An additional effect of de-bulking tumor is that you are decreasing the level of inherent immune suppression of the tumor (e.g.
• the present invention provides for a composition of an omega-3 polyunsaturated fatty acid (PUFA)-taxoid conjugate formulated in an oil-in-water nanoemulsion (NE) drug delivery system in combination with an immune-oncology (IO) agent to enhance therapeutic efficacy in refractory cancers, such as PDAC.
• PUFA omega-3 polyunsaturated fatty acid
• NE oil-in-water nanoemulsion
• IO immune-oncology
• the present invention also provides for a method of treating cancer, by administering an effective amount of a pharmaceutical composition including an omega-3 PUFA-taxoid conjugate in combination with an 10 agent encapsulated in an NE drug delivery system to a subject in need of treatment, and treating cancer.
• FIGURE l is a depiction of the molecule DHA-SBT-1214;
• FIGURE 2 is a graph of median tumor volume after CFPAC-1 tumor implant
• FIGURE 3 is a graph of median tumor volume after PANC-1 tumor implant
• FIGURE 4 is a graph of median tumor volume after Panc-02 tumor implant
• FIGURES 5A and 5B are graphs of the activity of different anti-cancer agents against Panc02 cells
• FIGURE 6A is a transmission electron microscopy (TEM) of nanoemulsion
• FIGURE 6B shows the oil droplet particle size determination in nm
• FIGURE 6C shows the measurement of zeta potential or surface charge on the oil droplets in mV
• FIGURE 6D shows the uptake of rhodamine-encapsulated nanoemulsion formulation in Panc02 cells
• FIGURE 7 is a graph of PD-L1 surface protein expression in response to different anti-cancer agent treatments in vitro and without any treatment in vivo;
• FIGURE 8 is a Western blot of untreated and IFN-gamma (20 ng/ml for 4 h) treated Panc02 cells compared to Panc02 mouse tumor;
• FIGURE 9 is a graph of tumor volume versus time for treatments tested;
• FIGURES 10A-10J are tumor images taken at the time of harvest from different treatment modalities
• FIGURE 10A shows tumors from mice treated with vehicle
• FIGURE 10B shows tumors from PD-L1 (200pg) treated mice
• FIGURE 10C and FIGURE 10D show tumors from ABRAXANETM plus IgG or PD-L1 (200pg) treated mice respectively
• FIGURE 10E shows tumors from NE-DHA-SBT-1214 (lOmg/kg) plus IgG (200pg) treated mice
• FIGURE 10F and FIGURE 10G show tumors from Gemcitabine plus IgG or PD-L1 (200pg) treated mice respectively
• FIGURE 10H shows tumors from NE-DHA-SBT-1214 (lOmg/kg) plus PD-L1 (200pg) treated mice
• FIGURE 101 and FIGURE 10J show tumors from NE-DHA-SBT-1214 plus IgG or PD-L1 (200pg) treated mice
• FIGURE 11 is a graph showing body weight alterations induced by treatment with different combination therapies
• FIGURE 12 is a graph of mRNA expression of PD-L1 from different mouse tumor treatment groups analyzed using RT-PCR and relative gene expression for RT-PCR data was calculated relative to murine b-actin;
• FIGURE IB is a graph of mRNA expression of PD-1 from different mouse tumor treatment groups analyzed using RT-PCR and relative gene expression for RT-PCR data was calculated relative to murine b-actin;
• FIGURE 14 is a graph of mRNA expression of CD-4 from different mouse tumor treatment groups analyzed using RT-PCR and relative gene expression for RT-PCR data was calculated relative to murine b-actin;
• FIGURE 15 is a graph of mRNA expression of CD-8 from different mouse tumor treatment groups analyzed using RT-PCR and relative gene expression for RT-PCR data was calculated relative to murine b-actin;
• FIGURE 16 is a graph of mRNA expression of Arginase-1 from different mouse tumor treatment groups analyzed using RT-PCR and relative gene expression for RT-PCR data was calculated relative to murine b-actin;
• FIGURE 17 shows tumor tissue lysate from different treated groups prepared and protein level of different proteins analyzed using western blotting
