NZ731669B2 - Phospholipid ether analogs as cancer-targeting drug vehicles - Google Patents

Phospholipid ether analogs as cancer-targeting drug vehicles Download PDF

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NZ731669B2
NZ731669B2 NZ731669A NZ73166915A NZ731669B2 NZ 731669 B2 NZ731669 B2 NZ 731669B2 NZ 731669 A NZ731669 A NZ 731669A NZ 73166915 A NZ73166915 A NZ 73166915A NZ 731669 B2 NZ731669 B2 NZ 731669B2
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cancer
compound
cells
tumor
paclitaxel
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NZ731669A
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NZ731669A (en
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Joseph Grudzinski
Kevin Kozak
Marc Longino
Chorom Pak
Anatoly Pinchuk
Nathan Stehle
Benjamin Titz
Jamey P Weichert
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Cellectar Biosciences Inc
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Priority claimed from PCT/US2015/059382 external-priority patent/WO2016081203A2/en
Publication of NZ731669A publication Critical patent/NZ731669A/en
Publication of NZ731669B2 publication Critical patent/NZ731669B2/en

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • 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
    • A61K31/537—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 spiro-condensed or forming part of bridged ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/665—Phosphorus compounds having oxygen as a ring hetero atom, e.g. fosfomycin
    • 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
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A61K47/544—Phospholipids
    • 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
    • A61K47/548—Phosphates or phosphonates, e.g. bone-seeking
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/655—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms
    • C07F9/6551—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a four-membered ring
    • C07F9/65512—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a four-membered ring condensed with carbocyclic rings or carbocyclic ring systems

Abstract

The present invention is directed to therapeutic compounds capable of targeting cancer cells and cancer stem cells. The present invention is further directed to compositions comprising these therapeutic compounds and methods of treating cancer comprising administering these therapeutic compounds. A therapeutic compound comprising the formula A-B-D wherein: A is at least one compound of formula (I). therapeutic compound comprising the formula A-B-D wherein: A is at least one compound of formula (I).

Description

Phospholipid Ether Analogs as Cancer-Targeting Drug Vehicles Background of the Invention In 2012, 14.1 million people were diagnosed with cancer worldwide and 8.2 million died of . In the United States, around 40% of all people will be sed with cancer during their lifetime. Despite receiving the best treatment available, 44% of those ans will die from cancer.
Cancer is the result of a cell dividing without limitation. Healthy cells have checkpoints that prevent unlimited cell division. A few examples of these checkpoints are nutrient bility, DNA damage and contact inhibition (i.e. a cell comes into contact with another cell). onally, most cells can replicate only a finite number of times and thus are programmed to die after a particular number of cell divisions.
Cancer is the result of a cell ming these built—in checkpoints and proliferating beyond control. This uncontrolled proliferation leads to the formation of a tumor. There are two types of tumors, benign and malignant. Benign tumors are incapable of crossing natural boundaries between tissue types. Malignant tumors, on the other hand, are e of invading nearby tissue or entering the bloodstream and metastasizing to a different location. Only malignant tumors are considered cancerous. It is this ability to infiltrate and metastasize that makes cancer such a deadly e.
To further complicate the fight against cancer, malignant tumors have distinct cell types.
One particularly troublesome type is cancer stem cells (“CSC‘S”). CSC’s are e of self- renewing and differentiating into the distinct types of cancer cells found in a malignant tumor.
Thus, CSC’s are a primary factor in the atic ability of a tumor. CSC’s often survive ion and chemotherapy. It is hypothesized that recurrence of cancer after radiation and chemotherapy is the result of the inability of radiation and chemotherapy to kill all CSC’s combined with the ability of CSC’s to establish a new tumor.
