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WO2021231762A2 - Combination therapy for the treatment of tnbc with a pi3k pathway inhibitor that targets pi3kdelta and pi3kgamma tnbc - Google Patents

Combination therapy for the treatment of tnbc with a pi3k pathway inhibitor that targets pi3kdelta and pi3kgamma tnbc Download PDF

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Publication number
WO2021231762A2
WO2021231762A2 PCT/US2021/032298 US2021032298W WO2021231762A2 WO 2021231762 A2 WO2021231762 A2 WO 2021231762A2 US 2021032298 W US2021032298 W US 2021032298W WO 2021231762 A2 WO2021231762 A2 WO 2021231762A2
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compound
ptk7
tnbc
inhibitor
ri3kg
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PCT/US2021/032298
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French (fr)
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WO2021231762A3 (en
Inventor
Milan Radovich
Jeffrey P SOLZAK
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The Trustees Of Indiana University
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Publication of WO2021231762A2 publication Critical patent/WO2021231762A2/en
Publication of WO2021231762A3 publication Critical patent/WO2021231762A3/en

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    • 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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links

Definitions

  • the present disclosure relates to methods of treating triple negative breast cancer in patients.
  • TNBC Triple-negative breast cancer
  • PI3K phosphatidylinositol 3-kinase pathway
  • PI3K is upregulated in most TNBC and so was naturally chosen as a target.
  • therapeutics when therapeutics are administered to inhibit PI3K, the clinical outcomes did not substantially improve.
  • the first issue is determining which isoform of PI3K has the greatest impact.
  • PTK7 Protein Tyrosine Kinase 7
  • the therapeutic method disclosed herein is two fold in that both PI3K and PTK7 need to be inhibited to treat TNBC.
  • the method is a targeted approach because it has now been discovered that inhibition or knockout of specific isoforms of PI3K, specifically PI3K5 and RI3Kg, is responsible for the PTK7 induced compensatory pathway.
  • the method disclosed herein results in a substantial improvement in treatment of TNBC.
  • a method of treating a patient diagnosed or suspected of having TNBC comprises administering a therapeutically effective amount of a first compound capable of inhibiting PI3K and a second compound capable of inhibiting PTK7 to the patient.
  • the first compound administered may be configured to inhibit PI3K5, RI3Kg, or both.
  • Fig. 1 shows a graph of TNBC cell lines treated with Gedatolisib
  • Fig. 2 is a table showing the IC50 values of cell lines treated with Gedatolisib in Fig. 1.
  • Fig. 3 is a graph showing the PTK7 RNA expression in cell lines treated and not treated with Gedatolisib.
  • Fig. 4 is a graph showing the relative Endogenous PTK7 RNA transcript expression level in TNBC cell lines.
  • Fig. 5 is a western blot to show the relative levels of PTK7 expression level to Actin.
  • Fig. 6 is a western showing the relative expression level of PTK7 in the MDA-MB-231 cell line after treatments with different PI3K inhibitors as shown in the key.
  • Fig. 7 is a bar graph showing the relative PTK7 transcript level in MDA-MB231 72 hours post treatment.
  • Fig. 8 is a western showing the relative expression level of PTK7 in the MDA-MB-468 cell line after treatments with different PI3K inhibitors as shown in the key.
  • Fig. 9 is a bar graph showing the relative PTK7 transcript level in MDA-MB46872 hours post treatment.
  • Fig. 10 is a western showing the relative expression level of PTK7 in the MDA-MB-453 cell line after treatments with different PI3K inhibitors as shown in the key.
  • Fig. 11 is a bar graph showing the relative PTK7 transcript level in MDA-MB453 72 hours post treatment.
  • Fig. 12 is a western showing the relative expression level of PTK7 in the Hs 578t cell line after treatments with different PI3K inhibitors as shown in the key.
  • Fig. 13 is a bar graph showing the relative PTK7 transcript level in Hs 578t 72 hours post treatment.
  • Fig. 14 is a bar graph showing the relative PTK7 transcript level of the MDA-MB-231 cell line at various dose levels.
  • Fig. 15 is a bar graph showing the relative PTK7 transcript level of the MDA-MB-468 cell line at various dose levels.
  • Fig. 16 is a bar graph showing the relative PTK7 transcript level of the MDA-MB-453 cell line at various dose levels.
  • Fig. 17 is a bar graph showing the relative PTK7 transcript level of the Hs 578t cell line at various dose levels.
  • Fig. 18 is a graph showing the relative PTK7 transcript level overtime in MDA-MB-231 cells.
  • Fig. 19 is a graph showing the relative PTK7 transcript level overtime in MDA-MB-468 cells.
  • Fig. 20 is a table showing the IC 50 value of the various PI3K inhibitors on each cell line.
  • Fig. 21 is a graph showing the relative IC 50 of nine PI3K inhibitors on two cell lines.
  • Fig. 22 is a western showing the endogenous expression of PI3K isoforms relative to actin in two different cell lines.
  • Fig. 23 is a western showing the endogenous PTK7 expression level compared to actin in several TNBC cell lines.
  • Fig. 24 is a bar graph showing the relative transcription level of PTK7 when knocking down the various isoforms of PI3K using siRNA.
  • Fig. 25 is a western showing the expression level of PTK7 as compared to actin after treating the various TNBC cells lines with the indicated siRNA. Scr is scrambled RNA and acts as a control.
  • Fig. 26 is a graph showing that the inhibition of PTK7 or RIK3g induced transcription of PI3K5.
  • Fig. 27 is a western showing that PTK7 expression is induced when knocking down either RIK3g, PI3K5, or both with siRNA.
  • Fig. 28 shows the synergistic effect of providing a PI3K inhibitor Duvelisib and an siRNA to inhibit PTK7 expression compared to Duvelisib alone in various TNBC cancer cell lines.
  • Fig. 29 shows the synergistic effect of providing a PI3K inhibitor Idelalisib and an siRNA to inhibit PTK7 expression compared to Idelalisib alone in various TNBC cancer cell lines.
  • Fig. 30 shows synergistic effect of providing a PI3K inhibitor Gedatolisib and an siRNA to inhibit PTK7 expression compared to Gedatolisib alone in various TNBC cancer cell lines.
  • Fig. 31 shows Buparlisib chemical structure.
  • Fig. 32 shows Duvelisib chemical structure.
  • Fig. 33 shows Gedatolisib chemical structure.
  • Fig. 34 shows Idelalisib chemical structure.
  • Fig. 35 shows CAY10505 chemical structure.
  • Fig. 36 shows GSK229276 chemical structure.
  • Fig. 37 shows Tyrphostin AG 527 chemical structure.
  • Fig. 38 shows Tyrphostin AG 112 chemical structure.
  • treat or treating refers to reducing the size of a tumor or eliminating it and resulting in an increase in life expectancy.
  • the term “patient” refers to any animal diagnosed with or suspected of having triple negative breast cancer (TNBC).
  • TNBC triple negative breast cancer
  • a patient is a human having been diagnosed with or suspected of having TNBC.
  • the TNBC diagnosis may be the original origin of the cancer or a secondary, subsequent cancer.
  • TNBC triple negative breast cancer, wherein the cancerous cells test negative for estrogen and progesterone receptors and do not make a significant amount of HER2 protein.
  • administering includes all known means of providing a treatment to a patient including orally, topically, intravenous, injection, transdermal, or through a port placed during surgery.
  • terapéuticaally effective amount refers to the amount of substance to illicit the desired effect. The amount will vary depending on the substance and the type, weight, height, of the subject, along with other variables to consider known by those skilled in the art.
  • ADC antibody-drug conjugate
  • ADCs refers to targeted biological therapies that are able to treat cancer cells with highly potent anticancer therapies with less severe side effects.
  • ADCs are generally made up of three main parts i) a monoclonal antibody that is specific to a protein, ii) a drug (also known as a treatment or payload) that is intended to treat or kill a cancer cell, and iii) a linker that connects the antibody and drug. This linker is cleaved inside the cell and once it occurs, the payload is activated.