• FIGURES 18A-18J show histopathological evaluation of the Panc02-induced tumor tissues collected from control and different combination treated mice (hematoxylin & eosin staining), FIGURE 18A is untreated, FIGURE 18B is PD-L1, FIGURE 18C is ABRAXANETM + IgG, FIGURE 18D is ABRAXANETM + PD-L1, FIGURE 18E is Gemcitabine + IgG, FIGURE 18F is Gemcitabine + PD-L1, FIGURE 18G is 10 mg/kg NE-DHA-SBT-1214 + IgG, FIGURE 18H is 10 mg/kg NE-DHA-SBT-1214 + PD-L1, FIGURE 181 is 25 mg/kg NE- DHA-SBT-1214 + IgG, and FIGURE 18J is 25 mg/kg NE-DHA-SBT-1214 + PD-L1;
• FIGURE 19A-19J show analysis of infiltrating CD4 cells by immunohistochemistry
• FIGURE 19A is untreated
• FIGURE 19B is PD-L1
• FIGURE 19C is ABRAXANETM + IgG
• FIGURE 19D is ABRAXANETM + PD-L1
• FIGURE 19E is Gemcitabine + IgG
• FIGURE 19F is Gemcitabine + PD-L1
• FIGURE 19G is 10 mg/kg NE-DHA-SBT-1214 + IgG
• FIGURE 19H is 10 mg/kg NE-DHA-SBT-1214 + PD-L1
• FIGURE 191 is 25 mg/kg NE- DHA-SBT-1214 + IgG
• FIGURE 19J is 25 mg/kg NE-DHA-SBT-1214 + PD-L1;
• FIGURE 20A-20J show analysis of infiltrating CD8 cells by immunohistochemistry
• FIGURE 20A is untreated
• FIGURE 20B is PD-L1
• FIGURE 20C is ABRAXANETM + IgG
• FIGURE 20D is ABRAXANETM +
• FIGURE 20E is Gemcitabine + IgG
• FIGURE 20F is Gemcitabine + PD-L1
• FIGURE 20G is 10 mg/kg NE-DHA-SBT-1214 + IgG
• FIGURE 20H is 10 mg/kg NE-DHA-SBT-1214 + PD-L1
• FIGURE 201 is 25 mg/kg NE-
• FIGURE 21A-21J show analysis of infiltrating PD1 cells by immunohistochemistry, FIGURE 21A is untreated, FIGURE 21B is PD-L1, FIGURE 21C is ABRAXANETM + IgG, FIGURE 21D is ABRAXANETM + PD-L1, FIGURE 21E is Gemcitabine + IgG, FIGURE 21F is Gemcitabine + PD-L1, FIGURE 21G is 10 mg/kg NE-DHA-SBT-1214 + IgG, FIGURE 21H is 10 mg/kg NE-DHA-SBT-1214 + PD-L1, FIGURE 211 is 25 mg/kg NE- DHA-SBT-1214 + IgG, and FIGURE 21J is 25 mg/kg NE-DHA-SBT-1214 + PD-L1.
• the present invention is generally directed to compositions for treating cancer.
• the composition is preferably an omega-3 polyunsaturated fatty acid (PUFA)-taxoid conjugate formulated in an oil-in-water nanoemulsion (NE) drug delivery system in combination with an immune-oncology (10) agent.
• PUFA polyunsaturated fatty acid
• NE oil-in-water nanoemulsion
• the preferred embodiment is NE-DHA-SBT-1214, in which the PUFA-taxoid conjugate is DHA-SBT- 1214, whose structure is shown in FIGURE 1, in combination with anti-PD-Ll antibody.
• second-generation taxoid will be used to refer to a first-generation taxanes, such as paclitaxel (taxol) and docetaxel (taxoid), in which (i) the C-3'-phenyl group is replaced with an alkenyl or alkyl group and (ii) the C-10 position is modified with certain acyl groups, and a C-3'N position is a t-Boc group.
• PUFA-taxoid conjugate will be used to refer to a taxoid conjugated to a polyunsaturated fatty acid (PUFA) at the C2' position.
• PUFA-taxoid conjugates are characterized by their ability to be preferentially accumulated in tumor and stay for long time, while exhibiting impressive efficacy especially against multidrug-resistant tumors (Ojima, I., Taxoid-Fatty Acid Conjugates and Pharmaceutical Compositions Thereof for Treatment of Cancer", US Patent 7,820,839 B2, 10/26/2010).
• NE nanoemulsion
• the term "nanoemulsion” (NE) will be used to refer to an oil-in-water emulsion with mean droplet diameters ranging from 50 to 1000 nm, with a diameter of >200 nm being preferred.
• the preferred NE oil phase is prepared as in U.S. Patent Application Publication US20070148194 to Amiji, et al.
• omega-3 fatty acid-rich edible oils such as fish oil or flax-seed oil.
• Other oils can be used such as, but not limited to, pine nut oil, safflower oil, primrose oil, black currant oil, borage oil, wheat germ oil, chia oil, hemp oil, perilla oil, grape oil, squalene oil, and fungal oil.
• the oil droplet is modified with surfactants, including phospholipids (e.g., LIPOID ® ) and polyethylene oxide)-containing non-ionic surfactants (e.g., Pluronic or Tween).
