EP3600430A1 - Folate conjugate for use in targeting tumor associated macrophages - Google Patents
Folate conjugate for use in targeting tumor associated macrophagesInfo
- Publication number
- EP3600430A1 EP3600430A1 EP17903862.5A EP17903862A EP3600430A1 EP 3600430 A1 EP3600430 A1 EP 3600430A1 EP 17903862 A EP17903862 A EP 17903862A EP 3600430 A1 EP3600430 A1 EP 3600430A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- tumor
- cancer
- conjugate
- associated macrophages
- host animal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
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- 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
- A61K47/55—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 the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
- A61K47/551—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 the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being a vitamin, e.g. niacinamide, vitamin B3, cobalamin, vitamin B12, folate, vitamin A or retinoic acid
-
- 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
- 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
- A61K47/55—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 the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- the invention described herein relates to methods for treating cancers using a conjugate having the formula
- Conjugate 5 (hereinafter referred to as "Conjugate 5") ⁇
- Conjugate 5 also relates to methods for treating cancers using Conjugate 5 to target tumor associated macrophages.
- cancer still remains the second leading cause of death following heart disease in the United States.
- cancer is treated with chemotherapy utilizing highly potent drugs, such as mitomycin, paclitaxel and camptothecin.
- highly potent drugs such as mitomycin, paclitaxel and camptothecin.
- these chemotherapeutic agents show a dose responsive effect, and cell killing is proportional to drug dose.
- a highly aggressive style of dosing is thus necessary to eradicate neoplasms; however, high-dose chemotherapy is hindered by poor selectivity for cancer cells and severe toxicity to normal cells. This lack of tumor- specific treatment is one of the many hurdles that needs to be overcome by current chemotherapy.
- Folic acid is a member of the B family of vitamins and plays an essential role in cell survival by participating in the biosynthesis of nucleic and amino acids.
- This essential vitamin is also a high affinity ligand that enhances the specificity of conjugated anti-cancer drugs by targeting folate receptor-positive cancer cells.
- FR folate receptor
- the folate receptor is also found at high to moderate levels in kidney, brain, lung, and breast carcinomas. At the same time, it has been reported that the folate receptor occurs at low levels in most normal tissues leading to a mechanism for selectively targeting the cancer cells.
- the folate receptor can be used to deliver agents to tumor tissue with very high specificity, there are a number of cancers that do not express the folate receptor at all, or not in sufficient numbers to provide the desired specificity. Thus, there is a need for developing targeted therapies to deliver agents to such folate receptor negative cancers.
- TAMs Tumor-associated macrophages
- tumors and cancers that either overexpress the folate receptor or do not express the folate receptor in sufficient numbers, or at all, can be treated by targeting drugs to TAMs.
- Described herein are methods for treating cancers by targeting TAMs using Conjugate 5, or a pharmaceutically acceptable salt thereof, as a TAM- targeting agent.
- Applicants have discovered that a subset of TAMs that is pro-tumorigenic expresses the folate receptor ⁇ , also known as folate receptor 2.
- these pro-tumorigenic TAMs can be targeted using folate as the targeting ligand to deliver the conjugate to these TAMs to deplete or inhibit the pro-tumorigenic TAMs to treat cancer in a host animal whether or not the cancer cells themselves express folate receptors. It is to be understood that the methods described herein can be used to treat cancers that do not express the folate receptor, as well as cancers that do express the folate receptor.
- a method for treating a cancer comprises the steps of identifying the presence of tumor-associated macrophages in a cancer in a host animal, and administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof.
- a method for treating a cancer comprises the step of administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof, wherein the host animal has previously been administered a folate imaging agent conjugate and the host animal's folate receptor status has been determined to be negative.
- a method for treating a cancer in a host animal by inhibiting or depleting tumor-associated macrophages in the host animal comprises the step of administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof, wherein the tumor- associated macrophages are inhibited or depleted.
- a method for targeting tumor-associated macrophages in a host animal comprises the step of administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof, wherein the tumor-associated macrophages are targeted.
- a method for treating a cancer in a host animal where tumor-associated macrophages are part of the cancer, tissue, or tumor comprises the steps of administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof, and treating the cancer having the tumor-associated macrophages.
- Conjugate 5, or a pharmaceutically acceptable salt thereof includes a folate that binds to the folate receptor-a and/or the folate receptor- ⁇ .
- a method for treating a folate receptor negative cancer comprises administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof, wherein tumor-associated macrophages are inhibited or depleted.
- a method for treating a folate receptor negative cancer comprises administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof, to deplete tumor- associated macrophages.
- a method for treating a folate receptor negative cancer comprises administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof, and treating the folate receptor negative cancer having tumor- associated macrophages.
- a method for treating a folate receptor negative cancer in a host animal comprises administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof, to target tumor associated macrophages.
- a method for treating a folate receptor negative cancer comprising administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof, wherein tumor-associated macrophages are inhibited or depleted.
- a method for treating a folate receptor negative cancer comprising administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof, to deplete tumor-associated macrophages.
- a method for treating a folate receptor negative cancer comprising administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof, and treating the folate receptor negative cancer having tumor-associated macrophages.
- a method for treating a folate receptor negative cancer in a host animal comprising administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof, to target tumor associated macrophages.
- a method for treating a cancer comprising the steps of identifying the presence of tumor-associated macrophages in the cancer in a host animal, and administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof.
- a method for treating a cancer in a host animal comprising the step of administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof, to inhibit or deplete tumor- associated
- a method for targeting tumor-associated macrophages in a host animal comprising the step of administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof, to target the tumor- associated macrophages.
- a method for treating a cancer in a host animal where tumor-associated macrophages are in the cancer and/or form part of the tissue or tumor comprising the steps of administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof, and treating the cancer having the tumor- associated macrophages.
- tumor-associated macrophages are in the cancer and the tumor-associated macrophages are pro-tumor M2-biased and express one or more markers selected from the group consisting of CD163(+), IL10(+), Argl(+), TGF-p(+), VEGF(+), CD206(+), CD1 lb(+), and F480(+) phenotype.
- tumor-associated macrophages are in the cancer and/or form part of the tissue or cancer and the tumor-associated macrophages are pro-tumor M2-biased and express one or more markers selected from the group consisting of CD163(+), IL10(+), Argl(+), TGF-p(+), VEGF(+), CD206(+), CDl lb(+), and F480(+) phenotype.
- Conjugate 5 or a pharmaceutically acceptable salt thereof, is capable of inhibiting, or inhibits the activity of the tumor-associated macrophages in the host animal.
- parenteral dosage form is selected from the group consisting of intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, and intrathecal dosage forms.
- tumor-associated macrophages are in the cancer and the tumor-associated macrophages have the pro-tumor M2- biased CD163(+), IL10(+), Argl(+), TGF-p(+), VEGF(+), CD206(+), CDl lb(+), and F480(+) phenotype.
- the cancer may express folate receptors, or may not express folate receptors.
- tumor associated macrophages are in the cancer and the tumor- associated macrophages may have the pro-tumor M2-biased CD163(+) phenotype, the pro-tumor M2- biased CD163(+) and TGF-p(+) phenotype, the pro-tumor M2-biased CD163(+), IL10(+),
- Argl(+), TGF- ⁇ (+), VEGF(+), and CD206(+) phenotype, or the tumor-associated macrophages are pro-tumor M2-biased and may express one or more markers selected from the group consisting of CD163(+), IL10(+), Argl(+), TGF-p(+), VEGF(+), CD206(+), CDl lb(+), F480(+), CD163(+)CDl lb(+), and F480(+)CDl lb(+).
- the cancer can be selected from the group consisting of non-small cell lung cancer, anaplastic thyroid cancer, pancreatic ductal adenocarcinoma, head and neck cancer, epidermal growth factor receptor negative breast cancer, mesothelioma, adult classical Hodgkins lymphoma, uveal melanoma, glioblastoma, renal carcinoma, leiomyosarcoma, and pigmented villonodular synovitis.
- Conjugate 5 or a pharmaceutically acceptable salt thereof can be administered to the host animal in a parenteral dosage form.
- the parenteral dosage form can be selected from the group consisting of intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, and intrathecal.
- the therapeutically effective amount can be from about 0.1 ⁇ /kg to about 6.0 ⁇ /kg of
- Conjugate 5 or a pharmaceutically acceptable salt thereof; from about 0.1 ⁇ /kg to about 4.0 ⁇ /kg of Conjugate 5, or a pharmaceutically acceptable salt thereof; or from about 0.1 ⁇ /kg to about 2.0 ⁇ /kg of Conjugate 5, or a pharmaceutically acceptable salt thereof.
- FIG. 1 is a chart that shows the percentage of H-thymidine incorporated into KB cells treated with Conjugate 5 ( ⁇ ) and with Conjugate 5 and excess folate ( ⁇ ).
- FIG. 2A is a chart that shows that Conjugate 5 dosed at 0.5 ⁇ /kg SIW for two weeks ( A ) decreased KB tumor size in test mice compared to untreated control ( ⁇ ). The dotted line indicates the last dosing day.
- FIG. 2B is a chart that shows % weight change for test mice dosed at 0.5 ⁇ /kg Conjugate 5 SIW for two weeks ( A ) compared to untreated control ( ⁇ ).