  • Each ADC is different as the antibody, linker, and payload are target, tissue, and cancer specific.
  • PI3K refers to phosphatidylinositol 3-kinases or phosphoinositide 3- kinases.
  • PI3K includes various isoforms including RI3Ka, RI3Kb, PI3K5, and RI3Kg.
  • PTK7 refers to Protein Tyrosine Kinase 7 and also known as Colon Carcinoma Kinase 4 (CCK-4).
  • nucleotide sequences described as SEQ ID NO.l, SEQ ID NO.2, and SEQ ID NO. 3 refer to silencing RNA sequences comprising a sequence that is complementary or having an identity of at least 90% up to 100%. “Nucleotide sequences” refers to both the sense and anti-sense strand and therefor are contemplated as within the scope of this disclosure.
  • a method may include administering a therapeutically effective amount of a first compound capable of inhibiting PI3K and a second compound capable of inhibiting PTK7 to the patient.
  • the first compound and/or the second compound may be administered as a small molecule inhibitor, an antibody-drug-conjugate, or as a silencing RNA such as a long non-coding RNA (IncRNA) or a small interfering RNA (siRNA).
  • IncRNA long non-coding RNA
  • siRNA small interfering RNA
  • the amount of the first and second compound administered will be dependent on several factors including the patient’s height, weight, type and stage of cancer, and age.
  • the first compound and the second compound may be administered in a single dose or in separate doses.
  • the amount of first compound and second compound may be the same amount or different amounts.
  • Each dose may include about 0.001 mg to about 1000 mg of the first compound or second compound per kg of the patient’s body weight.
  • the dose of the first compound or the second compound may include about 0.001 mg, about 0.005 mg, about 0.01 mg, about 0.05 mg, about 0.1 mg, about 0.5 mg, about 1 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, or about 100 mg per kg of the patient’s body weight.
  • the first compound may be administered before the second compound, or the first compound and the second compound may be administered simultaneously.
  • a first compound directed to inhibiting RKI3d, RKI3g, or both may be administered first at a dose suitable to substantially increase the PTK7 expression in the cells.
  • a second dose of an ADC targeted to PTK7 may be administered to the patient.
  • the first and second compound may be administered using different means, for example, one compound is administered orally, and the other compound is provided directly to the tumor environment via a port.
  • the first compound administered may be capable of inhibiting RI3Kd, RI3Kg, or both.
  • the first compound may target all PI3K isoforms, or may preferentially interact with PI3K5, RI3Kg, or both.
  • the first compound may directly bind with RI3Kd, RI3Kg, or both.
  • the first compound may indirectly bind with RI3Kd, RI3Kg, or both.
  • the first compound may comprise a single composition capable of inhibiting both isoforms.
  • the first compound may include a first composition capable of inhibiting PI3K5 and a second composition capable of inhibiting RI3Kg.
  • the first and second composition may each independently be selected from a small molecule inhibitor, an ADC, or a silencing RNA. Accordingly, the first and second compositions of the first compound may be administered by different means and at different times depending on the form.
  • the first composition and second composition may be present at a ratio of about 1 : 1 in the first compound.
  • the first composition and second composition may be present at a ratio of about 10:1, 9:1, 8:1, 7:1, 6:1, :5:1, 4:1, 3:2, 3:1, 2:1, 1:1, 1:2, 2:3, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, or any numerical value in between, in the first compound.
  • the endogenous levels of TNBC cell line 578 show a lower amount of gamma compared to delta, while the 231 cells line shows the opposite.
  • PI3K5 along with PTK7 may be induced to act as a compensatory pathway, as shown in FIG.
  • the amount of the first composition and the second composition in the first compound will be adjusted in response to the endogenous levels of the PI3K isoforms and the endogenous level of PTK7, as shown in FIG. 23. While a person having skill in the art can adjust the ratios of the first and second composition, as they do in all treatment regimens, the method comprises inhibiting PI3K isoforms and inhibiting or targeting PTK7 to treat TNBC in a patient.
  • the first compound may comprise or consist essentially of a PI3K pathway inhibitor.
  • the first compound may comprise or consist essentially of a PI3K inhibitor.
  • the PI3K inhibitor is selected from the group consisting of 5-(2,6-di-4- morpholinyl-4-pyrimidinyl)-4-(trifluoromethyl)-2-pyridinamine (buparlisib; BKM-120), 2-(lH- indazol-4-yl)-6-[[4-(methylsulfonyl)-l-piperazinyl]methyl]-4-(4-morpholinyl)-thieno[3,2- djpyrimidine (Pictilisib, GDC-0941), (lE,4S,4aR,5R,6aS,9aR)-5-(acetyloxy)-l-[(di-2-propen- l-ylamino)methylene]-4,4a,5,6,6a,8,9,
  • the first compound may comprise or may consist essentially of a PI3K5 inhibitor, a RI3Kg inhibitor, or both. In some embodiments, the first compound may comprise or may consist essentially of a PI3K5 inhibitor.
  • the first compound may be selected from the group consisting of GSK2292767, Idelalisib, Duvelisib, and Buparlisib. In some embodiments, the first compound comprises GSK2292767. In some embodiments, the first compound is GSK2292767. In some embodiments, the first compound comprises Idelalisib. In some embodiments, the first compound is Idelalisib. In some embodiments, the first compound comprises Duvelisib. In some embodiments, the first compound is Duvelisib. In some embodiments, the first compound comprises Buparlisib. In some embodiments, the first compound is Buparlisib. In some embodiments, the first compound is Buparlisib.
  • the first compound may comprise or consist essentially of a RI3Kg inhibitor.
  • the RI3Kg inhibitor may be selected from the group consisting of CAY10505, Idelalisib, Duvelisib, Gedatolisib, and Buparlisib.
  • the first compound comprises CAY10505.
  • the first compound is CAY 10505.
  • the first compound comprises Idelalisib.
  • the first compound is Idelalisib.
  • the first compound comprises Duvelisib.
  • the first compound is Duvelisib.
  • the first compound comprises Gedatolisib.
  • the first compound is Gedatolisib.
  • the first compound comprises Buparlisib.
  • the first compound is Buparlisib.
  • the first compound may comprise a PI3K5 inhibitor and/or a RI3Kg inhibitor.
  • the first compound may comprise CAY 10505 and one or more of GSK2292767, Idelalisib, Duvelisib, Gedatolisib, and Buparlisib.
  • the first compound may comprise GSK2292767 and one or more of CAY10505, Idelalisib, Duvelisib, Gedatolisib, and Buparlisib.
  • the first compound may comprise Idelalisib and one or more of CAY10505, GSK2292767, Duvelisib, Gedatolisib, and Buparlisib.
  • the first compound may comprise Duvelisib and one or more of CAY 10505, GSK2292767, Idelalisib, Gedatolisib, and Buparlisib.
  • the first compound may comprise Gedatolisib and one or more of CAY10505, GSK2292767, Idelalisib, Duvelisib, and Buparlisib.
  • the first compound may comprise Buparlisib and one or more of CAY 10505, GSK2292767, Idelalisib, Duvelisib, and Gedatolisib.
  • the first compound may comprise a silencing RNA such as a IncRNA or siRNA capable of inhibiting the expression of a PI3K5 inhibitor, a RI3Kg inhibitor, or both.
  • a silencing RNA such as a IncRNA or siRNA capable of inhibiting the expression of a PI3K5 inhibitor, a RI3Kg inhibitor, or both.
  • the siRNA may comprise an RNA nucleotide sequence having at least 90% sequence identity to SEQ ID NO. 2 or SEQ ID NO. 3.
  • the siRNA may have an RNA nucleotide sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 2 or SEQ ID NO. 3.
  • the siRNA may have a nucleotide sequence having at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO. 2 or SEQ ID NO. 3.
  • the first compound may include an siRNA having a nucleotide sequence having about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO. 2 or SEQ ID NO. 3.