• the surface of the oil droplet can also be modified for selective targeting to tumor cells with a targeting agent, including the use of folate, EGFR peptide, and other known targeting ligands.
• a targeting agent including the use of folate, EGFR peptide, and other known targeting ligands.
• the composition can also contain image contrast agents, including fluorophores, MRI contrast agents, or radioactive compounds.
• immuno-oncology agent or "10 agent” as used herein refers to any agent that targets the body's immune system to provide a response to cancer.
• Many cancer cells have tumor- associated antigens that can be recognized by the body's immune system, and these antigens can be targeted in active immunotherapy.
• Passive immunotherapies can enhance the body's existing anti tumor responses.
• An essential role of the immune system is protecting the body against the proliferation of malignant cells. Immune modulation is increasingly seen as pivotal to the treatment of many cancers. Regulatory approval has been achieved for several new cancer immunotherapies, and many others are in the developmental pipeline.
• the degree of immune infiltration and the ratio of effector T cells to regulatory T cells have been shown to be robust prognostic factors, regardless of therapy, in multivariate analyses in many different types of cancers. Cancers with high levels of immune infiltrate generally progress more slowly. Methodologies are now being validated for reproducible quantitation of immune infiltration.
• immunosuppression pathways are known to prevent T cells from effectively infiltrating malignancies and/or to suppress the function of infiltrating lymphocytes. These pathways include (1) generation of dysfunctional antigen-presenting cells; (2) polarization of the immune system toward a Th2 response, a less effective pathway for immune rejection of cancer; (3) induction of immune regulatory cells such as regulatory T cells and myeloid-derived suppressor cells; (4) induction or secretion of immunosuppressive cytokines such as IL10 and transforming growth factor (TGF); and (5) induction of T-cell anergy or T-cell exhaustion.
• TGF transforming growth factor
• the PUFA in the conjugate is preferably docosahexaenoic acid (DHA) (C-22), but can also be eicosapentaenoic acid (EPA, C-20), or alpha-linolenic acid (LNA, C-18).
• DHA docosahexaenoic acid
• EPA eicosapentaenoic acid
• LNA alpha-linolenic acid
• the present invention includes formulations of PUFA-taxoid conjugates, which are encapsulated into nanoparticles in NE as disclosed in U.S. Patent Application Publication US20070148194
• any taxoid, or combination of taxoids can be encapsulated in an NE, including, but limited to, any of the PUFA-taxoid conjugates described in U.S. Patent No. 7,820,839, to Ojima, and taxoids described in Ojima I and Das M, (2009), both of which are incorporated herein in their entirety.
• Taxoids which can be included in the present invention, as NE formulations include, but are not limited to, paclitaxel, docetaxel, SBT-1213, SBT-12854, SBT-121303; SBT-1216, SBT-11033, SBT-121313, SBT-121602, cabazitaxel, SBT-1212, SBT-1217, SBT-1102, SBT-1103, SBT-1104, SBT-1106, SBT-1107, SBT-121301, SBT-121302, SBT-121304, SBT-121403, SBT-11031, SBT-11032, SBT-11034, SBT- 12851, SBT-12852, SBT-12853, SBT-12855, SBT-12851-1, SBT-12851-3, SBT-12852-1, SBT-12852-3, SBT- 12853-1, SBT-12853-3,SBT-12854-1, SBT-12854-3, SBT-12855-1, and SBT-12855-3 (Ojima,
• DHA or LNA esters of any of the above second-generation taxoids can be used. One skilled in the art can easily make such esters. Working examples of their formulation and effectiveness are found within the indicated references, which are incorporated in their entirety herein.
• the 10 agent is an agent that uses the individual's immune system to attack and treat cancer, and is most preferably anti-PD-Ll antibody.
• any other 10 agent can also be used, such as, but not limited to, anti-PD-1 antibody, ipilumumab (CTLA-4 inhibitor), nivolumab (PD-1 checkpoint inhibitor), pembrolizumab (PD-1 checkpoint inhibitor), atezolizumab (PD-L1 checkpoint inhibitor), pidiluzumab, durvalumab, anti-CD47 antibodies, indoleamine (2,3)-dioxygenase inhibitors, anti-GD2 antibodies, alemtuzumab, ofatumumab, rituximab, or cytokines (interferon-oc, interferon-b, interferon- gamma, or interleukins (1-36).
• the present invention provides a method of treating cancer, by administering an effective amount of a pharmaceutical composition including a PUFA-taxoid conjugate encapsulated in an NE drug delivery system in combination with an 10 agent to a subject in need of treatment, and treating cancer.