A particularly troublesome type of cancer is brain cancer. Brain cancers, such as high- grade gliomas, are often treated with surgery followed by radiation therapy. Surgery for brain tumors is often very complicated. The surgeon must remove the tumor without damaging nearby brain tissue that could result in physical or cognitive lities. Often the surgeon is incapable of removing the boundaries of the tumor that contact the healthy tissue. Radiation therapy is often used to kill these remaining cancer cells. However, radiation doses are limited by the potential damage to healthy brain tissue. Unfortunately, brain cancer is usually chemotherapy resistant. This resistance is largely utable to the brain r (“BBB”). The BBB is a physical r that separates the fluid surrounding the brain from blood cells and other components in the blood stream. Most anti—cancer drugs are unable to cross the BBB.
One method of treating brain cancer is to inhibit the growth of new blood vessels that are necessary for tumor size progression. Bevacizumab marketed under the trademark Avastin® (Avastin is a registered trademark of Genentech, Inc.) is used to stop and even reverse tumor vasculai'ization. However, Rich .l., and colleagues, Canc Res, 2006, 66, 7843, found that when Avastin® was used to treat a glioma stem cell derivedbrain tumor it resulted in hypoxia and a lowered pH. Sathomsumetee 8., Phase II trial of bevacizumab and erlotinib in patients with recurrent ant glioma, Neuro—Oncol, 2010, Dec, 12(12), l300~l310. Hypoxia and low pH are both known to cause CSC propagation and can e CSC-driven tumor recurrence.
Chemotherapy is a term used to describe a particular type of cancer treatment that includes using cytotoxic anti-cancer drugs. Cytotoxic drugs used during chemotherapy can be broken down into several main categories including alkylating agents, antimetabolites, anti— tumor antibiotics, topoisomerase tors, and mitotic inhibitors. Cytotoxic anti-cancer drugs typically cause cell on to cease and thus affect healthy tissue as well as cancerous tissue.
Alkylating agents stop cancer cell division by damaging the DNA of the cancer cell. Some common alkylating agents used to treat cancer are nitrogen mustards (eg. hosphamide (Cytoxan®; Cytoxan is a registered trademark of Baxter ational), nitrosoureas, alkyl sulfonates, triazeines, and ethylenimines. Platinum drugs, such as cisplatin and carboplatin, work similarly to alkylating agents. Antimetabolites stop cancer cell division by inhibiting DNA and RNA synthesis. Some common antimetabolites used to treat cancer are 6—mercaptopurine, gemcitabine (Gemzar®; Gemzar is a registered trademark of Eli Lilly and Company), methotrexate and pemetrexed (Alimta®; Alimta is a registered trademark of Eli Lilly and Company). Topoisomerase inhibitors stop cancer cell division by inhibiting topoisomerase enzymes from separating the DNA for replication. Some common cmerase tors are topotecan, irinotecan, ide, and teniposide. Mitotic inhibitors stop cancer cell division by inhibiting key cell division enzymes. Some common mitotic inhibitors are taxanes (e.g. paclitaxel (Taxol®; Taxol is a registered trademark of Bristol—Myers Squibb Company) and docetaxel ere®; Taxotere is a registered trademark of Aventis Pharma SA)), lones, and vinca alkaloids.
One disadvantage of all of these anti-cancer drugs is the damage that they do to healthy tissue. Because the drugs treat cancer by inhibiting normal cell function, healthy tissue that also relies on constant cell division such as blood cells, mucosal surfaces and skin, can be severely damaged as well. This damage results in significant morbidity and can limit the amount of chemotherapy that can safely be delivered. Examples of side effects that occur during chemotherapy treatment include low blood count, hair loss, muscle and joint pain, nausea, vomiting, diarrhea, mouth sores, fever, and chills. To overcome this problem drugs are being developed that affect proteins and cellular functions that occur only in cancer cells. Some of these specific cancer drugs are ib (Gleevec®; Gleevec is a registered ark of Novartis AG), gefitinib (lressa®, Iressa is a registered trademark of AstraZeneca UK Limited), sunitinib (Sutent®; Sutent is a registered trademark of CF. Pharmaceuticals, International CV), and bortezomib (Velcadeag; Velcade is a registered trademark of Millennium ceuticals, Inc.) However, these drugs are not approved for the treatment of all cancer types and are universally associated with the development of treatment ance. Thus, a need exists in the art for an anti-cancer drug ry vehicle that can r potent, effective, broad spectrum anti-cancer drugs to cancer cells including CSC‘s while avoiding substantial uptake of the drug by healthy cells. Additionally, the anti—cancer drug delivery e should be able to cross the BBB and deliver the ancer drug to cancer cells of the brain.