  • the first compound may comprise an siRNA having at least about 90% sequence identity to SEQ ID NO. 2, and an siRNA having at least about 90% sequence identity to SEQ ID NO. 3.
  • the first compound may include a mixture of SEQ ID NO. 2 and SEQ ID NO. 3 each having at least a 90% sequence identity to SEQ ID NO. 2 and SEQ ID NO. 3, respectively.
  • the mixture of the two compositions may be about 50% to about 50%, about 60% to about 40%, about 70% to about 30%, about 80% to about 20%, or about 90% to 10%. In some aspects, the mixture of the compositions may be about 75% to about 25%.
  • the first compound comprises a PI3K inhibitor and the second compound comprises a compound that targets PTK7.
  • first compound comprises a PI3K5 inhibitor or a RI3Kg inhibitor and the second compound comprises a compound that targets PTK7.
  • the first compound comprises a PI3K5 inhibitor or a RI3Kg inhibitor and the second compound is a drug-antibody conjugate that targets PTK7.
  • the drug-antibody conjugate comprises a chemotherapeutic or a radioisotope.
  • the drug-antibody conjugate comprises a chemotherapeutic.
  • the first compound comprises a PI3K inhibitor and the second compound comprises a PTK7 inhibitor.
  • the first compound is a PI3K inhibitor and the second compound is a PTK7 inhibitor.
  • the first compound comprises a PI3K5 inhibitor or RI3Kg inhibitor and the second compound is a PTK7 inhibitor.
  • the second compound interacts with PTK7.
  • the second compound comprises an antibody-drug conjugate that targets PTK7 or a PTK7 inhibitor.
  • the second compound is an antibody-drug conjugate that targets PTK7 or a PTK7 inhibitor.
  • the second compound may be configured to interact with PTK7 directly or indirectly to inhibit or reduce its activity.
  • the second compound may be a small molecule inhibitor of PTK7 or may be an interfering RNA such as a IncRNA or an siRNA.
  • the siRNA may comprise an RNA nucleotide sequence having at least 90% sequence identity to SEQ ID NO. 1.
  • the siRNA may have an RNA nucleotide sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 1.
  • the siRNA may have an RNA nucleotide sequence having at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO. 1.
  • the second compound may include an siRNA a nucleotide sequence having about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO 1.
  • the second compound may include a small molecule inhibitor capable of inhibiting PTK7 activity.
  • the PTK7 inhibitor may be selected from (-)-(R)-N-(a-Methylbenzyl)-3,4- dihydroxybenzylidenecyanoacetamide (Tyrphostin AG 527) or 3-Amino-2,4-dicyano-5-(4'- hydroxyphenyl)penta-2,4-dienonitrile (Tyrphostin AG 112).
  • the second compound may be configured to target PTK7.
  • the second compound may include an antibody-drug conjugate that targets PTK7.
  • the antibody portion of the ADC would designed or selected from a monoclonal antibody capable of specifically binding to PTK7 as it can be expressed on the exterior of a cell.
  • the drug portion of the ADC may be any payload known to be toxic once activated inside a cell such as a chemotherapeutic or a radioisotope.
  • the drug is a chemotherapeutic.
  • the chemotherapeutic may be selected from any suitable agent that can be delivered to a cell by an antibody-drug conjugate delivery system.
  • a non-limiting example of a chemotherapeutic is monmethyl auristatin E.
  • the drug is a radioisotope.
  • the radioisotope may be selected from any suitable agent that can be delivered to a cell by an antibody-drug conjugate delivery system (also referred to as radioimmunotherapy).
  • a radioisotope are Yttrium-90 and Iodine- 131.
  • the method comprises treating a patient diagnosed or suspected of having TNBC.
  • the patient may be selected from a group consisting of stage 0, stage 1, stage 2, stage 3, and stage 4 TNBC.
  • the patient is selected from the group consisting of stage 1, stage 2, stage 3, and stage 4 TNBC.
  • the patient is selected from a group consisting of stage 2, stage 3, and stage 4 TNBC.
  • the patient is selected from a group consisting of stage 3 and stage 4 TNBC.
  • the patient is diagnosed as or suspected of having stage 0 TNBC.
  • the patient is diagnosed as or suspected of having stage 1 TNBC.
  • the patient is diagnosed as or suspected of having stage 2 TNBC.
  • is diagnosed as or suspected of having patient has stage 3 TNBC.
  • the patient is diagnosed as or suspected of having stage 4 TNBC.
  • EXAMPLE 1 PTK7 is upregulated in TNBC cell lines treated with a PI3K5 inhibitor, a RI3Kg inhibitor, or an inhibitor directed to both.
  • Total cell lysates were then separated by SDS-PAGE and subjected to Western blot analysis probed using antibodies against PTK7 (#25618, Cell Signaling), b-Actin (#5125, Cell Signaling), and rabbit IgG (#7074, Cell Signaling).
  • the signals were developed with ECL Reagent (32109, Pierce) and captured on ChemiDoc MP Imaging System.
  • CAY10505 a potent PI3K-gamma inhibitor, while not potent against cell viability displayed increases in PTK7 expression across cell lines (IC50: 86.1-146uM; PTK7 expression: 1.01-1.94 Fold). Similar results were observed with GSK2292767, a potent PI3K- delta inhibitor (IC 5 o:2.2-16uM; PTK7 expresssion: 1.09-2.42 Fold).
  • Idelalisib KNo:18.8- 99.6uM; PTK7 expression: 1.63-2.45 Fold
  • Duvelisib IC 5 o:6.3-53.7uM; PTK7 expression: 1.47-2.84 Fold
  • two dual PI3K-gamma and PI3K-delta inhibitors found the highest increase in PTK7 expression ranging from 150% to 300% increase in PTK7 expression.
  • the data also shows that the amount of induction of PTK7 is not the same across cell lines.
  • the 231 cell line takes more of the PI3K inhibitors to induce 50% cell death compared the 578 line.
  • FIG. 14 also shows that there is a large induction of PTK7 transcription level when the RI3Kg and PI3K5 are inhibited.
  • the induction of PTK7 in TNBCs compensates for the loss of PI3K resulting in a more resistant form of cancer.
  • the combination of inhibiting RI3Kg, PI3K5, or both and targeting PTK7 results in a more effective means of killing TNBC cells.
  • EXAMPLE 2 Inhibiting PTK7 or RI3Kg increases the transcription level of PI3K5.
  • three TNBC cell lines were treated with an siRNA to inhibit RI3Kg, PI3K5, and RI3Kd/g as indicated in the graph and the transcription level of PTK7 was analyzed.
  • the siRNA to inhibit both isoforms was a mixture of the two siRNAs at a ratio of about 1:1.
  • the amounts of PTK7 were normalized to the control.
  • the inhibition of RI3Kg, PI3K5, or both increased the expression level of PTK7 showing that it does act as a compensatory pathway in the TNBC cell line.
  • FIG. 24 and FIG. 25 the inhibition of RI3Kg, PI3K5, or both increased the expression level of PTK7 showing that it does act as a compensatory pathway in the TNBC cell line.
  • FIG. 26 and FIG. 27 are a graph and western blot that show when siRNA to inhibit RI3Kg activity or siRNA to inhibit the activity of PTK7, there was an increase in the transcription level for PI3K5.
  • EXAMPLE 3 Inhibiting RI3Kd, RI3Kg, or both PI3K d/g in combination with inhibiting PTK7 activity substantially increased the potency of the treatment and shows it is a viable options in methods of treating TNBC.
  • FIGS. 28, 29, and 30 when four different TNBC cells lines were treated with a PI3K inhibitor in combination with an siRNA directed to PTK7, the results show that the potency of the treatment increased substantially as opposed to the PI3K treatment alone. Scr is scrambled RNA and serves as a control to show the impact of the RNA procedure, if any, along with the PI3K inhibitor.