- FIG. 3 is a chart showing that mice bearing paclitaxel resistant KB tumors dosed at 0.5 ⁇ /kg SIW for two weeks with Conjugate 5 ( A ) had decreased tumor size compared to untreated control ( ⁇ ). The dotted line indicates the last dosing day.
- n 5, Conjugate 5 ⁇ 0,1,4 ⁇ as ⁇ partial response, complete response, cure ⁇ .
- FIG. 4 is a chart showing that mice bearing platinum resistant KB tumors dosed at 0.5 ⁇ /kg SIW for two weeks with Conjugate 5 ( ⁇ ), and dosed at 2.0 ⁇ /kg BIW for two weeks with EC 1456 ( T ) had decreased tumor size compared to untreated control ( ⁇ ). The dotted line indicates the last dosing day.
- n 4, Conjugate 5 ⁇ 0,0,4 ⁇ ; EC1446 ⁇ 0,2,2 ⁇ as ⁇ partial response, complete response, cure ⁇ .
- FIG. 5 is a chart showing that mice bearing ST502 TNBC PDX tumors dosed at 0.3 ⁇ /kg BIW for two weeks with Conjugate 5 ( A ) had decreased tumor size compared to untreated control ( ⁇ ), while mice dosed at 2.0 ⁇ /kg BIW for two weeks with EC1456 ( ⁇ ) did not have decreased tumor size compared to untreated control ( ⁇ ).
- the dotted line indicates the last dosing day.
- n 7, Conjugate 5 ⁇ 0,0,7 ⁇ as ⁇ partial response, complete response, cure ⁇ .
- FIG. 6 is a chart showing that mice bearing ST070 ovarian PDX tumors dosed at 0.5 ⁇ /kg SIW for two weeks with Conjugate 5 ( ⁇ ) had decreased tumor size compared to untreated control ( ⁇ ), while mice dosed at 4.0 ⁇ /kg SIW for two weeks with EC1456 (A) or dosed at 15.0 mg/kg SIW for two weeks with paclitaxel (T ) did not have decreased tumor size.
- the dotted line indicates the last dosing day.
- n 7, Conjugate 5 ⁇ 0,0,7 ⁇ as ⁇ partial response, complete response, cure ⁇ .
- FIG. 7 is a chart that shows the relative binding affinity of Conjugate 5 toward the folate receptor. The experiment shows that the relative binding affinity of Conjugate 5 was ⁇ 1.9-fold lower than that of folic acid. ( ⁇ ) folic acid (Control); ( ⁇ ) Conjugate 5.
- FIG. 8 is a graph that shows that intact Conjugate 5 is not able to crosslink DNA while the reduced form (treated with DTT) releases the active PBD molecule, which can then crosslink with DNA.
- FIG. 9A is a chart that shows that Conjugate 5 dosed at 0.1 ⁇ /kg SIW for two weeks ( ⁇ ) and Conjugate 5 dosed at 0.15 ⁇ /kg SIW for two weeks (A) decreased KB tumor size in test rats compared to untreated control ( ⁇ ). The dotted line indicates the last dosing day.
- FIG. 9B is a chart that shows % weight change for test rats dosed at 0.1 ⁇ /kg Conjugate 5 SrW for two weeks ( ⁇ ) and test mice dosed at 0.15 ⁇ /kg Conjugate 5 SrW for two weeks (A ) compared to untreated control ( ⁇ ).
- FIG. 10 is a chart that shows that Conjugate 5 dosed at 0.27 ⁇ /kg ⁇ for two weeks ( ⁇ ) decreased TNBC PDX tumor size in test mice compared to untreated control ( ⁇ ), whereas erubulin mesylate dosed at 1.0 ⁇ /kg SIW for two weeks ( A ) did not decrease TNBC PDX tumor size.
- FIG. 11 is a chart that shows that Conjugate 5 dosed at 0.27 ⁇ /kg ⁇ for two weeks ( ⁇ ) produced partial response in Endometrial PDX tumor size in test mice compared to untreated control ( ⁇ ), whereas paclitaxel dosed at 15.0 mg/kg SIW for two weeks (A ) did not produce a partial response.
- FIG. 12 is a chart showing a potent dose-dependent inhibition of cell
- FIG. 13 is a graph showing that Conjugate 5 demonstrated a potent activity at all concentrations tested (1 nM, 10 nM and 100 nM) after a 2 hourexposure and 9-day chase.
- the anti-tumor activity of Conjugate 5 was significantly reduced in the presence of excess amount of folic acid at both 1 nM and 10 nM concentrations.
- FIG. 15A is chart showing the presence of CD4+ and CD8+ T cells quantitated in total peritoneal cells of the immunocompetent C57BL6 mice at 7 day intervals post IP injection of the mouse ovarian cell line, ID8-CL15 (FIG. 15A).
- the CD45+ CD3e+ CD8+ CD4- T cells ( ⁇ ) slowly increased in number from day 7 to day 42 post implantation.
- the CD45+ CD3e+ CD4+ CD8- T cells ( ⁇ ) also increased in number from day 7 to day 35.
- FIG. 15B is a chart showing CD45- non bone-marrow derived ascites cells from ID8-CL15 implanted mice expressed very little functional FR (see FIG. 15B ( ⁇ )), whereas ascites macrophages expressed a significant amount of a functional FR (see FIG. 15B ( ⁇ )).
- FIG. 15C is a graph showing ascites macrophages expressed a significant amount of a functional FR.
- FIG. 16A is a chart that shows that Conjugate 5 dosed at 100 nmol/kg BIW, 6 doses, first dose at day 7 ( A ) increased survival time in test mice compared to untreated control ( ⁇ ) and anti-CTLA-5 alone dosed at 250 ⁇ g/dose BIW, 5 doses, and comparable to a significantly higher dose of comparator compound EC 1456 (T ) 2000nmol/kg BIW, 6 doses, first dose at day 7.
- FIG. 16A also shows that Conjugate 5 dosed with anti-CTLA-5, initiated at day 11, (o) increased survival time in test mice compared to all other test animals. The dotted line indicates the last dosing day.
- FIG. 16B is a chart that shows % weight change for test mice dosed with
- FIG. 17A is a chart that shows Conjugate 5 dosed at 0.1 ⁇ /kg, BIW x 3, 6 doses, first dose at 7 days (o) increased survival time in test mice compared to significantly higher dose of comparator compound EC 1456 dosed at 2 ⁇ /kg, BIW x 3, 6 doses, first dose at 7 days (T) and untreated control ( ⁇ ).
- FIG. 17B is a chart that shows % weight change for test mice dosed with Conjugate 5 (o), EC1456 (T ), and an untreated control ( ⁇ ) as described in 1A.
- FIG. 18B is a chart that shows % weight change for test mice dosed with
- FIG. 19A is a chart that shows Conjugate 5 dosed at 0.3 ⁇ /kg, D35, D42, SIW x 2 (o) increased survival time in test mice compared to significantly higher dose of comparator compound EC1456 dosed at 2 ⁇ /kg, DO-2 x 2 (T ), and untreated control ( ⁇ ).
- FIG. 19B is a chart that shows % weight change for test mice dosed with
- FIG. 20A is a chart that shows Conjugate 5 dosed at 0.3 ⁇ /kg, SIW x 2 (o) increased survival time in test mice compared to significantly higher dose of comparator compound EC1456 dosed at 2 ⁇ /kg, DO-2 x 1 (T), and untreated control ( ⁇ ).
- FIG. 20B is a chart that shows % weight change for test mice dosed with
- FIG. 21 is a comparison of Conjugate 5 and EC1456 against various stages of ID8-C115 tumor bearing mice.
- FIG. 22A is a comparison of Conjugate 5 in- vitro activity against 4T1-C12 tumor cells.
- FIG. 22B is comparison of Conjugate 5 in-vitro activity against 4Tlp tumor cells.
- FIG. 23 is a comparison of Conjugate 5 and EC 1456 in-vitro activity against human IGROV Cells after a 2 hour exposure and 9-day chase.
- FIG. 24A is an assessment of tumor-associated macrophages in 4Tlp and 4T1-
- FIG. 24B shows tumor-associated macrophages found in 4Tlp tumors expressed
- FIG. 24C shows tumor- associated macrophages found in 4Tlp tumors expressed FRP while other non-macrophage myeloid cells (MDSCs) were FR -negative.
- FIG. 25A is a chart showing P-1780 4T1P Balb/c mice tumor volume DOI 4/20/16 5xl0 5 mammary tumors with Conjugate 5 treatment at 200 nmol/kg (BIW x 2) (A) versus an untreated control (B).
- FIG. 25B is a chart that shows % weight change for test mice dosed with
- FIG. 26A is a chart showing P-1780 4T1P Balb/c mice tumor volume DOI 4/7/16 5xl0 6 mammary tumors with Conjugate 5 treatment at 200 nmol/kg ( ⁇ x 2) (A) versus an untreated control (B).
- FIG. 26B is a chart that shows % weight change for test mice dosed with
- FIG. 27 contains charts demonstrating apoptotic CD163-CDl lb-, CD163- CDl lb+, and CD163+CDl lb+ when treatment of untreated control ( ⁇ ), Conjugate 5 ( ⁇ ), Conjugate 5 + EC0923 ( A ), and EC0923 (T).