Currently, there are few chemical compounds that preferentially target cancer cells. One such compound is CLR1404. Generally, CLR1404 is a promising new tumor—selective diagnostic imaging agent used to r the treatment se of several tumor treatment modalities. Radioiodinated CLRl404, a second—generation phospholipid ether (“PLE”) analog ll s (CH3)lgOPUCHgCHENMeg, , with the following structure, 0e displayed remarkable tumor selectivity in 55/60 xenograft, orthotopic and transgenic cancer and cancer stem cell derived animal models making the core molecule an ideal platform for an anti— cancer drug delivery vehicle. See U.S. Patent No. 8535641; U.S. Patent Application Publication No. 2014/0030187 and Weichert, J.P., et al., Alkylphosphocholine s for broad—spectrum cancer imaging and therapy, Sci Transl Med, 2014, Jun 11, , 240ra75; each of which incorporated by reference herein in its entirety, What is not known is whether a compound that is selectively sequestered and retained by cancer cells and cancer stem cells is capable of ring an anti~cancer drug to these same cells. Further, it is not known r this compound is also capable of transporting anti—cancer drugs across the BBB to treat brain cancers. Finally, it is unknown whether this or similar compounds can cause the cancer cell to retain the anti-cancer drug in sufficient quantities and for a sufficient period of time to eradicate the tumor and prevent further growth and metastasis. The present invention adapts the CLR1404 core molecule for use as an anti-cancer drug delivery vehicle capable of targeting the anti—cancer drug to cancer cells and cancer stem cells ing brain cancer cells. Further, the compounds of the present invention are retained in cancer cells.
Summary of the Invention The present invention is directed to therapeutic compounds capable of targeting cancer cells and cancer stem cells including brain tumor cells. The present invention is also directed to therapeutic nds e of being sequestered and retained by cancer cells and cancer stem cells including brain tumor cells in sufficient quantity and for sufficient duration to treat the cancer and prevent metastasis and recurrence.
In one embodiment, the present invention is directed to a therapeutic compound of the formula A—B—D n: A is at least one compound of a (I), ‘3 e W*"'>‘*(Cl~t2}rg;r‘0"“‘ij’hrOCl-{ECHENtCHfig ‘ (I), at least one compound of formula (II), WW (engimwww-QCHmi—lgcs—igmowe}“avocmcugmcagh ac.)i” (H), at least one nd of formula (III), 2015/059382 ‘1?“R 9. :: w~w~‘»(CH;,),;wncugechgmo«gmoeugeuzmtemg (111), or a combination thereof, wherein W is selected from the group consisting of an aryl, a C1-C6 alkyl, an alkenyl, an optionally substituted C3—C6 cycloalkyl and an optionally substituted C3—C6 cycloalkyl, wherein R is H or an alkyl and wherein m is an integer from 12 to 24; B is a linker compound, preferably a bond or a compound of formula (IV), Y~(CH2)n—Z (IV), wherein: Y is bound to A; Z is bound to D; Y is selected from the group consisting of a bond, 0, NH, C=O, NHSOzO, and OC(=O)O; Z is selected from the group consisting of O, NH, C==O, C(=O)O, C(=O)NH, SOZ, OCH2, and ~S-S-; and n is an integer from 0 to 6; and D is an anti—cancer drug, wherein the ratio ofA to D is from 1:2 to 2:1.