  • This data indicates that the combination of a PI3K inhibitor and a PTK7 inhibitor can be viable treatment option for patients diagnosed with TNBC. Specifically, the data shows that inhibiting specific PI3K isoforms in combination with inhibiting PTK7 could increase the effectiveness of treating patients diagnosed with TNBC. Finally, because this data demonstrates that inhibiting certain isoforms of PI3K increases the expression level of PTK7, a protein expressed on the outside of the cancer cell, a method of treating a patient with TNBC first with an inhibitor of PI3K5, RI3Kg, or both would substantially increase the protein expression level of PTK7 in the TNBC cells, making the TNBC cells perfect targets for an ADC directed to PTK7.
  • ADCs that carry a drug such as a chemotherapeutic or radioisotope that become active once taken up by the cancer cell are known in the art, rather it is the combinatorial method of targeting both PI3K5, RI3Kg, both and PTK7 to the benefit of treating TNBC that is unknown.
  • SEQ ID NO 1 PTK7 siRNA: Chromosome 6 band 12, 43146486-43146752 Homo sapiens chromosome 6, GRCh38.pl3 Primary Assembly
  • SEQ ID NO:2 PI3K5 siRNA: Chromosome 1, band 11, 9724290-9724517 Homo sapiens chromosome 1, GRCh38.pl3 Primary Assembly
  • SEQ ID NO: 3 RI3Kg siRNA: Chromosome 6, band 12, 43146486-43146752 Homo sapiens chromosome 6, GRCh38.pl3 Primary Assembly

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Abstract

Methods of treating patients diagnosed with or suspected of having triple negative breast cancer with a combination of compounds that target PI3K isoforms and target PTK7.

Description

COMBINATION THERAPY FOR THE TREATMENT OF TNBC WITH A PI3K PATHWAY INHIBITOR THAT TARGETS PI3K5 AND RI3Kg TNBC
CROSS-REFERENCE TO RELATED APPLICATIONS
[001] This application claims priority to U.S. Provisional Application No. 63/024,171, filed May 13, 2020, the contents of which are hereby incorporated herein by reference in their entirety.
TECHNICAL FIELD
[002] The present disclosure relates to methods of treating triple negative breast cancer in patients.
BACKGROUND
[003] Triple-negative breast cancer (TNBC) accounts for about 10-15% of all breast cancers.
It is a devastating disease accompanied with substantial morbidity and mortality with very few tools available to clinicians to improve outcomes. The phosphatidylinositol 3-kinase (PI3K) pathway is activated in the majority of TNBCs. However, the use of a PI3K inhibitor to inhibit PI3K has had limited clinical efficacy. A need exists for a more effective treatment to improve outcomes for patients diagnosed with triple-negative breast cancer.
SUMMARY
[004] Disclosed herein is a method for treating triple negative breast cancer in patients. PI3K is upregulated in most TNBC and so was naturally chosen as a target. However, when therapeutics are administered to inhibit PI3K, the clinical outcomes did not substantially improve. The first issue is determining which isoform of PI3K has the greatest impact. Second, it was recently discovered that when PI3K pathway is inhibited, the Protein Tyrosine Kinase 7 (PTK7) compensates for its loss. Accordingly, the therapeutic method disclosed herein is two fold in that both PI3K and PTK7 need to be inhibited to treat TNBC.
[005] The method is a targeted approach because it has now been discovered that inhibition or knockout of specific isoforms of PI3K, specifically PI3K5 and RI3Kg, is responsible for the PTK7 induced compensatory pathway. With this understanding, the method disclosed herein results in a substantial improvement in treatment of TNBC.
[006] In some embodiments, a method of treating a patient diagnosed or suspected of having TNBC comprises administering a therapeutically effective amount of a first compound capable of inhibiting PI3K and a second compound capable of inhibiting PTK7 to the patient. The first compound administered may be configured to inhibit PI3K5, RI3Kg, or both. BRIEF DESCRIPTION OF THE DRAWINGS
[007] Fig. 1 shows a graph of TNBC cell lines treated with Gedatolisib [008] Fig. 2 is a table showing the IC50 values of cell lines treated with Gedatolisib in Fig. 1. [009] Fig. 3 is a graph showing the PTK7 RNA expression in cell lines treated and not treated with Gedatolisib.
[010] Fig. 4 is a graph showing the relative Endogenous PTK7 RNA transcript expression level in TNBC cell lines.
[Oil] Fig. 5 is a western blot to show the relative levels of PTK7 expression level to Actin. [012] Fig. 6 is a western showing the relative expression level of PTK7 in the MDA-MB-231 cell line after treatments with different PI3K inhibitors as shown in the key.
[013] Fig. 7 is a bar graph showing the relative PTK7 transcript level in MDA-MB231 72 hours post treatment.
[014] Fig. 8 is a western showing the relative expression level of PTK7 in the MDA-MB-468 cell line after treatments with different PI3K inhibitors as shown in the key.
[015] Fig. 9 is a bar graph showing the relative PTK7 transcript level in MDA-MB46872 hours post treatment.
[016] Fig. 10 is a western showing the relative expression level of PTK7 in the MDA-MB-453 cell line after treatments with different PI3K inhibitors as shown in the key.
[017] Fig. 11 is a bar graph showing the relative PTK7 transcript level in MDA-MB453 72 hours post treatment.
[018] Fig. 12 is a western showing the relative expression level of PTK7 in the Hs 578t cell line after treatments with different PI3K inhibitors as shown in the key.
[019] Fig. 13 is a bar graph showing the relative PTK7 transcript level in Hs 578t 72 hours post treatment.
[020] Fig. 14 is a bar graph showing the relative PTK7 transcript level of the MDA-MB-231 cell line at various dose levels.
[021] Fig. 15 is a bar graph showing the relative PTK7 transcript level of the MDA-MB-468 cell line at various dose levels.
[022] Fig. 16 is a bar graph showing the relative PTK7 transcript level of the MDA-MB-453 cell line at various dose levels.
[023] Fig. 17 is a bar graph showing the relative PTK7 transcript level of the Hs 578t cell line at various dose levels. [024] Fig. 18 is a graph showing the relative PTK7 transcript level overtime in MDA-MB-231 cells.
[025] Fig. 19 is a graph showing the relative PTK7 transcript level overtime in MDA-MB-468 cells.
[026] Fig. 20 is a table showing the IC50 value of the various PI3K inhibitors on each cell line. [027] Fig. 21 is a graph showing the relative IC50 of nine PI3K inhibitors on two cell lines. [028] Fig. 22 is a western showing the endogenous expression of PI3K isoforms relative to actin in two different cell lines.
[029] Fig. 23 is a western showing the endogenous PTK7 expression level compared to actin in several TNBC cell lines.
[030] Fig. 24 is a bar graph showing the relative transcription level of PTK7 when knocking down the various isoforms of PI3K using siRNA.
[031] Fig. 25 is a western showing the expression level of PTK7 as compared to actin after treating the various TNBC cells lines with the indicated siRNA. Scr is scrambled RNA and acts as a control.
[032] Fig. 26 is a graph showing that the inhibition of PTK7 or RIK3g induced transcription of PI3K5.
[033] Fig. 27 is a western showing that PTK7 expression is induced when knocking down either RIK3g, PI3K5, or both with siRNA.
[034] Fig. 28 shows the synergistic effect of providing a PI3K inhibitor Duvelisib and an siRNA to inhibit PTK7 expression compared to Duvelisib alone in various TNBC cancer cell lines. [035] Fig. 29 shows the synergistic effect of providing a PI3K inhibitor Idelalisib and an siRNA to inhibit PTK7 expression compared to Idelalisib alone in various TNBC cancer cell lines.
[036] Fig. 30 shows synergistic effect of providing a PI3K inhibitor Gedatolisib and an siRNA to inhibit PTK7 expression compared to Gedatolisib alone in various TNBC cancer cell lines.
[037] Fig. 31 shows Buparlisib chemical structure.
[038] Fig. 32 shows Duvelisib chemical structure.
[039] Fig. 33 shows Gedatolisib chemical structure.
[040] Fig. 34 shows Idelalisib chemical structure.
[041] Fig. 35 shows CAY10505 chemical structure.