• a pharmaceutical composition including a PUFA-taxoid conjugate encapsulated in an NE drug delivery system in combination with an 10 agent to a subject in need of treatment, and treating cancer.
• the combinatorial effect is driven by the ability of the PUFA-taxoid conjugate increasing the expression of PD-L1 in the tumor microenvironment as well causing an increase of both CD4 + and CD8 + tumor- infiltrating lymphocytes.
• the cancer being treated in the methods herein can be any type of cancer, such as, but not limited to, breast, ovary, lung, head and neck, colon, rectal, pancreatic, melanoma, brain, prostate, leukemia, sarcomas, thyroid, Non-Hodgkin Lymphoma, bladder, gliomas, endometrial, and renal cancer.
• the PUFA-taxoid conjugate can be any of those described herein, and especially DHA-SBT-1214. Because the PUFA-taxoid conjugate is encapsulated in the NE, it is actively taken up by the body and DHA is cleaved more efficiently than in normal delivery methods.
• the 10 agent is preferably anti-PD-Ll antibody, but can be any 10 agent described above. When anti-PD-Ll is administered, the method further includes the step of upregulating PD-L1.
• DHA-conjugated SBT-1214 (Figure 1), exerts a remarkable efficacy against highly drug resistant tumor xenografts in mice, wherein DHA conjugated paclitaxel (TAXOPREXIN ® ); Luitpold Pharmaceuticals; human Phase l-lll clinical trials) paclitaxel, and nab-paclitaxel (ABRAXANE ® ) do not show meaningful activity.
• DHA- was linked to SBT-1214, based on the hypothesis that the omega-3 fatty acid conjugate would have the beneficial properties of DHA-paclitaxel (e.g., much reduced toxicity, prolonged residence time in the tumor as compared to paclitaxel, and higher stability in plasma).
• DHA conjugation also aids in the incorporation into the omega-3 fatty acid based nanoemulsion, where a 5- fold increase in drug concentration can be achieved.
• DHA-SBT-1214 shares some activities with paclitaxel such as stabilizing microtubules; however, it has additional anti-tumor mechanisms.
• the effect of these compounds on the microtubule network is different from those observed with the classical taxanes (docetaxel and paclitaxel), inducing different bundling in cells with microtubules being very short, indicating a very fast nucleation effect and reflecting their high assembly induction power and the ability to inhibit cell division in various cell lines harboring tubulin mutations.
• DHA-SBT-1214 has been shown to down-regulate many survival genes in three colon cancer stem cell lines and activate p53 and p21. Taken together, these data suggest that there are several mechanisms that differentiate DHA-SBT-1214 from paclitaxel, docetaxel or ABRAXANE ® . The observed remarkable efficacy of DHA-SBT-1214 against several tumor xenografts, especially, Panc-1 and CFPAC-1 (pancreatic) ( Figures 2 and 3), respectively, as well as CSCs clearly demonstrates that this is not just an incremental improvement, but a profound shift in chemotherapy paradigm.
• DHA-SBT-1214 showing minor weight loss ( ⁇ 4%) until day 20, while the weight loss was negligible for DHA-SBT-1214 at either the 240 mg/kg or BOO mg/kg total dose.
• the nanoemulsions (NEs) technology developed is simple, versatile, and clinically- translatable colloidal carriers formed by dispersion of PUFA rich edible oils in water and stabilized with an amphiphilic phospholipid monolayer.
• These NEs have a hydrodynamic diameter of ⁇ 200 nm, can incorporate considerable amounts of hydrophobic drugs in the high volume fraction of the oil phase, and are suitable for both systemic and oral delivery.
• the NEs are highly flexible vehicles for the incorporation of drugs and are composed entirely of generally regarded as safe (GRAS) materials, which have highly favorable safety profiles and amenable for large scale GMP manufacturing using high pressure homogenizers, a significant advantage for rapid clinical adoption.
• GRAS generally regarded as safe
• One of the Improvements of the nanoemulsion in the present invention as compared to earlier formulations is how it is taken up the cell.
• Traditional formulations are taken up by the cell by passive diffusion through the lipid bi-layer.
• the nanoemulsion is taken up by receptor- mediated endocytosis, bypassing P-glycoprotein/mdr-l mediated drug efflux.
• ester bond between taxoid and fatty acid tail is cleaved, resulting in the release of an active compound.
• Applicants have successfully formulated NE-DHA-SBT-1214 with concentrations as high as 30 mg/ml as compared to 6 mg/ml in Tween ® 80 or Solutol-HS15.
• the droplet size is consistently less than 200 nm in diameter allowing one to filter sterilize the final formulation.