In another embodiment, the present invention is directed to a therapeutic compound of the formula A-B-D selected from the group consisting of ’ ‘ S? @ D“Z"(CH2)n_—‘Y"W—(CH2)m"O-i‘?—OCH2CH2N(CH3)3 \@/____J ”.ng Q S) D “‘2 “‘*‘”’(CH2 )fiW’Y " V?! “W (C H2)m'WOC HECHQCHQ “G ”i? ”DCHQCH§N{CH3)3 l Ox: 2 “x.mm,mwmmmw , ”WWW/z" K /" \!.i , and a combination f, wherein: W is selected from the group consisting of an aryl, a C1-C6 alkyl, an alkenyl, an optionally substituted C3-C6 cycloalkyl and an optionally tuted C3-C6 heterocycloalkyl; R is H or an alkyl; m is an integer from 12 to 24; Y is selected from the group consisting of a bond, O, NH, C=O, NHSO2O, and OC(=O)O; Z is selected from the group consisting of O, NH, C=O, C(=O)O, C(=O)NH, SO2, OC(=O)OCH2, and –S-S-; n is an integer from 0 to 6; and D is an anti-cancer drug, wherein B is optionally a bond n A and D.
In a red embodiment, the present invention is directed to a therapeutic compound of the formula A-B-D wherein: A is compound of a (I), wherein W is selected from the group consisting of a C1 alkyl, , , and and wherein m is 18; B is a linker compound selected from a bond and a compound of formula (IV), Y-(CH2)n- Z (IV), wherein n is an integer from 0 to 6, Y is bound to A, Z is bound to D, Y is selected from the group consisting of a bond and C=O and Z is selected from the group consisting of NH, C=O, C(=O)NH and C(=O)O; and 1003091162 D is selected from the group consisting of paclitaxel, irinotecan, topotecan, gemcitabine, cisplatin, geldanamycin and mertansine.
In a more preferred ment, the present invention is directed to a therapeutic compound of the formula A-B-D wherein: A is a compound of formula (I), wherein W is and m is 18; B is a compound of formula (IV), wherein Y is C=O and Z is C=O, C(=O)NH or C(=O)O and n is 3 or 4; and D is paclitaxel, n the ratio of A to D is 1:1.
In another more preferred embodiment, the present invention is directed to a eutic compound of the formula A-B-D wherein: A is a nd of formula (I), wherein W is and m is 18; B is a bond or a compound of a (IV), n Y is C=O, Z is NH and n is 1 or 3; D is geldanamycin, wherein the ratio of A to D is 1:1.
In another more preferred embodiment, the present invention is directed to a therapeutic compound of the formula A-B-D wherein: A is a compound of a (I), wherein W is and m is 18; B is a bond; and D is mertansine, wherein the ratio of A to D is 1:1.
In another aspect, the present invention provides a pharmaceutical composition containing a compound of the present invention in combination with one or more pharmaceutically acceptable carriers. 1003091162 WO 81203 In another aspect, the present invention provides a method of treating cancer comprising administering an effective amount of a therapeutic compound of the present invention to a subject with .
In one embodiment, the present invention es a method of treating cancer comprising administering an ive amount of a therapeutic nd of the present invention to a subject with cancer wherein the cancer comprises cancer stem cells.
In another embodiment, the present invention provides a method of treating cancer comprising administering an effective amount of a therapeutic compound of the t invention to a subject with cancer n the cancer is recurrent.
Brief Description of the s Figure l. Phoslpholipid ether (”PLE”) s are sequestered via lipid rafts.
Figure 2. Preferential uptake of CLRlSOl by cancer cells. e uptake of CLRlSOl by cancer cell lines in (A) and (C)—(F) with normal cells in (B). (A) Renal (Caki—Z). (B) Normal human skin fibroblast. (C) Ovarian (OVcar—3). (D) Pancreatic (Pane-l). (E) Melanoma (A—375).
(F) Prostate (PC-3).