[042] Fig. 36 shows GSK229276 chemical structure.
[043] Fig. 37 shows Tyrphostin AG 527 chemical structure. [044] Fig. 38 shows Tyrphostin AG 112 chemical structure.
DETAILED DESCRIPTION
[045] As used herein the terms “treat or treating” refers to reducing the size of a tumor or eliminating it and resulting in an increase in life expectancy.
[046] As used herein the term “patient” refers to any animal diagnosed with or suspected of having triple negative breast cancer (TNBC). In particular, a patient is a human having been diagnosed with or suspected of having TNBC. In some aspects, the TNBC diagnosis may be the original origin of the cancer or a secondary, subsequent cancer.
[047] As used herein the term “TNBC” refers to triple negative breast cancer, wherein the cancerous cells test negative for estrogen and progesterone receptors and do not make a significant amount of HER2 protein.
[048] As used herein, the term “administering” includes all known means of providing a treatment to a patient including orally, topically, intravenous, injection, transdermal, or through a port placed during surgery.
[049] As used herein the phrase “therapeutically effective amount” refers to the amount of substance to illicit the desired effect. The amount will vary depending on the substance and the type, weight, height, of the subject, along with other variables to consider known by those skilled in the art.
[050] As used herein the phrase “antibody-drug conjugate” (ADC) refers to targeted biological therapies that are able to treat cancer cells with highly potent anticancer therapies with less severe side effects. ADCs are generally made up of three main parts i) a monoclonal antibody that is specific to a protein, ii) a drug (also known as a treatment or payload) that is intended to treat or kill a cancer cell, and iii) a linker that connects the antibody and drug. This linker is cleaved inside the cell and once it occurs, the payload is activated. Each ADC is different as the antibody, linker, and payload are target, tissue, and cancer specific.
[051] As used herein “PI3K” refers to phosphatidylinositol 3-kinases or phosphoinositide 3- kinases. PI3K includes various isoforms including RI3Ka, RI3Kb, PI3K5, and RI3Kg.
[052] As used herein “PTK7” refers to Protein Tyrosine Kinase 7 and also known as Colon Carcinoma Kinase 4 (CCK-4).
[053] As used herein, the “nucleotide sequences” described as SEQ ID NO.l, SEQ ID NO.2, and SEQ ID NO. 3 refer to silencing RNA sequences comprising a sequence that is complementary or having an identity of at least 90% up to 100%. “Nucleotide sequences” refers to both the sense and anti-sense strand and therefor are contemplated as within the scope of this disclosure.
[054] In some aspects, a method may include administering a therapeutically effective amount of a first compound capable of inhibiting PI3K and a second compound capable of inhibiting PTK7 to the patient. The first compound and/or the second compound may be administered as a small molecule inhibitor, an antibody-drug-conjugate, or as a silencing RNA such as a long non-coding RNA (IncRNA) or a small interfering RNA (siRNA).
[055] The amount of the first and second compound administered will be dependent on several factors including the patient’s height, weight, type and stage of cancer, and age. The first compound and the second compound may be administered in a single dose or in separate doses. The amount of first compound and second compound may be the same amount or different amounts. Each dose may include about 0.001 mg to about 1000 mg of the first compound or second compound per kg of the patient’s body weight. The first compound or second compound may be administered at a dose may include about 0.001 mg, about 0.01 mg, about 0.1 mg, about 1 mg, about 10 mg, or about 100 mg per kg of the patient’s body weight. The dose of the first compound or the second compound may include about 0.001 mg, about 0.005 mg, about 0.01 mg, about 0.05 mg, about 0.1 mg, about 0.5 mg, about 1 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, or about 100 mg per kg of the patient’s body weight.
[056] The first compound may be administered before the second compound, or the first compound and the second compound may be administered simultaneously. In one aspect, a first compound directed to inhibiting RKI3d, RKI3g, or both may be administered first at a dose suitable to substantially increase the PTK7 expression in the cells. Once the PTK7 protein expression has increased in response to the PI3K inhibition, a second dose of an ADC targeted to PTK7 may be administered to the patient. The first and second compound may be administered using different means, for example, one compound is administered orally, and the other compound is provided directly to the tumor environment via a port.
[057] The first compound administered may be capable of inhibiting RI3Kd, RI3Kg, or both. The first compound may target all PI3K isoforms, or may preferentially interact with PI3K5, RI3Kg, or both. When administered, the first compound may directly bind with RI3Kd, RI3Kg, or both. In other aspects, the first compound may indirectly bind with RI3Kd, RI3Kg, or both. [058] When the first compound is capable of inhibiting both PI3K5 and RI3Kg, the first compound may comprise a single composition capable of inhibiting both isoforms. Alternatively, the first compound may include a first composition capable of inhibiting PI3K5 and a second composition capable of inhibiting RI3Kg. The first and second composition may each independently be selected from a small molecule inhibitor, an ADC, or a silencing RNA. Accordingly, the first and second compositions of the first compound may be administered by different means and at different times depending on the form. The first composition and second composition may be present at a ratio of about 1 : 1 in the first compound. This may vary depending on the cancer type, however, the first composition and second composition may be present at a ratio of about 10:1, 9:1, 8:1, 7:1, 6:1, :5:1, 4:1, 3:2, 3:1, 2:1, 1:1, 1:2, 2:3, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, or any numerical value in between, in the first compound. For example, as shown in FIG. 22, the endogenous levels of TNBC cell line 578 show a lower amount of gamma compared to delta, while the 231 cells line shows the opposite. However, it was also observed that when RI3Kg is inhibited, PI3K5, along with PTK7 may be induced to act as a compensatory pathway, as shown in FIG. 26. Accordingly, the amount of the first composition and the second composition in the first compound will be adjusted in response to the endogenous levels of the PI3K isoforms and the endogenous level of PTK7, as shown in FIG. 23. While a person having skill in the art can adjust the ratios of the first and second composition, as they do in all treatment regimens, the method comprises inhibiting PI3K isoforms and inhibiting or targeting PTK7 to treat TNBC in a patient.