• the zeta potential of the nanoemulsion is in the range of negative 23 mV to 33m. This is a critical point because the negative charges on the lipid layer ensures that the nanoemulsion droplets do not coalesce and Applicants have stability data for up to a year at 4°C versus less than 24 hours in Solutol-HS15.
• Initial toxicology studies have provided a safe and efficacious dose of 25 mg/kg in an aggressive patient-derived xenograft model.
• the 72 hours IC 50 of NE-DHA-SBT-1214 against the Panc-1 cell line is 2.3 nM, a 25-fold reduction as compared to Tween-80 formulated DHA-SBT-1214 and the efficacy is at least a 3-fold higher in vivo.
• DHA-SBT-1214 induced complete regression of drug- resistant colon tumor xenografts in all surviving mice with unusually long-term tumor growth delay (>167 days). Perhaps more significant was the effect of exposure to relatively low concentrations of DHA-SBT-
• NE-DHA-SBT-1214 (100 nM to 1 mM) for 24 hours on the expression of genes associated with "sternness" in several colon and prostate cancer cells lines grown as microspheres (6,7). Additionally, NE-DHA-SBT-1214 is superior to ABRAXANE ® in a patient-derived PPT2 prostate cancer stem cell xenograft model, and equally effective in pancreatic cancer organoids.
• DHA-SBT-1214 In studies with DHA-SBT-1214 and prostate cancer stem cells, Applicants previously determined that low concentrations of DHA-SBT-1214 (0.1-1 mM) induced up to 80-90% death of the highly tumorigenic and highly drug-resistant prostate CD133 + cells maintained under sternness- promoting culture conditions. In addition, treatment resulted in the significant up-regulation of the previously absent expression of the pro-apoptotic proteins, p53 and p21 ("gene wake-up" effect), and as a result, a dramatic increase in sensitivity to treatment. In a patient-derived prostate CSC xenograft model, DHA-SBT-1214 was superior to ABRAXANE ® and resulted in tumor clearance. This shows the long term efficacy of DHA-SBT-1214 against drug resistant pancreatic, prostate and breast cancer tumors in vivo can be explained by its effects on both the bulk tumor and cancer stem cell sub-population.
• Lipoid GMBH Lipoid GMBH (Ludwigshafen, Germany), DSPE PEG2000 from Avanti Polar Lipids, Inc. (Alabaster, AL), Tween 80 from Sigma Chemicals, Inc. (St. Louis, MO), Dulbecco's Modified Eagle Medium (DMEM) and LAL chromogenic endotoxin quantitation kit from Thermo Scientific (Rockford, IL). Penicillin, streptomycin and Trypsin were obtained from Invitrogen (Grand Island, NY, USA). Female C57BL/6 mice (4-6 weeks old) were purchased from Charles River Laboratories (Frederick Research Model Facility-NCI) (Cambridge, MA, USA).
• Panc02 The murine pancreatic cancer cell line Panc02, which is syngeneic to C57BI/6 mice was a kind gift from Professor Michael A. Hollingsworth, UNMC, Omaha, NE. Panc02 cells were grown in 75 cm 2 cell culture flasks and maintained in DMEM medium supplemented with 10% fetal bovine serum (FBS), L-glutamine and penicillin (100 U/ml)/streptomycin (100 pg/ml) (both from Gibco Life
• FBS fetal bovine serum
• L-glutamine and penicillin 100 U/ml
• streptomycin 100 pg/ml
• Panc02 cells (0.5xl0 6 ) were seeded onto glass cover-slips in 6-well plates for overnight at 37°C in a humidified atmosphere containing 5% C02. Then cells were incubated with 2 mM of NE- Rhodamine nanoparticles for different time points ranging from 0.5 hours to 4 hours to allow uptake of nanoparticles by cells. After last incubation time point, the glass cover-slips were washed with PBS before fixing in formalin for 15 minutes at room temperature. Nuclei of the fixed cells were stained with 4', 6- diamidino-2-phenylindole (DAPI). Uptake of rhodamine nanoemulsion was studied by a fluorescence confocal microscope (Zeiss LSM 700) with fixed parameters to have comparable uptake among different time points.
• DAPI 6- diamidino-2-phenylindole
• MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
• cells harvested from in vitro cultures were washed twice with 3%BSA/PBS and then incubated with rat anti-PD-Ll or isotype control antibodies (mouse, BioXcell, West Riverside, NH, USA) for 30 minutes at 4C, then washed three times and incubated with anti-rat AlexaFluor 488 conjugated Antibody.
• the cells were washed once with 3%BSA/PBS and analyzed by flow cytometer on a FACSCalibur flow cytometer and CellQuestTM Pro version 6.0 software (both from Becton-Dickinson and Co.).