- untreated control ⁇
- Conjugate 5 ⁇
- Conjugate 5 + EC0923 A
- EC0923 T
- TAMs for example, pro-tumor M2-biased TAMs
- Conjugate 5 or a pharmaceutically acceptable salt thereof
- a subset of TAMs that is pro-tumorigenic expresses the folate receptor ⁇ which is useful for targeting TAMs with Conjugate 5, or a pharmaceutically acceptable salt thereof, using folates as targeting agents.
- targeting of the pro-tumorigenic TAMs to deplete TAMs or to inhibit the activity of TAMs can result in inhibition of tumor growth, elimination of a tumor, or stable disease, and like therapeutic effects for the host animal.
- the methods described herein can be used to treat cancers that do not express the folate receptor, as well as cancers that do express the folate receptor.
- the tumor-associated macrophages described herein are pro- tumor and M2-biased, and, if depleted or inhibited, the host animal's condition may be improved.
- TAMs may have a phenotype resulting from the expression of one or more markers selected from CD163(+), IL10(+), Argl(+), TGF-p(+), VEGF(+), CD206(+), CDl lb(+), F480(+), CD163(+)CDl lb(+), F480(+)CDl lb(+) and combinations thereof.
- the tumor-associated macrophages described herein that are pro- tumor and M2-biased have a CD163(+) phenotype.
- the tumor- associated macrophages described herein that are pro-tumor and M2-biased have a CD163(+) and TGF-P(+) phenotype.
- the tumor- associated macrophages described herein that are pro-tumor and M2-biased have a CD163(+) and CD1 lb(+) phenotype.
- the tumor-associated macrophages described herein that are pro- tumor and M2-biased have a F480(+) and CD1 lb(+) phenotype.
- the tumor- associated macrophages described herein that are pro-tumor and M2-biased have a phenotype resulting from the expression of CD163(+), IL10(+), Argl(+), TGF-p(+), VEGF(+),
- the tumor-associated macrophages described herein that are pro-tumor and M2-biased have a phenotype resulting from the expression of one or more markers selected from the group consisting of CD163(+), IL10(+), Argl(+), TGF-p(+), VEGF(+), and CD206(+), CDl lb(+), and F480(+).
- the presence of the tumor-associated macrophages (e.g., pro-tumor M2-biased TAMs) in the tumor indicates a poor prognosis for the host animal without the therapy described herein.
- the method comprises the steps of identifying the presence of tumor- associated macrophages (e.g., pro-tumor M2-biased TAMs) in a cancer in a host animal, and administering to the host animal a therapeutically effective amount of Conjugate 5 or a pharmaceutically acceptable salt thereof.
- tumor-associated macrophages e.g., pro-tumor M2-biased TAMs
- a method for treating a cancer by targeting TAMs comprises the step of administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof, wherein the host animal has previously been administered a folate imaging agent conjugate and the host animal's folate receptor status has been determined to be negative.
- a method for treating a cancer in a host animal by inhibiting or depleting tumor-associated macrophages e.g., pro-tumor M2-biased TAMs
- the method comprises the step of administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof, wherein the tumor-associated macrophages are inhibited or depleted.
- a method of targeting tumor-associated macrophages e.g., pro-tumor M2-biased TAMs
- the method comprises the step of administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof, wherein the tumor-associated macrophages are targeted.
- a method for treating a cancer in a host animal wherein tumor- associated macrophages are in the cancer comprises the steps of administering to the host animal a therapeutically effective amount Conjugate 5, or a pharmaceutically acceptable salt thereof, and treating the cancer having the tumor-associated macrophages (e.g., pro-tumor M2-biased TAMs).
- Conjugate 5 includes a folate that binds the folate receptor-a and/or the folate receptor- ⁇ .
- tumor-associated macrophages are in the cancer
- the tumor associated macrophages e.g., pro-tumor M2-biased TAMs
- a cancer e.g., a tumor
- cancerous tissue e.g., tumor tissue
- the methods described herein are used to treat a "host animal" with cancer in need of such treatment.
- the methods described herein can be used for both human clinical medicine and veterinary applications.
- a "host animal" can be
- the host animal can be human (e.g., a human patient) or, in the case of veterinary applications, can be a laboratory, agricultural, domestic, or wild animal.
- the host animal can be a human, a laboratory animal such as a rodent (e.g.
- mice, rats, hamsters, etc. mice, rats, hamsters, etc.
- a rabbit, a monkey, a chimpanzee domestic animals such as dogs, cats, and rabbits
- agricultural animals such as cows, horses, pigs, sheep, goats, and wild animals in captivity such as bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins, and whales.
- the cancer described herein can be a cancer cell population that is tumorigenic, including benign tumors and malignant tumors, or the cancer can be non-tumorigenic.
- the cancer can arise spontaneously or by such processes as mutations present in the germline of the host animal or by somatic mutations, or the cancer can be chemically-, virally-, or radiation-induced.
- Cancers applicable to the invention described herein include, but are not limited to, a carcinoma, a sarcoma, a lymphoma, a melanoma, a mesothelioma, a nasopharyngeal carcinoma, a leukemia, an adenocarcinoma, and a myeloma.
- the cancer can be lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head, cancer of the neck, cutaneous melanoma, intraocular melanoma uterine cancer, ovarian cancer, endometrial cancer, rectal cancer, stomach cancer, colon cancer, breast cancer, triple negative breast cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, non-small cell lung cancer, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, prostate cancer, leukemia, lymphoma, pleural mesothelioma, cancer of the bladder, Burkitt's lymphoma, cancer of the ureter, cancer of the kidney, neoplasms of the central nervous system, brain cancer, pituitary
- the cancers can be selected from the group consisting of non- small cell lung cancer, anaplastic thyroid cancer, pancreatic ductal adenocarcinoma, head and neck cancer, epidermal growth factor receptor negative breast cancer, mesothelioma, adult classical Hodgkins lymphoma, uveal melanoma, glioblastoma, renal carcinoma,
- Any cancer that has tumor-associated macrophages e.g., pro-tumor M2-biased TAMs
- TAMs tumor-associated macrophages
- Conjugate 5 described herein is the compound having the formula
- a pharmaceutically acceptable salt of Conjugate 5 can also be used.
- a method for treating a folate receptor negative cancer comprising administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof, wherein tumor-associated macrophages are inhibited or depleted.
- a method for treating a folate receptor negative cancer comprising administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof, to deplete tumor-associated macrophages.
- a method for treating a folate receptor negative cancer comprising administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof, and treating the folate receptor negative cancer having tumor-associated macrophages.
- a method for treating a folate receptor negative cancer in a host animal comprising administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof, to target tumor associated macrophages.
- a method for treating a cancer comprising the steps of identifying the presence of tumor-associated macrophages in the cancer in a host animal, and administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof.
- a method for treating a cancer in a host animal comprising the step of administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof, to inhibit or deplete tumor- associated
- a method for targeting tumor-associated macrophages in a host animal comprising the step of administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof, to target the tumor- associated macrophages.
- a method for treating a cancer in a host animal where tumor-associated macrophages are in the cancer and/or form part of the tissue or tumor comprising the steps of administering to the host animal a therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof, and treating the cancer having the tumor- associated macrophages.
- tumor-associated macrophages are in the cancer and the tumor-associated macrophages are pro-tumor M2-biased and express one or more markers selected from the group consisting of CD163(+), IL10(+), Argl(+), TGF-p(+), VEGF(+), CD206(+), CDl lb(+), and F480(+) phenotype.
- tumor-associated macrophages are in the cancer and/or form part of the tissue or cancer and the tumor-associated macrophages are pro-tumor M2-biased and express one or more markers selected from the group consisting of CD163(+), IL10(+), Argl(+), TGF-p(+), VEGF(+), CD206(+), CD1 lb(+), and F480(+) phenotype.
- the cancer is selected from the group consisting of non- small cell lung cancer, anaplastic thyroid cancer, pancreatic ductal adenocarcinoma, head and neck cancer, epidermal growth factor receptor negative breast cancer, mesothelioma, adult classical Hodgkin's lymphoma, uveal melanoma, glioblastoma, renal carcinoma, leiomyosarcoma, and pigmented villonodular synovitis.
- the cancer is selected from the group consisting of non- small cell lung cancer, anaplastic thyroid cancer, pancreatic ductal adenocarcinoma, head and neck cancer, epidermal growth factor receptor negative breast cancer, mesothelioma, adult classical Hodgkin's lymphoma, uveal melanoma, glioblastoma, renal carcinoma, leiomyosarcoma, and pigmented villonodular synovitis.
- Conjugate 5 or a pharmaceutically acceptable salt thereof, is capable of inhibiting, or inhibits the activity of the tumor-associated macrophages in the host animal.
- tumor-associated macrophages are in the cancer and the tumor-associated macrophages have the pro-tumor M2- biased CD163(+), IL10(+), Argl(+), TGF-p(+), VEGF(+), CD206(+), CDl lb(+), and F480(+) phenotype.
- the dosage of Conjugate 5, or a pharmaceutically acceptable salt thereof can vary significantly depending on the condition of the host animal, the cancer being treated, the route of administration of Conjugate 5, or a pharmaceutically acceptable salt thereof, and tissue distribution, and the possibility of co-usage of other therapeutic treatments, such as radiation therapy or additional drugs in combination therapies.
- the therapeutically effective amount to be administered to a host animal is based on body surface area, mass, and physician assessment of condition of the host animal.