[059] The first compound may comprise or consist essentially of a PI3K pathway inhibitor. The first compound may comprise or consist essentially of a PI3K inhibitor. In some embodiments, the PI3K inhibitor is selected from the group consisting of 5-(2,6-di-4- morpholinyl-4-pyrimidinyl)-4-(trifluoromethyl)-2-pyridinamine (buparlisib; BKM-120), 2-(lH- indazol-4-yl)-6-[[4-(methylsulfonyl)-l-piperazinyl]methyl]-4-(4-morpholinyl)-thieno[3,2- djpyrimidine (Pictilisib, GDC-0941), (lE,4S,4aR,5R,6aS,9aR)-5-(acetyloxy)-l-[(di-2-propen- l-ylamino)methylene]-4,4a,5,6,6a,8,9,9a-octahydro-ll-hydroxy-4-(methoxymethyl)-4a,6a- dimethyl-cyclopenta[5,6]naphtho[l,2-c]pyran-2,7,10(lH)-trione (PX866), 2-methyl-2-[4-(3- methyl-2-oxo-8-quinolin-3-ylimidazo[4,5-c]quinolin-l-yl)phenyl]propanenitrile (dactoblisib; BEZ-235), (Z)-but-2-enedioic acid;8-(6-methoxypyridin-3-yl)-3-methyl-l-[4-piperazin-l-yl-3- (trifluoromethyl)phenyl]imidazo[4,5-c]quinolin-2-one (BGT-226), (2S)-l-[4-[[2-(2- aminopyrimidin-5-yl)-7-methyl-4-morpholin-4-ylthieno[3,2-d]pyrimidin-6- yl]methyl]piperazin-l-yl]-2-hydroxypropan-l-one (apitolisib; GDC-0980), N-[4-[[[3-[(3,5- dimethoxyphenyl)amino]-2-quinoxalinyl]amino]sulfonyl]phenyl]-3-methoxy-4-methyl- benzamide (voxtalisib; XL-765), 2-amino-N-[3-[[3-(2-chloro-5-methoxyanilino)quinoxalin-2- yl]sulfamoyl]phenyl]-2-methylpropanamide (pilaralisib; XL-147), 3-(2,4-diaminopteridin-6- yl)phenol (TG100713); l-[4-(3-ethyl-7-morpholin-4-yltriazolo[4,5-d]pyrimidin-5-yl)phenyl]-3- [4-(4-methylpiperazine-l-carbonyl)phenyl]urea (PKI-402), 5-Fluoro-3-phenyl-2-[(lS)-l-(7H- purin-6-ylamino)propyl]-4(3H)-quinazolinone (idelalisib), N-[4-[[4-(dimethylamino)-l- piperidinyl]carbonyl]phenyl]-N'-[4-(4,6-di-4-morpholinyl-l,3,5-triazin-2-yl)phenyl]-urea (Gedatolisib), 5-pyrimidinecarboxamide, 2-amino-N-(2,3-dihydro-7-methoxy-8-(3-(4- morpholinyl)propoxy)imidazo(l,2-C)quinazolin-5-yl)-, hydrochloride (1:2) (Copanlisib), l(2H)-isoquinolinone, 8-chloro-2-phenyl-3-((lS)-l-(9H-purin-6-ylamino)ethyl)-8-chloro-2- phenyl-3-((lS)-l-(7H-purin-6-ylamino)ethyl)isoquinolin-l(2H)-one (Duvelisib), (8S,14S,17S)- 14-(carboxymethyl)-8-(3-guanidinopropyl)-17-(hydroxymethyl)-3,6,9,12,15-pentaoxo-l-(4-(4- oxo- 8-phenyl-4H-chromen-2-yl)morpholino-4-ium)-2-oxa-7, 10,13, 16-tetraazaoctadecan- 18- oate (SF1126), 3-(3-fluorophenyl)-2-[(lS)-l-(7H-purin-6-ylamino)propyl]chromen-4-one (Tenalisib), 5-[8,9-dihydro-6,6-dimethyl-4-(4-morpholinyl)-6H-[l,4]oxazino[4,3-e]purin-2-yl]- 2-pyrimidinamine (GDC-0084), 5-[8-methyl-9-(l-methylethyl)-2-(4-morpholinyl)-9H-purin-6- yl]-2-pyrimidinamine (VS-5584),l-[(3S)-3-[[5,6,7,8-tetrahydro-6-[6-methoxy-5- (trifluoromethyl)-3 -pyridinyl]pyrido [4,3 -d]pyrimidin-4-yl] amino] - 1 -pyrrolidinyl] - 1 -propanone (CDZ173), and 5-[[5-(4-fluorophenyl)-2-furanyl]methylene]-2,4-thiazolidinedione (CAY 10505), and re/-N-|5-|4-|5-| | (2R,6S)-2,6-di methyl -4-morphol i nyl | methyl |-2-oxazolyl |- lH-indazol-6-yl] -2-methoxy-3 -pyridinyl] -methanesulfonamide (GSK2292767) .
[060] The first compound may comprise or may consist essentially of a PI3K5 inhibitor, a RI3Kg inhibitor, or both. In some embodiments, the first compound may comprise or may consist essentially of a PI3K5 inhibitor. The first compound may be selected from the group consisting of GSK2292767, Idelalisib, Duvelisib, and Buparlisib. In some embodiments, the first compound comprises GSK2292767. In some embodiments, the first compound is GSK2292767. In some embodiments, the first compound comprises Idelalisib. In some embodiments, the first compound is Idelalisib. In some embodiments, the first compound comprises Duvelisib. In some embodiments, the first compound is Duvelisib. In some embodiments, the first compound comprises Buparlisib. In some embodiments, the first compound is Buparlisib.
[061] The first compound may comprise or consist essentially of a RI3Kg inhibitor. The RI3Kg inhibitor may be selected from the group consisting of CAY10505, Idelalisib, Duvelisib, Gedatolisib, and Buparlisib. In some embodiments, the first compound comprises CAY10505. In some embodiments, the first compound is CAY 10505. In some embodiments, the first compound comprises Idelalisib. In some embodiments, the first compound is Idelalisib. In some embodiments, the first compound comprises Duvelisib. In some embodiments, the first compound is Duvelisib. In some embodiments, the first compound comprises Gedatolisib. In some embodiments, the first compound is Gedatolisib. In some embodiments, the first compound comprises Buparlisib. In some embodiments, the first compound is Buparlisib.
[062] The first compound may comprise a PI3K5 inhibitor and/or a RI3Kg inhibitor. The first compound may comprise CAY 10505 and one or more of GSK2292767, Idelalisib, Duvelisib, Gedatolisib, and Buparlisib. The first compound may comprise GSK2292767 and one or more of CAY10505, Idelalisib, Duvelisib, Gedatolisib, and Buparlisib. The first compound may comprise Idelalisib and one or more of CAY10505, GSK2292767, Duvelisib, Gedatolisib, and Buparlisib. The first compound may comprise Duvelisib and one or more of CAY 10505, GSK2292767, Idelalisib, Gedatolisib, and Buparlisib. The first compound may comprise Gedatolisib and one or more of CAY10505, GSK2292767, Idelalisib, Duvelisib, and Buparlisib. In other aspects, the first compound may comprise Buparlisib and one or more of CAY 10505, GSK2292767, Idelalisib, Duvelisib, and Gedatolisib.
[063] The first compound may comprise a silencing RNA such as a IncRNA or siRNA capable of inhibiting the expression of a PI3K5 inhibitor, a RI3Kg inhibitor, or both. When the first compound is an siRNA that targets PI3K5 inhibitor, a RI3Kg inhibitor, or both the siRNA may comprise an RNA nucleotide sequence having at least 90% sequence identity to SEQ ID NO. 2 or SEQ ID NO. 3. The siRNA may have an RNA nucleotide sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 2 or SEQ ID NO. 3. In some embodiments, the siRNA may have a nucleotide sequence having at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO. 2 or SEQ ID NO. 3. The first compound may include an siRNA having a nucleotide sequence having about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO. 2 or SEQ ID NO. 3. The first compound may comprise an siRNA having at least about 90% sequence identity to SEQ ID NO. 2, and an siRNA having at least about 90% sequence identity to SEQ ID NO. 3. In some aspects, the first compound may include a mixture of SEQ ID NO. 2 and SEQ ID NO. 3 each having at least a 90% sequence identity to SEQ ID NO. 2 and SEQ ID NO. 3, respectively. The mixture of the two compositions may be about 50% to about 50%, about 60% to about 40%, about 70% to about 30%, about 80% to about 20%, or about 90% to 10%. In some aspects, the mixture of the compositions may be about 75% to about 25%.
[064] In some embodiments, the first compound comprises a PI3K inhibitor and the second compound comprises a compound that targets PTK7. In some embodiments, first compound comprises a PI3K5 inhibitor or a RI3Kg inhibitor and the second compound comprises a compound that targets PTK7. In some embodiments, the first compound comprises a PI3K5 inhibitor or a RI3Kg inhibitor and the second compound is a drug-antibody conjugate that targets PTK7. In some embodiments, the drug-antibody conjugate comprises a chemotherapeutic or a radioisotope. In some embodiments, the drug-antibody conjugate comprises a chemotherapeutic.
[065] In some embodiments, the first compound comprises a PI3K inhibitor and the second compound comprises a PTK7 inhibitor. In some embodiments, the first compound is a PI3K inhibitor and the second compound is a PTK7 inhibitor. In some embodiments, the first compound comprises a PI3K5 inhibitor or RI3Kg inhibitor and the second compound is a PTK7 inhibitor. In some embodiments, the second compound interacts with PTK7. In some embodiments, the second compound comprises an antibody-drug conjugate that targets PTK7 or a PTK7 inhibitor. In some embodiments, the second compound is an antibody-drug conjugate that targets PTK7 or a PTK7 inhibitor.