• RT-PCR Real time-polymerase chain reaction
• the expression level of PD-L1 and mRNA for other proteins was determined using real-time PCR as previously described.
• the samples used for mRNA isolation were removed from the pancreatic cancer cells (Pan02) or tumor tissues.
• Total mRNA was extracted using commercially available RNA extraction kit according to mentioned protocol (Thermo Fisher Scientific (Rockford, IL).
• the isolated RNA was stored at -80 ° C until use for real-time PCR. In the latter, 1 pg of extracted RNA was reverse-transcribed using commercial cDNA synthesis kit (Thermo Fisher Scientific (Rockford, IL).
• mouse PD-L1 (forward primer, 5 '-AAAGTC AAT G CCCC AT ACCG -3 ' (SEQ ID NO: 1) and reverse primer, 5'-TTCTCTTCCCACTCACGGGT-3' (SEQ ID NO: 2)); mouse PD-1 (forward primer, 5'-TTCACCTGCAGCTTGTCCAA-3' (SEQ ID NO:4) and reverse primer, 5'-TGGGCAGCTGTATGATCTGG-3' (SEQ ID NO: 5)); CD4: (forward primer, 5'- ACACACCTGTGCAAGAAGCA-3' (SEQ ID NO:6) and reverse primer, 5'-GCTCTTGTTGGTTGGGAATC-3' (SEQ ID NO:
• mouse CD8 forward primer, 5'-CTCACCTGTGCACCCTACC-3' (SEQ ID NO: 8) and reverse primer, 5'-ATCCGGTCCCCTTCACTG-3' (SEQ ID NO:9)
• mouse Arginase-1 forward primer, 5'-
• PCR was performed using a real-time PCR system (7300; Applied Biosystems, Foster City, CA, US A). Relative quantifications of gene expression with qRT-PCR data were calculated relative to murine b-actin.
• Mouse Antibody against PD-L1 (10F.9G2) and relevant isotype IgG control was purchased from Bio X Cell. Two hundred micrograms of antibody against PD-L1 and relevant isotype IgG control was injected through IP per mice twice a week for 3 weeks. Gemcitabine solution and abraxane 120mg/kg was injected through i.p. once a week. Paclitaxel 120mg/kg and NE-DHA-SBT-1214 either lOmg/kg or 25mg/kg was injected once a week through i.v. All chemotherapy drugs were either injected in combination to anti PD-L1 antibody or isotype IgG control. In total, three treatments were given per experiment.
• H&E hematoxylin and eosin
• Nanoemulsion delivery approach has shown enhanced therapeutic potential in Applicants' previous studies.
• Applicants have formulated an oil-in-water nanoemulsion of DHA-SBT- 1214, a new-generation taxoid using fish oil which is rich in PUFAs such as omega-3 and omega-6 fatty acids.
• This taxoid encapsulated nanoemulsion was used to study its therapeutic efficacy in combination with immune check point inhibitor in a pancreatic cancer preclinical mouse model.
• Applicants have used a high pressure homogenization technique to formulate this uniform, milky-white and stable nanoemulsion.
• the nanoemulsion droplets were near spherical in morphology with an average diameter of approximately 220 nm, as observed by light scattering and transmission electron microscopy (TEM). Fluorescence microscopy images showing the blue (nucleus), red (rhodamine encapsulated nanoemulsion) and overlay images in purple color. The images were taken at 63x magnification. The image scale bar is 100 pm. Along with particle size, uniformity and charge of the nanoemulsions also predicts their bioavailability. Uniformity is represented by polydispersity index (PDI) and the lower value of PDI ( ⁇ 0.2) indicates uniform and stable form of nanoemulsions.
• PDI polydispersity index
• ⁇ 0.2 indicates uniform and stable form of nanoemulsions.
• PDI values of drug encapsulated nanoemulsions were less than 0.1.
• the average surface charge of the oil droplets in the nanoemulsions was -28.9 mV (FIGURE 6C).
• the negative charge of the nanoemulsion could be due to the presence of free fatty acids of the fish oil used in the preparation of these nanoemulsions.
• Panc02 cells were treated with gemcitabine, AbraxaneTM, paclitaxel and DHA-SBT-1214 both in solution and nanoemulsion for 48 hours to determine whether they can induce PD-L1 protein expression.
• PD-L1 expression levels on tumor cells were determined by flow cytometry and is expressed as the D mean fluorescence intensity (AMFI; MFI using anti-PD-Ll subtracted from the isotype control).
• DHA-SBT-1214 treatment even at lOmg/kg in combination to PD-L1 antibody was more effective in suppressing tumor growth compared to standard chemotherapy drug, gemcitabine.