- Therapeutically effective amounts can range, for example, from about 0.05 mg/kg of host animal weight to about 30.0 mg/kg of host animal weight, or from about 0.01 mg/kg of host animal weight to about 5.0 mg/kg of host animal weight, including but not limited to 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 3.0 mg/kg, 3.5 mg/kg, 4.0 mg/kg, 4.5 mg/kg, and 5.0 mg/kg, all of which are kg of host animal weight.
- pharmaceutically acceptable salt thereof may be administered in single or divided doses and may, at the physician's discretion, fall outside of the typical range given herein.
- Conjugate 5, or a pharmaceutically acceptable salt thereof can be administered in a therapeutically effective amount of from about 0.5 ⁇ g/m to about 500 mg/m 2 , from about 0.5 ⁇ g/m 2 to about 300 mg/m 2 , or from about 100 ⁇ g/m 2 to about
- the amounts can be from about 0.5 mg/m 2 to about 500 mg/m 2 , from about 0.5 mg/m 2 to about 300 mg/m 2 , from about 0.5 mg/m 2 to about 200 mg/m 2 , from about 0.5 mg/m 2 to about 100 mg/m 2 , from about 0.5 mg/m 2 to about 50 mg/m 2 , from about 0.5 mg/m 2 to about 600 mg/m 2 , from about 0.5 mg/m 2 to about 6.0 mg/m 2 , from about
- the total amount may be administered in single or divided doses and may, at the physician's discretion, fall outside of the typical range given herein. These amounts are based on m of host animal surface area.
- Conjugate 5, or a pharmaceutically acceptable salt thereof can be administered in a therapeutically effective amount of from about 0.05 ⁇ /kg to about 6.0 ⁇ /kg, from about 0.05 ⁇ /kg to about 5.0 ⁇ /kg, from about 0.05 ⁇ /kg to about 4.0 ⁇ /kg, from about 0.05 ⁇ /kg to about 3.0 ⁇ /kg, from about 0.05 ⁇ /kg to about 2.0 ⁇ /kg, from about 0.05 ⁇ /kg to about 1.0 ⁇ /kg, from about 0.05 ⁇ /kg to about 0.5 ⁇ /kg, from about 0.05 ⁇ /kg to about 0.4 ⁇ /kg, from about 0.05 ⁇ /kg to about 0.3 ⁇ /kg, from about 0.05 ⁇ /kg to about 0.2 ⁇ /kg, or from about 0.05 ⁇ /kg to about 0.1 ⁇ /kg.
- the total therapeutically effective amount of Conjugate 5, or a pharmaceutically acceptable salt thereof may be administered in single or divided doses and may, at the physician's discretion, fall outside of the typical range given herein. In each case, these amounts are "kg" of host animal weight.
- Conjugate 5, or a pharmaceutically acceptable salt thereof can be administered as single doses, or it can be divided and
- the host animal is treated with multiple injections of Conjugate 5, or a pharmaceutically acceptable salt thereof.
- the host animal for example, may be injected multiple times with Conjugate 5, or a pharmaceutically acceptable salt thereof, for example, at 12-72 hour intervals or at 48-72 hour intervals.
- Additional injections of Conjugate 5, or a pharmaceutically acceptable salt thereof can be administered to the host animal at intervals of days or months after the initial injections, and the additional injections prevent recurrence of disease.
- Conjugate 5, or a pharmaceutically acceptable salt thereof can be administered to the host animal, for example, for at least one hour, at least four hours, at least six hours, at least eight hours, at least ten hours, at least twelve hours, or at least twenty-four hours, or can be administered daily or weekly, such as once a day, two times a day, three times a day, every day, every other day, two times weekly, three times weekly, or any other suitable regimen may be used.
- an imaging agent linked to a folate can be used to determine folate receptor status, and/or whether the cancer expresses folate receptors, and/or to identify the presence of TAMs associated with cancers.
- Exemplary folate-linked imaging agents are described in U.S. Patent Nos. 7,128,893 and 9,731,035, incorporated herein by reference.
- TAMs tumor associated macrophages
- TAMs tumor associated macrophages
- tumor associated macrophages generally refers to reducing the activity or eliminating the activity of TAMs, such as by reducing or eliminating the ability of TAMs to stimulate angiogenesis in tumor tissue.
- tumor associated macrophages generally refers to reducing the number of TAMs, eliminating TAMs, or repolarizing TAMs, including causing TAMs to shift from an M2 to an Ml phenotype.
- pro-tumor with reference to TAMs generally refers to TAMs that enhance tumorgenesis, such as, for example, by inhibiting B and/or T cell activation, inhibiting tumor-associated antigen presentation, inhibiting cytotoxic granule release, and/or increasing angiogenesis.
- M2-biased generally refers to TAMs that are pro- tumor TAMs which may include TAMS that are Ml and that may shift from an Ml to M2 phenotype.
- composition generally refers to any product comprising more than one ingredient. It is to be understood that the compositions described herein may be prepared from isolated Conjugate 5 described herein or from salts, solutions, hydrates, solvates, and other forms of Conjugate 5 described herein. It is appreciated that certain functional groups, such as the hydroxy, amino, and like groups may form complexes with water and/or various solvents, in the various physical forms of Conjugate 5. It is also to be understood that the compositions may be prepared from various amorphous, non- amorphous, partially crystalline, crystalline, and/or other morphological forms of Conjugate 5, or a pharmaceutically acceptable salt thereof, described herein.
- compositions may be prepared from various hydrates and/or solvates of Conjugate 5, or a pharmaceutically acceptable salt thereof, described herein. Accordingly, such pharmaceutical compositions that recite Conjugate 5, or a pharmaceutically acceptable salt thereof, described herein are to be understood to include each of, or any combination of, the various morphological forms and/or solvate or hydrate forms of Conjugate 5, or a pharmaceutically acceptable salt thereof, described herein.
- the term "therapeutically effective amount” refers to an amount of the conjugate, or pharmaceutically acceptable salt thereof, that elicits the biological or medicinal response in a subject (i.e. a tissue system, animal or human) that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes, but is not limited to, alleviation of the symptoms of the disease or disorder being treated.
- the therapeutically effective amount is that amount of an active which may treat or alleviate the disease or symptoms of the disease at a reasonable benefit/risk ratio applicable to any medical treatment.
- the therapeutically effective amount is that amount of an inactive prodrug of Conjugate 5, which when converted through normal metabolic processes to produce an amount of active Conjugate 5, or a pharmaceutically acceptable salt thereof, capable of eliciting the biological or medicinal response in a subject that is being sought.
- the co-therapies described herein may allow for the administration of lower doses of Conjugate 5, or a pharmaceutically acceptable salt thereof, that show such toxicity, or other undesirable side effect, where those lower doses are below thresholds of toxicity or lower in the therapeutic window than would otherwise be administered in the absence of a cotherapy.
- administering includes all means of introducing Conjugate 5, or a pharmaceutically acceptable salt thereof, described herein to the host animal, including, but are not limited to, oral (po), intravenous (iv), intramuscular (im), subcutaneous (sc),
- conjugates and compositions described herein may be administered in unit dosage forms and/or
- formulations containing conventional nontoxic pharmaceutically-acceptable carriers, adjuvants, and/or vehicles.
- composition refers to a mixture of Conjugate 5 described herein, or pharmaceutically acceptable salts, solvates, hydrates thereof, with other chemical components, such as pharmaceutically acceptable excipients.
- the purpose of a pharmaceutical composition is to facilitate administration of a conjugate to a host animal.
- Pharmaceutical compositions suitable for the delivery of Conjugate 5, or a pharmaceutically acceptable salt thereof, described herein and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, for example, in 'Remington's Pharmaceutical Sciences', 19th Edition (Mack Publishing Company, 1995).
- a “pharmaceutically acceptable excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of Conjugate 5, or a
- Conjugate 5, or a pharmaceutically acceptable salt thereof, described herein can be utilized to treat such cancers as carcinomas, sarcomas, lymphomas, Hodgekin's disease, melanomas, mesotheliomas, Burkitt's lymphoma, nasopharyngeal carcinomas, leukemias, and myelomas; including associated cancers resistant to treatment modalities, such as therapeutic agents.
- Resistant cancers include, but are not limited to paclitaxel resistant cancers, and platinum resistant cancers, such as those cancers resistant to platinum drugs, such as cisplatin, carboplatin, oxaplatin, nedaplatin, and the like.
- the cancer cell population can include, but is not limited to, oral, thyroid, endocrine, skin, gastric, esophageal, laryngeal, pancreatic, colon, bladder, bone, ovarian, cervical, uterine, breast, testicular, prostate, rectal, kidney, liver, stomach and lung cancers.
- the cancer cell population produces a cancer, such as lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, triple negative breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, chronic or acute leukemia, lymphocytic
- a cancer such as lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, ovarian cancer
- lymphomas cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, spinal axis tumors, brain stem glioma and pituitary adenoma.
- CNS central nervous system
- the Conjugate 5, or a pharmaceutically acceptable salt thereof, or compositions described herein may be administered orally.
- Oral administration may involve swallowing, so that the Conjugate 5, or a pharmaceutically acceptable salt thereof, or composition enters the gastrointestinal tract, or buccal or sublingual administration may be employed by which the Conjugate 5, or a pharmaceutically acceptable salt thereof, or composition enters the blood stream directly from the mouth.