[066] The second compound may be configured to interact with PTK7 directly or indirectly to inhibit or reduce its activity. The second compound may be a small molecule inhibitor of PTK7 or may be an interfering RNA such as a IncRNA or an siRNA. When the second compound is an siRNA that targets PTK7, the siRNA may comprise an RNA nucleotide sequence having at least 90% sequence identity to SEQ ID NO. 1. The siRNA may have an RNA nucleotide sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 1. In some embodiments, the siRNA may have an RNA nucleotide sequence having at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO. 1. The second compound may include an siRNA a nucleotide sequence having about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO 1.
[067] The second compound may include a small molecule inhibitor capable of inhibiting PTK7 activity. The PTK7 inhibitor may be selected from (-)-(R)-N-(a-Methylbenzyl)-3,4- dihydroxybenzylidenecyanoacetamide (Tyrphostin AG 527) or 3-Amino-2,4-dicyano-5-(4'- hydroxyphenyl)penta-2,4-dienonitrile (Tyrphostin AG 112).
[068] The second compound may be configured to target PTK7. For example, the second compound may include an antibody-drug conjugate that targets PTK7. The antibody portion of the ADC would designed or selected from a monoclonal antibody capable of specifically binding to PTK7 as it can be expressed on the exterior of a cell. The drug portion of the ADC may be any payload known to be toxic once activated inside a cell such as a chemotherapeutic or a radioisotope. In some embodiments, the drug is a chemotherapeutic. The chemotherapeutic may be selected from any suitable agent that can be delivered to a cell by an antibody-drug conjugate delivery system. A non-limiting example of a chemotherapeutic is monmethyl auristatin E. In some embodiments, the drug is a radioisotope. The radioisotope may be selected from any suitable agent that can be delivered to a cell by an antibody-drug conjugate delivery system (also referred to as radioimmunotherapy). Non- limiting examples of a radioisotope are Yttrium-90 and Iodine- 131.
[069] In some embodiment, the method comprises treating a patient diagnosed or suspected of having TNBC. In some embodiments, the patient may be selected from a group consisting of stage 0, stage 1, stage 2, stage 3, and stage 4 TNBC. In some embodiments, the patient is selected from the group consisting of stage 1, stage 2, stage 3, and stage 4 TNBC. In some embodiments, the patient is selected from a group consisting of stage 2, stage 3, and stage 4 TNBC. In some embodiments, the patient is selected from a group consisting of stage 3 and stage 4 TNBC. In some embodiments, the patient is diagnosed as or suspected of having stage 0 TNBC. In some embodiments, the patient is diagnosed as or suspected of having stage 1 TNBC. In some embodiments, the patient is diagnosed as or suspected of having stage 2 TNBC. In some embodiments, is diagnosed as or suspected of having patient has stage 3 TNBC. In some embodiments, the patient is diagnosed as or suspected of having stage 4 TNBC.
EXAMPLES
[070] EXAMPLE 1: PTK7 is upregulated in TNBC cell lines treated with a PI3K5 inhibitor, a RI3Kg inhibitor, or an inhibitor directed to both.
[071] Cell lines cultured. Human breast cancer cell lines MDA-MB-231, MDA-MB-453, MDA-MB-468, and Hs578t were purchased from ATCC and cultured at 37°C with 5% CO2 in DMEM supplemented with 10% FBS. [072] Cell viability assays were performed using CellTiter-Glo 2.0 (G9242, Promega). Referring to FIG. 1, breast cancer cells were seeded in 96 well plates at 2500 cells/well, cultured overnight, and treated with inhibitor at different concentrations or vehicle for 3 days. 100 pL of CellTiter-Glo 2.0 reagent was then added to each well, and luminescent was measured with a Cytation5 cell imaging multi-mode reader. Referring to FIGs 2, 20, and 21, the concentration-response curve and IC50 of each inhibitor were generated using Prism 8 (GraphPad).
[073] Combination studies were performed in the presence of different concentrations of two inhibitors at potency ratios of 1: 1. Referring to FIGs 6-13, for each combination, IC50 and combination index were calculated according to the Chou and Talalay method.
[074] Cells were plated at 105 cells/well in 6 well plates and treated with single inhibitor or in combination at their IC50 for 3 days for RT-PCR and Western blot analysis.
[075] Real-Time Quantitative PCR Analysis. Cells were harvested, and total RNA was extracted using a RNeasy Mini Kit (74104, Qiagen) followed by reverse transcription using a high capacity cDNA reverse transcription kit (4387406, Applied Biosystems) according to the manufacturer’s instructions. RT-PCR was performed using a QuantStudio 3 Real-Time PCR System and TaqMan Gene Expression Master Mix (4369016, Applied Biosystems). Human PTK7 Taqman primer (Hs00177173_ml, Applied Biosystems) and human IP08 Taqman primer (Hs00914057_ml, Applied Biosystems) were used. Referring to FIGs 3, 4, 7, 9, 11, and 13-19, expression levels of PTK7 were calculated as a ratio to expression of IP08.
[076] Cell Lysate Preparation and Western Blot Analysis. Cells were harvested and then lysed in RIPA buffer (89900, Thermo Scientific) with Halt Protease Inhibitor Cocktail (78430, Thermo Scientific) at 4°C with occasional agitation. Referring to FIGs 5, 6, 8, 10, 12, 22, and 23, the cells were sonicated briefly and total proteins were harvested after centrifugation of the lysate at 16,000g for 15 minutes followed by determination of protein concentration using Pierce 660-nm Protein Assay (22662, Thermo Scientific). Total cell lysates were then separated by SDS-PAGE and subjected to Western blot analysis probed using antibodies against PTK7 (#25618, Cell Signaling), b-Actin (#5125, Cell Signaling), and rabbit IgG (#7074, Cell Signaling). The signals were developed with ECL Reagent (32109, Pierce) and captured on ChemiDoc MP Imaging System.
[077] The data shows that specific PI3K isoforms are responsible for upregulating PTK7. Alpelisib, a PI3K-alpha inhibitor, while potent in reducing cell viability (IC50: 0.16-3.8uM), saw the least PTK7 induction across the TNBC cell lines (.85-1.15 Fold). GSK2636771, a PI3K-beta inhibitor, had limited efficacy in reducing cell viability and did not induce PTK7 across the TNBC cell lines (IC50: 16.6-166.8uM; PTK7 expression: 0.65-1.09 Fold). Interestingly, CAY10505, a potent PI3K-gamma inhibitor, while not potent against cell viability displayed increases in PTK7 expression across cell lines (IC50: 86.1-146uM; PTK7 expression: 1.01-1.94 Fold). Similar results were observed with GSK2292767, a potent PI3K- delta inhibitor (IC5o:2.2-16uM; PTK7 expresssion: 1.09-2.42 Fold). Idelalisib (KNo:18.8- 99.6uM; PTK7 expression: 1.63-2.45 Fold) and Duvelisib (IC5o:6.3-53.7uM; PTK7 expression: 1.47-2.84 Fold), two dual PI3K-gamma and PI3K-delta inhibitors, found the highest increase in PTK7 expression ranging from 150% to 300% increase in PTK7 expression.
[078] The data also shows that the amount of induction of PTK7 is not the same across cell lines. Turning to FIG. 21, the 231 cell line takes more of the PI3K inhibitors to induce 50% cell death compared the 578 line. FIG. 14 also shows that there is a large induction of PTK7 transcription level when the RI3Kg and PI3K5 are inhibited. Without being limited by theory, it is proposed that the induction of PTK7 in TNBCs compensates for the loss of PI3K resulting in a more resistant form of cancer. As the methods provided herein disclose, the combination of inhibiting RI3Kg, PI3K5, or both and targeting PTK7 results in a more effective means of killing TNBC cells.
[079] EXAMPLE 2: Inhibiting PTK7 or RI3Kg increases the transcription level of PI3K5. [080] Referring to FIGs 24 and 25, three TNBC cell lines were treated with an siRNA to inhibit RI3Kg, PI3K5, and RI3Kd/g as indicated in the graph and the transcription level of PTK7 was analyzed. The siRNA to inhibit both isoforms was a mixture of the two siRNAs at a ratio of about 1:1. The amounts of PTK7 were normalized to the control. As shown in FIG. 24 and FIG. 25, the inhibition of RI3Kg, PI3K5, or both increased the expression level of PTK7 showing that it does act as a compensatory pathway in the TNBC cell line. As shown in FIG.