• Treatment with lOmg/kg DHA-SBT-1214 was superior to abraxane treatment at 120 mg/kg.
• a combinational treatment of NE-DHA-SBT-1214 with anti-PD-Ll antibody showed a synergistic effect compared with single treatment. As a crude proxy for toxicity there was no significant weight change within each treatment group as shown in FIGURE 11.
• PD-L1, PD-1, CD4, CD8 and Arginase-1 mRNA expression in pancreatic cancer tumor tissues the mRNA level of PD-L1, PD-1, CD4, CD8, and Arginase-1 either alone or in combination of immune checkpoint inhibitor was determined by RT-PCR.
• PD-L1 mRNA level was upregulated in combination therapy among all the anticancer agents compared to their respective IgG control groups as shown in FIGURE 12.
• PD-1 and CD4 mRNA level was lower in anti-PD-Ll plus anticancer agents except gemcitabine which was not significantly higher compared to its IgG treated group as shown in FIGURES 13 and 14 respectively.
• CD8 mRNA level was upregulated in response to combination treatment of all anticancer agents when combined with immune check point inhibitor compared to their IgG treated groups as shown in FIGURE 15 respectively.
• Arginase-1 level was significantly higher in IgG treatment group compared to their immune check point inhibitor as described in FIGURE 16.
• treatment of anticancer agents in combination to immune check point inhibitor also enhance PD-L1 protein expression level as shown in FIGURE 17.
• Data represent the mean ⁇ standard deviation of at least 3 independent experiments; *p ⁇ 0.05, **p ⁇ 0.01.
• PD-1 expression was also up-regulated except higher dose of NE-DHA-SBT-1214 compared to its IgG treatment group. Higher PD-L1 protein level might be attributed to presence of macrophages in this higher dose NE-DHA- SBT-1214 treated group, which is evident due to higher protein level of F4/80 in FIGURE 17.
• Applicants then examined the infiltration of CD4+, CD8+, and PD-1 cells in tumor tissues on day 21 by histology (FIGURES 18A-18J) and by immunohistochemistry (FIGURES 19A-19J, 20A-20J, and 21A-21J).
• the tumor tissue histology from different treatment groups showed that tumor from NE-DHA- SBT-1214 treated group has less dense stroma compared to solid tumor mass from other treatment groups (FIGURES 18A-18J).
• the images were taken at 63x magnification.
• Pancreatic cancer remains an intractable disease due to development of resistance to conventional anticancer agents.
• immune checkpoint inhibitors have not shown promising results when used as a single treatment regimen in many tumor types, especially in certain solid tumors, such as PDAC.
• PDAC chimeric antigen receptor
• Paclitaxel is still a front-line treatment for many solid tumor types, it initiates the apoptosis and causes cell cycle arrest at the G2/M stage. Taxanes, particularly paclitaxel in Cremophor-ethanol formulation (TAXOL ® ), have some toxicity issues due to its delivery vehicle and lack of tumor specific delivery.
• paclitaxel To further improve the efficiency of paclitaxel, numerous formulations as well as prodrugs of paclitaxel have been developed that increase its aqueous solubility, such as cyclodextrin, liposomes and albumin-bound nanoparticle (ABRAXANETM) formulations.
• ABRAXANETM albumin-bound nanoparticle
• some cancers including colon and prostate overexpress P-glycoprotein (Pgp), an effective ATP- binding cassette (ABC) transporter and effluxes paclitaxel, that is why paclitaxel is not effective against these cancers.
• Pgp colon and prostate overexpress P-glycoprotein
• ABSC effective ATP- binding cassette
• DHA conjugated drug has higher affinity for human serum albumin which is the primary carrier for PUFAs in the bloodstream, but in cancers which overexpress Pgp and/or other ABC transporters, when paclitaxel free itself from DHA in the presence of esterase, even though it will be released slowly but still be caught by the efflux pump(s) and eliminated from the cancer cells.
• SBT-1214 a new-generation taxoid, named SBT-1214, had showed excellent activity against drug-resistant cancer cells, which express MDR phenotypes.
• DHA- conjugated SBT-1214 improved therapeutic efficacy by increased accumulation of drug at the tumor site through the EPR effect.
• This colloidal system has desired particle size and zeta potential to preserve the stability of formulation in vitro and enhance its performance in vivo.
• the surface morphology DHA-SBT-1214 nanoemulsion formulation was spherical in morphology with no visible drug crystals. The qualitative cellular uptake analysis demonstrated that the nanoemulsion formulations were efficiently internalized in Panc02 cells.
• nanoemulsions did efficiently deliver the payload to the subcellular sites in the cell and was more potent than its drug solution.