- Formulations suitable for oral administration include solid formulations such as tablets, capsules containing particulates, liquids, or powders, lozenges (including liquid-filled), chews, multi- and nano-particulates, gels, solid solution, liposome, films, ovules, sprays and liquid formulations.
- Liquid formulations include suspensions, solutions, syrups and elixirs. Such formulations may be employed as fillers in soft or hard capsules and typically comprise a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents.
- a carrier for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents.
- formulations may also be prepared by the reconstitution of a solid, for example, from a sachet.
- the Conjugate 5, or a pharmaceutically acceptable salt thereof, described herein may also be used in fast-dissolving, fast disintegrating dosage forms such as those described in Expert Opinion in Therapeutic Patents, 11 (6), 981-986, by Liang and Chen (2001).
- the Conjugate 5, or a pharmaceutically acceptable salt thereof may make up from 1 weight % to 80 weight % of the dosage form, more typically from 5 weight % to 60 weight % of the dosage form.
- the Conjugate 5, or a pharmaceutically acceptable salt thereof may make up from 1 weight % to 80 weight % of the dosage form, more typically from 5 weight % to 60 weight % of the dosage form.
- tablets generally contain a disintegrant.
- disintegrants include sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methyl cellulose, microcrystalline cellulose, lower alkyl- substituted hydroxypropyl cellulose, starch, pregelatinised starch and sodium alginate.
- the disintegrant will comprise from 1 weight % to 25 weight %, preferably from 5 weight % to 20 weight % of the dosage form.
- Binders are generally used to impart cohesive qualities to a tablet formulation. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinised starch, hydroxypropyl cellulose and hydroxypropyl methylcellulose. Tablets may also contain diluents, such as lactose
- Tablets may also optionally comprise surface active agents, such as sodium lauryl sulfate and polysorbate 80, and glidants such as silicon dioxide and talc.
- surface active agents such as sodium lauryl sulfate and polysorbate 80
- glidants such as silicon dioxide and talc.
- surface active agents may comprise from 0.2 weight % to 5 weight % of the tablet, and glidants may comprise from 0.2 weight % to 1 weight % of the tablet.
- Tablets also generally contain lubricants such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulphate.
- Lubricants generally comprise from 0.25 weight % to 10 weight %, preferably from 0.5 weight % to 3 weight % of the tablet.
- Other possible ingredients include anti-oxidants, colorants, flavoring agents, preservatives and taste-masking agents.
- Exemplary tablets contain up to about 80% drug, from about 10 weight % to 25 about 90 weight % binder, from about 0 weight % to about 85 weight % diluent, from about 2 weight % to about 10 weight % disintegrant, and from about 0.25 weight % to about 10 weight % lubricant.
- Tablet blends may be compressed directly or by roller to form tablets. Tablet blends or portions of blends may alternatively be wet-, dry-, or melt-granulated, melt congealed, or extruded before tableting.
- the final formulation may comprise one or more layers and may be coated or uncoated; it may even be encapsulated.
- the formulation of tablets is discussed in Pharmaceutical Dosage Forms: Tablets, Vol. 1, by H. Lieberman and L. Lachman (Marcel Dekker, New York, 1980).
- Consumable oral films for human or veterinary use are typically pliable water- soluble or water-swellable thin film dosage forms which may be rapidly dissolving or mucoadhesive and typically comprise Conjugate 5, or a pharmaceutically acceptable salt thereof, as described herein, a film-forming polymer, a binder, a solvent, a humectant, a plasticizer, a stabilizer or emulsifier, a viscosity-modifying agent and a solvent.
- Some components of the formulation may perform more than one function.
- Solid formulations for oral administration may be formulated to be immediate and/or modified release.
- Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
- Conjugate 5, or a pharmaceutically acceptable salt thereof, described herein can be formulated as a solid, semi- solid, or thixotropic liquid for administration as an implanted depot providing modified release of the active compound.
- formulations include drug-coated stents and poly(lactic-coglycolic)acid (PGLA) microspheres.
- PGLA poly(lactic-coglycolic)acid
- Other suitable modified release formulations for the purposes of the disclosure are described in US Patent No.6, 106,864. Details of other suitable release technologies such as high energy dispersions and osmotic and coated particles are to be found in Pharmaceutical Technology Online, 25(2), 1-14, by Verma et al (2001). The use of chewing gum to achieve controlled release is described in WO 00/35298.
- the Conjugate 5, or a pharmaceutically acceptable salt thereof, described herein can also be administered directly into the blood stream, into muscle, or into an internal organ.
- Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular and subcutaneous.
- Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques.
- Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.
- excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9)
- a suitable vehicle such as sterile, pyrogen-free water.
- parenteral formulations under sterile conditions may readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art.
- solubility of Conjugate 5, or a pharmaceutically acceptable salt thereof, described herein used in the preparation of parenteral solutions may be increased by the use of appropriate formulation techniques, such as the incorporation of solubility-enhancing agents.
- the Conjugate 5, or a pharmaceutically acceptable salt thereof, described herein can also be administered topically to the skin or mucosa, that is, dermally or transdermally.
- Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibres, bandages and microemulsions. Liposomes may also be used.
- Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol and propylene glycol. Penetration enhancers may be incorporated - see, for example, J.
- Formulations for topical administration may be formulated to be immediate and/or modified release.
- Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
- the Conjugate 5, or a pharmaceutically acceptable salt thereof, described herein can also be administered intranasally or by inhalation, typically in the form of a dry powder (either alone, as a mixture, for example, in a dry blend with lactose, or as a mixed component particle, for example, mixed with phospholipids, such as phosphatidylcholine) from a dry powder inhaler or as an aerosol spray from a pressurized container, pump, spray, atomizer (preferably an atomizer using electrohydrodynamics to produce a fine mist), or nebulizer, with or without the use of a suitable propellant, such as 1,1, 1,2-tetrafluoroethane or 1,1, 1,2,3,3, 3-heptafluoropropane.
- a suitable propellant such as 1,1,
- the powder may comprise a bioadhesive agent, for example, chitosan or cyclodextrin.
- a bioadhesive agent for example, chitosan or cyclodextrin.
- the pressurized container, pump, spray, atomizer, or nebulizer contains a solution or suspension of the
- Conjugate 5 or a pharmaceutically acceptable salt thereof, of the present disclosure comprising, for example, ethanol, aqueous ethanol, or a suitable alternative agent for dispersing,
- the Conjugate 5, or a pharmaceutically acceptable salt thereof may be micronized to a size suitable for delivery by inhalation (typically less than 5 microns). This may be achieved by any appropriate comminuting method, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenization, or spray drying.
- Capsules made, for example, from gelatin or
- blisters and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of the Conjugate 5, or a pharmaceutically acceptable salt thereof, described herein, a suitable powder base such as lactose or starch and a performance modifier such as Iso-leucine, mannitol, or magnesium stearate.
- the lactose may be anhydrous or in the form of the monohydrate, preferably the latter.
- suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.
- a typical formulation may comprise Conjugate 5, or a pharmaceutically acceptable salt thereof, of the present disclosure, propylene glycol, sterile water, ethanol and sodium chloride.
- Alternative solvents which may be used instead of propylene glycol include glycerol and polyethylene glycol.
- Conjugate 5, or a pharmaceutically acceptable salt thereof, described here can be combined with soluble macromolecular entities, such as cyclodextrin and suitable derivatives thereof or polyethylene glycolcontaining polymers, in order to improve their solubility, dissolution rate, taste-masking, bioavailability and/or stability for use in any of the aforementioned modes of administration.
- soluble macromolecular entities such as cyclodextrin and suitable derivatives thereof or polyethylene glycolcontaining polymers
- n is 0, or n is 1, or n is 2, etc.
- n is an integer from 0 to 8 also describes each and every subrange, each of which may for the basis of a further embodiment, such as n is an integer from 1 to 8, from 1 to 7, from 1 to 6, from 2 to 8, from 2 to
- Conjugate 5, or a pharmaceutically acceptable salt thereof, described herein may exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention.
- compositions and/or dosage forms for administration of the Conjugate 5, or a pharmaceutically acceptable salt thereof are prepared from the Conjugate 5, or a pharmaceutically acceptable salt thereof, with purity of at least about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or about 99.5%.
- compositions and or dosage forms for administration of the Conjugate 5, or a pharmaceutically acceptable salt thereof are prepared from the Conjugate 5, or a
- the stereocenters of the conjugates described herein may be substantially pure (S), the substantially pure (R), or any mixture of (S) and (R) at any asymmetric carbon atom, and each may be used in the processes described herein.
- the processes described in these illustrative examples may be adapted to prepare other conjuagtes described herein by carrying out variations of the processes described herein with routine selection of alternative starting materials and reagents.
- Step 1 Preparation of Compound 3. Methyl vanillate (2.18g, 11.98 mmol) and Ph 3 P (4.71 g, 17.97 mmol) in THF (20 niL) was cooled to 0°C and to which was added DIAD (2.59 niL, 13.18 mmol) dropwise. The reaction was stirred at 0°C for 1 hr. 1,5-petanediol (0.6 mL, 5.75 mmol) in THF (20 mL) was added over 30 min. The reaction was stirred overnight and precipitate formed and was collected with filtration. The filtrate was concentrated to form more solid.