24, when both were knocked out the PTK7 expression levels increased higher compared to knocking out only one of the PI3K isoforms. FIG. 26 and FIG. 27 are a graph and western blot that show when siRNA to inhibit RI3Kg activity or siRNA to inhibit the activity of PTK7, there was an increase in the transcription level for PI3K5.
[081] EXAMPLE 3: Inhibiting RI3Kd, RI3Kg, or both PI3K d/g in combination with inhibiting PTK7 activity substantially increased the potency of the treatment and shows it is a viable options in methods of treating TNBC. [082] Referring to FIGS. 28, 29, and 30, when four different TNBC cells lines were treated with a PI3K inhibitor in combination with an siRNA directed to PTK7, the results show that the potency of the treatment increased substantially as opposed to the PI3K treatment alone. Scr is scrambled RNA and serves as a control to show the impact of the RNA procedure, if any, along with the PI3K inhibitor. This data indicates that the combination of a PI3K inhibitor and a PTK7 inhibitor can be viable treatment option for patients diagnosed with TNBC. Specifically, the data shows that inhibiting specific PI3K isoforms in combination with inhibiting PTK7 could increase the effectiveness of treating patients diagnosed with TNBC. Finally, because this data demonstrates that inhibiting certain isoforms of PI3K increases the expression level of PTK7, a protein expressed on the outside of the cancer cell, a method of treating a patient with TNBC first with an inhibitor of PI3K5, RI3Kg, or both would substantially increase the protein expression level of PTK7 in the TNBC cells, making the TNBC cells perfect targets for an ADC directed to PTK7. ADCs that carry a drug such as a chemotherapeutic or radioisotope that become active once taken up by the cancer cell are known in the art, rather it is the combinatorial method of targeting both PI3K5, RI3Kg, both and PTK7 to the benefit of treating TNBC that is unknown.
[083] SEQUENCE INFORMATION:
[084] SEQ ID NO 1 : PTK7 siRNA: Chromosome 6 band 12, 43146486-43146752 Homo sapiens chromosome 6, GRCh38.pl3 Primary Assembly
[085] AAGGGGCCCAGGCUAGCUUAGGCCUUCUCACUGCCUCCCCAGGCAGGAAU UCU GCG AGC ACCU GCUU GGUCU G AGGCUUU AC AGCC A AU GGCU GUGC ACU G ACC UGAGCGAGAUGCCUGGCUUUUCCCUUAGGGAGACCUCAAGCAGUUCCUGAGGA UUUCCAAGAGCAAGGAUGAAAAAUUGAAGUCACAGCCCCUCAGCACCAAGCAG AAGGUGAGGACAGGGAGUGAAGGAGGGAGGGAGAGGGUGCCCAGGGGUGGGCC AGGA
[086] SEQ ID NO:2: PI3K5 siRNA: Chromosome 1, band 11, 9724290-9724517 Homo sapiens chromosome 1, GRCh38.pl3 Primary Assembly
[087] UUUUGGCCACUUUCUGGGGAAUUUCAAGACCAAGUUUGGAAUCAACCGCG
AGCGUGUCCCAUUCAUCCUCACCUACGACUUUGUCCAUGUGAUUCAGCAGGGGA
AGACUAAUAAUAGUGAGAAAUUUGAACGGUGAGAGUGCCUGAGCCCCACCAGAU
GCCCCUCGGUGUGGGGCCCCAGGGAACAGGGCAGAGGUUCCCAGGCAGGGUGCA
GGAUGGGGCUCAGGUC [088] SEQ ID NO: 3: RI3Kg siRNA: Chromosome 6, band 12, 43146486-43146752 Homo sapiens chromosome 6, GRCh38.pl3 Primary Assembly
[089] AAGGGGCCCAGGCUAGCUUAGGCCUUCUCACUGCCUCCCCAGGCAGGAAU
UCU GCG AGC ACCU GCUU GGUCU G AGGCUUU AC AGCC A AU GGCU GU GC ACU G ACC
UGAGCGAGAUGCCUGGCUUUUCCCUUAGGGAGACCUCAAGCAGUUCCUGAGGAU
UUCCAAGAGCAAGGAUGAAAAAUUGAAGUCACAGCCCCUCAGCACCAAGCAGAA
GGUGAGGACAGGGAGUGAAGGAGGGAGGGAGAGGGUGCCCAGGGGUGGGCCAG
GA

Claims

We claim:
1. A method of treating TNBC in a patient comprising: administering a therapeutically effective amount of a first compound capable of inhibiting PI3K and a second compound capable of inhibiting PTK7 to the patient.
2. The method of claim 1 , wherein the first compound is capable of inhibiting PI3K5, RI3Kg, or both.
3. The method of claim 2, wherein the first compound is selected from the group consisting of CAY10505, Idelalisib, Duvelisib, Gedatolisib, Buparlisib, and GSK2292767.
4. The method of claim 2, wherein the first compound is capable of inhibiting PI3K5.
5. The method of claim 4, wherein the first compound is selected from the group consisting of GSK2292767, Idelalisib, Duvelisib, and Buparlisib.
6. The method of claim 2, wherein the first compound is capable of inhibiting RI3Kg.
7. The method of claim 6, wherein the compound is selected from the group consisting of CAY10505, Idelalisib, Duvelisib, Gedatolisib, and Buparlisib.
8. The method of claim 1, wherein the second compound is selected from a small molecule inhibitor, an antibody-drug-conjugate, or an siRNA.
9. The method of claim 8, wherein the small molecule inhibitor is selected from (-)- (R)-N-(a-Methylbenzyl)-3,4-dihydroxybenzylidenecyanoacetamide (Tyrphostin AG 527) and 3- Amino-2,4-dicyano-5-(4'-hydroxyphenyl)penta-2,4-dienonitrile (Tyrphostin AG 112).
10. The method of claim 1 further comprising administering a therapeutically effective amount of a drug selected from a chemotherapeutic or radioisotope.
11. The method of claim 10, wherein the drug is a chemotherapeutic.
12. The method of claim 1, wherein the patient is selected from a group consisting of stage 0, stage 1, stage 2, stage 3, and stage 4 TNBC.
13. The method of claim 1, wherein the first compound is an inhibitor of PI3K5, RI3Kg, or both and the second compound is an siRNA of PTK7.
14. The method of claim 13, wherein the siRNA has at least a 95% sequence identity to a nucleotide sequence selected from SEQ ID NO 1.
15. The method of claim 13, wherein the first compound comprises an siRNA capable of targeting PI3K5, RI3Kg, or both and has at least 95% sequence identity to a nucleotide sequence selected from SEQ ID NO. 2 or SEQ ID NO.3.
16. The method of claim 2, wherein the first compound is an siRNA capable of binding to the mRNA of PI3K5, RI3Kg, or both.
17. The method of claim 16, wherein the first compound comprises at least a 95% sequence identity to a nucleotide sequence selected from SEQ ID NO 2 or SEQ ID NO 3.
18. The method of claim 1, wherein the second compound comprises an antibody- drug-conjugate, wherein the antibody portion is capable of binding to PTK7 and the drug portion is a chemotherapeutic or a radioisotope.
19. The method of claim 18, wherein the first compound comprises an siRNA capable of binding to the mRNA of PI3K5, RI3Kg, or both.
20. The method of claim 18, wherein the first compound is selected from the group consisting of CAY10505, Idelalisib, Duvelisib, Gedatolisib, Buparlisib, and GSK2292767.
PCT/US2021/032298 2020-05-13 2021-05-13 Combination therapy for the treatment of tnbc with a pi3k pathway inhibitor that targets pi3kdelta and pi3kgamma tnbc WO2021231762A2 (en)

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