• DHA-SBT-1214 suppressed tumor growth to a higher extent when delivered in nanoemulsion formulations emphasizing its higher therapeutic efficacy when used as stand alone therapy.
• nanoemulsion of the DHA-SBT-1214 conjugate induces superior regression and tumor growth inhibition and has high potential as a novel anti-cancer drug candidate.
• pancreatic cancer cell lines Panc02 were upregulated by paclitaxel, ABRAXANETM, DHA-SBT-1214 and gemcitabine.
• Peng reported that PD-L1 expression in ovarian cancer cell lines was augmented via NF-kB signaling by paclitaxel, gemcitabine or carboplatin treatment.
• Ghebeh, et al. reported that doxorubicin downregulated the surface expression of PD-L1 in breast cancer cells and upregulated nuclear expression of PD-L1.
• anticancer agents not only cause cytotoxicity, but also alter the tumor immune response, which may induce tumor immune escape.
• Applicants demonstrated anti-tumor effects of different anticancer agents in combination to PD-L1 blockade in vivo by using a syngeneic murine pancreas cancer model. It is well known that PD-1/PD-L1 interactions induce a negative regulation, which is critical for immune homeostasis after activation of T cells. This negative regulation is thought to be beneficial for cancer cells to escape from tumor-specific T-cell immunity.
• pancreas cancer cell line that showed PD-L1 blocking inhibited tumor development, although these studies have not used anticancer agents along with immune check point inhibitor.
• pancreas cancer model established by subcutaneous injection of murine pancreatic cancer cells into the mouse pancreas because cancer immunity is highly regulated by specie-specific leukocyte recruitment.
• blocking of PD-L1 reduced rate of tumor growth in our pancreas cancer model when used as a single treatment option or when used in combination with commonly used anticancer agents (Paclitaxel, Abraxane and Gemcitabine) for pancreatic cancer.
• combination of NE-DHA-SBT-1214 with PD- L1 blockade showed significant tumor suppression and kept tumors regressed even after treatment, showing that PD-L1 is a possible target for treatment of pancreas cancer.
• the increased IFN-gamma from infiltrating CD8+ cells in tumor tissue might contribute to the antitumor effect, because a large amount of IFN- gamma expression from effector T cells for a long period can induce infiltration of inflammatory cells such as Ml macrophages which enhance anti-tumor immunity. Macrophages in tumor microenvironment overexpress Arginase-1 indicating that these macrophages are Ml in addition to possible presence of MDSC.
• the suppressive effect of anti-PD-Ll antibody on tumor growth can be mainly explained by the increased number of tumor-infiltrating effector cells in NE- DHA-SBT-1214 combination treatment group.
• PD-L1 might attenuate tumor immunity in this cancer model by decreasing the infiltration of IFN-gamma-producing T cells and Ml macrophages.
• the same cells that were injected into mice to form a pancreatic tumor expressed very high levels of PD-L1 after IFN-gamma treatment in vitro.
• the number of tumor-infiltrating CD4+ T cells did not decrease after PD-L1 blockade.
• the results show that PD-L1 blockade can decrease the pancreatic tumor burden through synergistic effects of NE-DHA- SBT-1214.
• NE-DHA-SBT-1214 treated group has less dense stroma compared to the solid tumor mass from other treatment groups.
• the single therapy and the combination therapy of most commonly used anticancer agents unexpectedly did not show an additive anti-tumor effect except NE-DHA-SBT- 1214.
• One possible explanation for better efficacy of NE-DHA-SBT-1214 is its role in treating cancer stem cells as compared to other anti-cancer agents.
• Applicants' results indicate a significant tumor suppression by blocking PD-L1 in combination to NE-DHA-SBT-1214.
• Blockade of PD-L1 increased intra-tumoral IFN-gamma producing T cells and infiltration of inflammatory macrophages, which directly leads to the anti-tumor effect.
• both PD-1 and PD-L1 level was high in combination of commonly used anti-cancer agents emphasizing increased tumor infiltration of Treg cells, which might be primarily responsible for the non- anti-tumor effect.
• Taxol a unique antineoplastic agent with significant activity in advanced ovarian epithelial neoplasms. Ann Intern Med 1989;111: 273-9.
• Pardoll DM The blockade of immune checkpoints in cancer immunotherapy. Nature reviews Cancer 2012;12: 252-64.
• Nishimura H, Honjo T. PD-1 an inhibitory immunoreceptor involved in peripheral tolerance.
• PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting src homology 2-domain-containing tyrosine phosphatase 2 to phosphotyrosine. Proc Natl Acad Sci U S A 2001;98: 13866-71.

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