- Step 2 Preparation of aldehyde intermediate.
- the crude aldehyde was redis solved in dry DCM (10 mL) and treated with ethanolamine (106 ⁇ , 1.75 mmol) in the presence of anhydrous MgS0 4 (5 mmol, mg) at r.t. (room temperature) under Ar. The reaction was stirred for 1 hour. Then into this reaction mixture was added FmocCl (755.4 mg, 2.92 mmol) and TEA (611 ⁇ , 4.38 mmol) and the reaction was stirred for overnight at r.t. under Ar. The reaction was purified with CombiFlash in 0-50% EtOAc/petroleum ether to provide Compound 8 334.2 mg, 46% for 3 steps.
- Step 1 Preparation of 3-(2-Pyridyldithio)propionic acid.
- PR partial response
- CR complete response
- SIW once weekly
- M/F biweekly
- TIW three times per week
- a PR is observed where tumor volume, as defined herein, decreases from a previous high during the observation period, though regrowth may occur.
- a CR is observed where tumor volume, as defined herein, decreases to zero during the observation period, though regrowth may occur.
- a cure is observed where tumor volume, as defined herein, decreases to zero, and does not regrow during the observation period.
- conjugates described herein were evaluated using an in vitro cytotoxicity assay that predicted the ability of the drug to inhibit the growth of the corresponding targeted cells, such as, but not limited to the following
- test conjugates were conjugates of a cell surface receptor or target antigen binding compound and PBD prodrugs, poly-PBD prodrugs, and mixed PBDs, as described herein.
- the test cells were exposed to varying concentrations of the conjugates, and optionally also in the absence or presence of at least a 100-fold excess of the unconjugated cell surface receptor or target antigen binding compound for competition studies to assess activity as being specific to the cell surface receptor or target antigen.
- METHOD 2 In Vitro Folate Receptor Specific Activity Assay of Folate conjugates. KB cells were seeded in individual 24-well Falcon plates and allowed to form nearly confluent monolayers overnight in folate free Roswell Park Memorial Institute (FFRPMI)/Heat- Inactivated Fetal Calf Serum (HIFCS). Thirty minutes prior to the addition of folate-conjugate, spent medium was aspirated from all wells and replaced with either fresh FFRPMI or FFRPMI supplemented with 100 ⁇ folic acid. Each well then received 1 mL of medium containing increasing concentrations of folate-conjugate (3 wells per sample).
- FFRPMI Roswell Park Memorial Institute
- HFCS Heat- Inactivated Fetal Calf Serum
- Cells were pulsed for 2 hours at 37°C, rinsed 4 times with 0.5 mL of medium and then chased in 1 mL of fresh medium up to 72 h. Spent medium was aspirated from all wells and replaced with fresh medium containing 5 ⁇ /mL of H-thymidine. Following a 2 hour incubation at 37°C, cells were washed 3 times with 0.5 mL of PBS and then treated with 0.5 mL of ice-cold 5% trichloroacetic acid per well. After 15 minutes, the trichloroacetic acid was aspirated and the cells solubilized by the addition of 0.5 mL of 0.25 N sodium hydroxide for 15 minutes at room temperature.
- FR-positive KB cells were seeded in 24-well Falcon plates and allowed to form adherent monolayers (>90% confluent) overnight in FFRPMI/HIFCS. Spent incubation medium was replaced with FFRPMI supplemented with 10% HIFCS and containing 100 nmol/L of [ 3 H]FA in the absence and presence of increasing concentrations of unlabeled FA or the test conjugate.
- DPM Disintegrations per minute
- CT-DNA Calf thymus DNA
- Conjugate 5 (1.1 to 75 ⁇ ) or Conjugate 5 +/- DTT. These solutions were incubated at 37 °C for 2 hours. The solutions were then mixed with ethidium bromide and incubated for 2 hours at room temperature. Fluorescence (Ex: 535 nm, Em: 605 nm) from these samples was measured on the Fluoroskan II fluorimeter. Next, the samples were heated to 104 °C for 5 minutes, cooled on ice for 5 minutes, kept at RT for 15 minutes and fluorescence measured. % crosslinking of each sample was calculated using the fluorescence values from the positive and negative controls. Results are shown in FIG. 8.
- EXAMPLE 4 Conjugate 5 in vivo activity against tumors.
- Conjugate 5 dosed at 0.5 ⁇ /kg SIW for two weeks (A ) decreased KB tumor size in test mice compared to untreated control ( ⁇ ).
- Treatment with 0.5 ⁇ /kg of Conjugate 5, once a week for two weeks also produced maximal anti-tumor activity with 100% cures.
- Change in weight is shown in FIG. 2B for test mice dosed at 0.5 ⁇ /kg Conjugate 5 SIW for two weeks (A ) compared to untreated control ( ⁇ ).
- EXAMPLE 5 Conjugate 5 in vivo activity against paclitaxel resistant tumors.
- KB-PR10 (paclitaxel resistant) tumor cells were inoculated subcutaneously at the right flank of each mouse. Mice were dosed through the lateral tail vein under sterile conditions in a volume of 200 mL of phosphate-buffered saline (PBS).
- PBS phosphate-buffered saline
- Conjugate 5 dosed at 0.5 ⁇ /kg SrW for two weeks (A) decreased paclitacel resistant KB tumor size in test mice compared to untreated control ( ⁇ ).
- EXAMPLE 6 Conjugate 5 in vivo activity against platinum resistant tumors.
- KB-CR2000 (platin resistant) tumor cells were inoculated subcutaneously at the right flank of each mouse. Mice were dosed through the lateral tail vein under sterile conditions in a volume of 200 mL of phosphate-buffered saline (PBS). As shown in FIG. 4, Conjugate 5 dosed at 0.5 ⁇ /kg SIW for two weeks ( ⁇ ) and EC 1456 (EC 1456 is a conjugate folate linked to a tubulysin that is known in the art) dosed at 2.0 ⁇ /kg BIW for two weeks (T ) decreased paclitacel resistant KB tumor size in test mice compared to untreated control ( ⁇ ).
- PBS phosphate-buffered saline
- EXAMPLE 7 Conjugate 5 in vivo activity against triple negative breast tumors.
- Primary human TNBC model ST502 (2-4 mm in diameter) or primary human TNBC model ST738 (2-4 mm in diameter) were inoculated subcutaneously at the right flank of each mouse. Mice were randomized into experimental groups of 7 mice each and test articles were injected through the lateral tail vein under sterile conditions in a volume of 200 mL of phosphate-buffered saline (PBS).
- PBS phosphate-buffered saline
- Conjugate 5 dosed at 0.3 ⁇ /kg BIW for two weeks decreased TNBC PDX tumor size in test mice compared to untreated control ( ⁇ ), whereas EC1456 dosed at 2.0 ⁇ /kg BIW for two weeks ( ⁇ ) did not decrease TNBC PDX tumor size.
- EXAMPLE 8 Conjugate 5 in vivo activity against ovarian tumors.
- mice Female Balb/c nu/nu rats were fed ad libitum with folate-deficient chow (Harlan diet).
- Rats were dosed through the lateral tail vein under sterile conditions in a volume of 200 mL of phosphate-buffered saline (PBS).
- PBS phosphate-buffered saline
- mice Female Balb/c nu/nu mice were fed ad libitum with folate-deficient chow (Harlan diet).
- FIG. 11 shows that treatment with paclitaxel at 15 mg/kg SrW for two weeks produced 0% partial response subjects, while Compound 5 dosed at 0.27 mmol/kg BIW for two weeks produced 43% partial response subjects.
- the mouse and human folate binding protein 1 (FBP1, FOLR1) PicoKineTM ELSIA kits were purchased from Boster Biological Technology (Pleasanton, CA).
- Antibodies used for surface marker staining were purchased from eBioscience: PD-Ll (clone MIH5; cat# 25-5982), F4/80 (clone BM8; cat# 12-4801), CDl lb (clone Ml/70; cat# 48-0112), CD3s (clone 145- 2C11; cat# 25-0031), CD4 (clone GK1.5; cat# 46-0041), and CD8p (clone H3517.2; cat# 11- 0083).
- the FR-oc expressing cell lines utilized to evaluate Conjugate 5 activity in in-vitro and ex-vivo studies were (1) ID8-C115, an ovarian carcinoma cell line transfected with the murine FR-oc, and (2) IGROV1, a human ovarian carcinoma cell line that expresses the human FR-oc.
- the FR-oc negative ID8 parent (ID8p) cell line was used as controls in-vivo.
- ID8p and ID8-C115 cells were grown respectively in a folate-replete or folate-free RPMI1640 medium (Gibco BRL) (FFRPMI) containing 10% heat-inactivated fetal calf serum (HIFCS) and antibiotics, and maintained under a 5% C0 2 atmosphere using standard cell culture techniques.
- IGROV1 cells were grown in the same medium as ID8-C115 except that Corning® ultra-low attachment culture flasks (VWR, Cat. #89089-878) were used.
- ELISA plates that were pre-coated with a rat anti-FOLRl monoclonal antibody.
- a biotinylated goat anti-FOLRl polyclonal antibody was added and followed by a buffer wash.
- the avidin- biotin-peroxidase complex was then added and unbound conjugates were washed away.
- a horseradish peroxidase substrate 3,3',5,5'-Tetramethylbenzidine was added and catalyzed to produce a blue color product.
- the absorbance was read at 375 nm in a microplate reader at least two different time points.
- IGROV1 cells seeded in 6-well plates 1000 cells/well were exposed for 2 hours to Conjugate 5 at 1, 10, and 100 nM and followed by a 9-day chase in drug-free medium.
- the cells were washed with PBS and fixed for 5 minutes in a 3:1 methanol: acetic acid solution. The cells were then stained with 0.5% crystal violet/methanol solution for 15 minutes and washed with tap water. After a drying step, the colonies were photographed and counted using the ImageJ software.
- the single-cell suspensions prepared from ascites were blocked in a FACS stain solution on ice for 20 minutes prior to staining for flow cytometry.
- the FACS stain solution consisted of 1% bovine serum albumin fraction V (Fisher scientific, cat# BP1600), 0.5 mg/mL human immunoglobulin (Equitech-Bio, cat# SLH66) and 0.05% sodium azide in PBS.
- a FACS stain solution consisted of 1% bovine serum albumin fraction V (Fisher scientific, cat# BP1600), 0.5 mg/mL human immunoglobulin (Equitech-Bio, cat# SLH66) and 0.05% sodium azide in PBS.
- PD-L1, F4/80, CDl lb, CD3, CD4, CD8 For surface marker detections (PD-L1, F4/80, CDl lb, CD3, CD4, CD8), the tumor cells were stained in the FACS stain solution containing various fluorophore conjugated antibodies purchased from
- the tumor cells were washed with PBS and re-suspended in PBS containing 3 ⁇ propidium iodide for dead cell exclusion. Data was collected on the Gallios flow cytometer (Beckman Coulter) and analyzed using the Kaluza vl.2 software (Beckman Coulter).
- Conjugate 5 activity against ID8-C115 tumor cells was assessed using the XTT cell viability assay. The cells were exposed for 2 hours to 10-fold serial dilutions of Conjugate 5 (up to 1 ⁇ ) and followed by a 72-120 hours chase in drug-free medium. As determined by the XTT assay, Conjugate 5 showed a potent dose-dependent inhibition of cell proliferation with relative IC 50 values of -0.52 (72 h), 0.61 (96 h), and 0.17 (120 h) (FIG. 12). Importantly, the maximal cell kill was observed after 96-120 hours chase, supporting the mechanism of action of this class of DNA-crosslinking compound.
- Conjugate 5 activity against the slow-growing IGROV tumor cells was assessed using a clonogenic assay. After a 2 hour exposure and 9-day chase (FIG. 13), Conjugate 5 demonstrated a potent activity at all concentrations (1 - 100 nM) tested. More importantly, Conjugate 5 antitumor activity was significantly reduced in the presence of excess amount of folic acid at both 1 and 10 nM concentrations.
- mice Female C57BL/6 (ID8p, ID8-C115) and nu/nu (IGROV1) mice were purchased from Envigo (Indianapolis, IN) and used when they reached 6-8 weeks of age. The mice were fed folate-deficient diet (TestDiet, St. Louis, MO) on the day of arrival.
- mice were generated by intra-peritoneal implantation of cultured cells at 5 x 10 6 in C57BL/6 (ID8p, ID8-C115) and nu/nu (IGROV1) mice respectively.
- Ascites was collected via an I.P. injection of 5 mL of cold PBS containing 5 mM EDTA then removal of the intra-peritoneal fluid containing ascitic tumor cells. The cells were then collected by a 5 minute 400 x g centrifugation, followed by an RBC lysis step, then a cold PBS wash and finally a 40 ⁇ nylon filtration to remove tissue and large cellular aggregates.
- total ascitic fluid was collected via an I.P. lavage of the intraperitoneal fluid containing ascitic tumor cells.
- the acellular fraction of the ascitic fluid was obtained by a 5-minute 2200 x g centrifugation and stored at -80°C until future use.
- Conjugate 5 plus Anti-CTLA-4 Combination Study To test the effect of Conjugate 5 alone and in combination with anti-CTLA-4 antibody, ID8-C115 tumor cells (5 x 10 6 cells per animal in 1% syngeneic mouse serum/folate-deficient RPMI1640 medium) were inoculated intraperitoneally 13 days post the date of arrival and start of the folate deficient diet. For comparison, EC 1456 alone and in combination with the same regimen of anti-CTLA-4 antibody was also evaluated. Starting 7 days after tumor implant, mice were intravenously dosed BIW for a total of 6 doses with Conjugate 5 at 0.1 ⁇ /kg or EC 1456 at 2 ⁇ /kg.
- the anti-CTLA-4 antibody dosing solution was prepared by diluting the stock solution (BioXcell, Clone UC10-4F10-11) to 1.25 mg/mL in PBS, pH 7.4.
- Anti-CTLA-4 250 ⁇ g/dose was i.p. administered BrW for a total of 5 doses starting 11 days after the tumor implant.
- Conjugate 5 plus anti-CTLA-4 and EC 1456 plus anti-CTLA-4 combination groups all compounds were dose- and schedule-matched with the single-agent dosing groups.
- Mice were weighed 3 times/week and assessed for any clinical sign of swollen bellies indicative of ascites formation and for the evidence of toxicity such as respiratory distress, mobility, weight loss, diarrhea, hunched posture, and failure to eat. Once the animals developed ascites, they were monitored daily and euthanized when ascites became severe (rounded and walking on tip toes). Healthy animals from the same cohort of mice were used as controls for normal weight gain.
- acellular ascitic fluid samples collected from ID8p, ID8-C115 and IGROV1 tumor- bearing mice at the time of euthanasia were assayed for soluble murine (ID8p, ID8-C115) and human (IGROV1) FBPl levels.
- Murine FBPl was detected in the ascitic fluid derived from mice intraperitoneally implanted with ID8-C115 tumor cells at 0.93 - 4.6 nM (Table 1).
- CD45+ and CD8+ T cells were also quantitated in total peritoneal cells of the immunocompetent C57BL6 mice at 7 day intervals post IP injection of the mouse ovarian cell line, ID8-CL15 (FIG. 15A).
- the CD45+ CD3e+ CD8+ CD4- T cells ( ⁇ ) slowly increased in number from day 7 to day 42 post implantation.
- the CD45+ CD3e+ CD4+ CD8- T cells (A) also increased in number from day 7 to day 35 with a more significant increase from day 35 to day 42 post implantation suggesting an immune response to the ovarian cancer cell had occurred.
- CD45- non bone-marrow derived ascites cells from ID8-CL15 implanted mice expressed very little functional FR (see FIG.
- CTLA-4 (CD 152) is a protein receptor that functions as an immune checkpoint to downregulate immune responses.
- CTLA-4 competes with CD28 for binding to B7 on antigen presentation cells in order to shut down T-cell activation.
- CTLA4 antagonists can enhance the activity of chemotherapy in certain tumor types.
- Fig. 16A syngeneic intraperitoneal ID8-C115 tumor bearing mice
- EC 1456 was also tested as single agent or in combination with anti-CTLA-4 antibody.
- untreated control mice had a median survival time of -46 days post tumor implant. Both EC 1456 alone (i.v.
- ID8-C115 cells were grown in a folate-free RPMI1640 medium (Gibco BRL) (FFRPMI) containing 10% heat-inactivated fetal calf serum (HIFCS) and antibiotics, and maintained under a 5% C0 2 atmosphere using standard cell culture techniques.
- FFRPMI folate-free RPMI1640 medium
- HFCS heat-inactivated fetal calf serum
- mice Female C57BL/6 mice were purchased from Envigo (Indianapolis, IN) and used when they reached 6-8 weeks of age. The mice were fed a folate-deficient diet (TestDiet, St. Louis, MO) on the day of arrival.
- a folate-deficient diet TeestDiet, St. Louis, MO
- Mouse ascites tumors were generated by intra-peritoneal implantation of cultured cells at 5 x 10 6 in C57BL/6 mice.
- mice were intravenously dosed with Conjugate 5 at 0.1 ⁇ /kg for 3 consecutive days each week for 3 weeks (DO-2 x 3, 9 doses).
- EC 1456 was dosed at 2 ⁇ /kg for 3 consecutive days each week for 3 weeks (DO-2 x 3, 9 doses).
- Conjugate 5 treatment started 35 days after the tumors were implanted.
- the mice were intravenously dosed with Conjugate 5 at 0.3 ⁇ /kg once-a-week for 2 consecutive weeks (SrW x 2, 2 doses).
- EC 1456 was dosed at 2 ⁇ /kg for 3 consecutive days each week for 2 weeks (DO-2 x 2, 6 doses).
- untreated control mice had a median survival time of -46 days post tumor implant.
- Conjugate 5 (0.1 ⁇ /kg, DO-2 x 3, 9 doses) produced a significant anti-tumor effect in 5 animals in each group, with -65% increase in the median survival time (-76 days post tumor implant).
- all Conjugate 5 treated animals displayed mild ataxia at the end of study and one animal did not develop ascites (an outlier).
- EC 1456 (2 ⁇ /kg, DO-2 x, 9 doses) treated mice developed severe dermatitis and two animals were euthanized on Day 44 due to the skin condition. The two remaining animals developed ascites and had a median survival time of 59 days, -28% increase from the untreated controls.
- Conjugate 5 (0.3 ⁇ /kg, SIW x 2, 2 doses) produced a significant anti-tumor effect in 5 animals in each group, with -52% increase in the median survival time (-64 days post tumor implant).
- EC 1456 (2 ⁇ /kg, DO-2 x 2, 4 doses) produced no anti-tumor effects with a median survival time of -44 days post tumor implant, similar to that of untreated controls.
- FIG. 21 summarizes the results of each experiment where ID8-C115 tumor-bearing mice at various stages of the disease were treated with Conjugate 5 and EC 1456 at respective dosing regimens (some toxicity were observed as described above).
- Conjugate 5 was consistently more active. More importantly, from the onset of ascites (days 35) to end-stage of the disease that required euthanasia in untreated animals (day 43), Conjugate 5 provided a therapeutic benefit while EC 1456 was completely inactive.
- EC 1456 (M.W. 2626) and Conjugate 5 (M.W. 2369) were synthesized in house.
- Antibodies used for surface marker staining were purchased from eBioscience: F4/80 (clone BM8; cat# 12-4801), CDl lb (clone Ml/70; cat# 48-0112). In- Vitro Methods
- the FRoc- and FRoc+ expressing cell lines utilized to evaluate Conjugate 5 activity in- vitro and/or ex- vivo studies were (1) 4Tlp, a mouse breast cancer cell line that resembles triple negative breast cancer in humans, (2) 4T1-C12, 4Tlp stably transfected with a mouse FRoc, and (3) IGROV1, a human ovarian carcinoma cell line that expresses the human FRoc.
- 4Tlp and 4T1-C12 cells were grown respectively in a folate-replete or folate-free RPMI1640 medium (Gibco BRL) (FFRPMI) containing 10% heat-inactivated fetal calf serum (HIFCS) and antibiotics, and maintained under a 5% C0 2 atmosphere using standard cell culture techniques.
- IGROV1 cells were grown in the same medium as 4T1-C12.
- 4Tlp and 4T1-C12 tumor cells in 96-well plates were treated with 10-fold serial dilutions of Conjugate 5 ( ⁇ 100 nM) in FFRPMI medium. After a 2 hour exposure, the drug-containing media were replaced and the cells were washed and allowed to incubate further for 96 hours. The cell viability was assessed by adding XTT (2,3-bis(2-methoxy-4-nitro- 5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide) to the culture medium for 2 hours following the manufacturer's instructions. All results were expressed as % absorbance (minus background) relative to the untreated control cells.
- IGROV1 cells seeded in 6-well plates (1000 cells/well) were exposed for 2 hours to Conjugate 5 and EC 1456 at 1, 10, 100, and 1000 nM and followed by a 9-day chase in drug- free medium. Afterwards, the cells were washed with PBS and fixed for 5 minutes in a 3: 1 methanol: acetic acid solution. The cells were then stained with 0.5% crystal violet/methanol solution for 15 minutes and washed with tap water. After a drying step, the colonies were photographed and counted using the ImageJ software.
- the single-cell suspensions prepared from 4Tlp and 4T1-C12 tumors were blocked in a FACS stain solution on ice for 20 minutes prior to staining for flow cytometry.
- the FACS stain solution consisted of 1% bovine serum albumin fraction V (Fisher scientific, cat# BP 1600), 0.5 mg/mL human immunoglobulin (Equitech-Bio, cat# SLH66) and 0.05% sodium azide in PBS.
- F4/80, CD1 lb the tumor cells were stained in the FACS stain solution containing various fluorophore conjugated antibodies purchased from eBioscience at optimized concentrations (0.4 - 2.5 ⁇ g/mL).
- the tumor cells were washed with PBS and re-suspended in PBS containing 3 ⁇ propidium iodide for dead cell exclusion. Data was collected on the Gallios flow cytometer (Beckman Coulter) and analyzed using the Kaluza vl.2 software (Beckman Coulter).
- mice Female Balb/c mice (4Tlp, 4T1-C12) were purchased from Envigo (Indianapolis, IN) and used when they reached 6-8 weeks of age. The mice were fed a folate-deficient diet (TestDiet, St. Louis, MO) on the day of arrival.
- Solid tumors in Balb/c mice were generated by subcutaneous implantation of cultured cells at 5 x 10 5 (4Tlp) and 2 x 10 6 (4T1-C12) per animal in the mammary region.
- Tumor digestion solution was prepared by adding type IV collagenase (Sigma cat# C5138 at 0.5mg/mL final), hyaluronidase (Sigma cat# H3506 at 0.5mg/mL final) and DNase I (Sigma cat# DN25 at O.lmg/mL final) in serum and folate free RPMI1640 and was then warmed to 37C.
- Single cell preparations of 4T1 and 4T1-CL2 orthotopic tumors were prepared by excision of each tumor from the Balb/c mice and by washing in cold PBS. After the cold PBS wash, subcutaneous fat became visible on the surface of the excised tumors and was carefully peeled away prior to tumor digestion.
- the solid tumors were minced and incubated in 10 mL of tumor digestion solution for 1 hour at 37C with vigorous shaking. After digestion, the single cell preparation was pelleted down at 400 X g for 5 minutes and supernatant was discarded. The pellet was treated with 5 mL of room temperature sterile IX RBC lysis solution (VWR cat# 420301-BL) for 5 minutes to lyse any red blood cells. An equal volume of cold PBS was added to the solution and the tumor cells were pelleted again at 400 x g for 5 minutes and the supernatant was discarded.
- VWR cat# 420301-BL room temperature sterile IX RBC lysis solution
- mice with mammary 4Tlp (-78.3 + 12.1 mm 3 ) and 4T1-C12 (-70.1 + 14.1 mm ) were scheduled to receive Conjugate 5 at 200 nmol/kg, biweekly for two weeks.
- 4Tlp tumor bearing mice received a total of 3 doses only and 4T1-C12 tumor bearing mice received a total of 4 doses as planned.
- Mice were weighed and measured for tumor size 3 times a week.
- mice were euthanized when the tumor volume reached -1500 mm . Mice were also monitored closely for the evidence of toxicity such as respiratory distress, mobility, weight loss, diarrhea, hunched posture, and failure to eat. The last Conjugate 5 dose in the 4Tlp tumor-bearing mice was skipped due to weight loss.
- Conjugate 5 activity against 4T1-C12 and 4Tlp tumor cells was assessed using the XTT cell viability assay. The cells were exposed for 2 hours to 10-fold serial dilutions of Conjugate 5 (up to 100 nM) and followed by a 96 hour chase in drug-free medium. In the FRoc-positive 4T1- C12 tumor cell line, Conjugate 5 showed a dose-dependent inhibition of cell proliferation with a relative IC 50 value of -8.7 nM (FIGS. 22A and 22B). The activity against 4T1-C12 was partially reversible in the presence of excess folic acid under this testing condition. In comparison, Conjugate 5 was found completely inactive against the FR a- negative 4Tlp tumor cell in- vitro.
- Conjugate 5 activity against the slow-growing IGROV tumor cells was compared against that of EC 1456 in a standard clonogenic assay. After a 2 hour exposure and 9-day chase (FIG. 23), Conjugate 5 demonstrated a potent activity at all concentrations tested (1 - 1000 nM). On the other hand, significant EC 1456 activity was only observed at 1 ⁇ .
- orthotopic tumors derived from the 4T1 mouse breast cancer cell line (open squares), possessed little detectable functional FR, while tumors grown from a FRoc-transduced 4T1 subclone (4T1-C12; filled squares) contained significant levels.
- Tumor-associated macrophages found in 4T1 parent (B, -16%) and 4T1-C12 (C, -24%) tumors expressed FRP while other non-macrophage myeloid cells (MDSCs) were FR - negative.
- Conjugate 5 anti-TAM activity alone was assessed in the FRoc-negative 4Tlp tumor model.
- Conjugate 5 anti-tumor and anti-TAM dual activity was assessed in the FRa-positive 4T1-C12 tumor model.
- Flow cytometric analysis showed similar TAM content in both 4Tlp and 4T1-C12 mammary tumors established in Balb/c mice.
- 4Tlp tumors showed a partial sensitivity to Conjugate 5 at 0.2 ⁇ /kg (i.v., BIW x 3 doses) with a significant tumor growth delay (FIGS. 25A and 25B).
- Conjugate 5 at 0.2 ⁇ /kg produced 3 out 5 complete responders in the FRa-positive 4T1- C12 tumor model (FIGS. 26A and 26B).
- Conjugate 5 treatment caused significant weight loss in animals.
- Conjugate 5 activity against FRa-positive tumor models could be enhanced by the presence of FR -positive TAMs.
- Conjugate 5 demonstrated in- vivo selectivity for FR+ 4T1 TAMs over FR- 4T1 tumor cells (FIG. 27). With a single administration, Conjugate 5 was shown to significantly decrease the CD163+CD1 lb+ TAM population in these 4T1 tumors. While the folate competitor EC0923 (a folate not linked to a drug) alone did not have any effect on 4T1 TAMs, Conjugate 5 anti-TAM activity was also not blocked by the 500-fold excess of EC0923. Further analysis revealed that Conjugate 5 had no effect against FR- cell populations including CD163-CD1 lb+ 4T1 TAMs and 4T1 tumor cell themselves. These data show that the maximum apoptosis (killing) of TAMs occurred with Conjugate 5 treatment.
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18D | Application deemed to be withdrawn |
Effective date: 20210629 |