Figure 3. Prolonged retention of 131l—CLR1404 by human RL—251 tumor xenograft in SCID mouse.
Figure 4. Detection of C6—glioma in rat brain using 125LNM404. (A) Bioscan of sham control rat brain. (B) Bioscan image of rat brain from (A) superimposed over digital photo showing background levels of 125I-NM404 in normal brain tissue. (A’) Digital photo of C6- glioma bearing rat brain 4 days post 1251—NM404 injection. (B’) Bioscan image of rat brain from (A’). (C’) Position and size—matched images of (A’) and (B’) superimposed to show intense localization ofNM404 in tumor. (D’) H&E stained sample confirming presence of tumor.
Figure 5. 124I—CLR14O4 uptake in a broad range of malignant tumors. (A)-(I) are rodent models with human cancer xenografts. ) are rodent cancer models. (A) Orthotopic glioma U87 (rat). (B) Colon HGT-116. (C) Colon . Arrow indicates location of tumor.
(D) Breast MDA-MB—23 1. Arrow indicates location of tumor. (E) Prostate PC-3. (F) Metastatic PC-3. (G) PC—3 tibial xenograft. (H) Pancreatic BXPC3. Lower arrow indicates location of tumor. Upper arrow indicates liver metastasis. (I) Ewing’s sarcoma. Arrow indicates location of tumor. (J) Mouse SV4O bladder. (K) Mouse Breast 4T1. (L) Mouse pancreatic c-myc. (M) Rat brain CNS-l.
Figure 6. Detection of non~small cell lung cancer (“NSCLC”) tumors in human t using 131l-CLRI404. (A) shows gamma camera images of Patient 1 at 4 and 11 days post 131[- CLR1404 injection. Note intense and prolonged retention of CLR1404 in the NSCLC tumors (arrows). (B and C) show the on and size of focal 3cm lesion in left lung (A) and large infiltrative mass in right lung (B) s). (D and E) Show whole body planar nuclear ne images of Patient 2 1, 2 and 4 days post ” II-CLR1404 IV administration. (F and G) show axial (F) and coronal (G) CT scans ting location of large 6cm NSCLC tumor (arrows).
Figure 7. Detection of 3 previously n brain tumor metastases in NSCLC patient using 124I-CLR1404. Arrows indicate on of tumors as imaged using PET/CT after uptake of 124l-CLR1404 by cancer cells.
Figure 8. Detection of tumor recurrence of a right frontal falcine metastasis using 1241— CLR1404. (A) MRI of brain following radiosurgery. Arrow indicates lesion which was interpreted as radiation necrosis. (B) PET image using 124I—CLR1404 shows uptake of 124I~ CLR1404 by lesion. (C) MR1 of brain 8 months after stereotactic radiosurgery shows se in size of lesion indicating possible recurrence.
Figure 9. PLE—Paclitaxel Conjugates Ing for MDA-MB—468. (A) free paclitaxel, (B) CLR1601 and (C) CLR1603.
Figure 10. PLE—Paclitaxel Conjugates IC50 for NCl—H1299. (A) free paclitaxel, (B) CLR1601 and (C) CLR1603.
Figure 11. PLE-Paclitaxel Conjugates ICso for NCl—H460. (A) free paelitaxel, (B) CLR1601 and (C) CLR1603.
Figure 12. PLE—Paclitaxel Conjugates leo for Capan—2. (A) free paclitaxel, (B) CLR1601 and (C) CLR1603.
Figure 13. clitaxel Conjugates 1C50 for MiaPaCa—l. (A) free paclitaxel, (B) CLR1601 and (C) CLR1603.
Figure 14. PLE-Paclitaxel Conjugates ICso for HT29. (A) free paclitaxei, (B) CLR1601 and (C) CLR1603.
Figure 15. PLE-Paclitaxel Conjugates leo for . (A) free paclitaxel, (B) CLR1601 and (C) CLR1603. [037} Figure 16. PLE—Paclitaxel Conjugates IC50 for PC-3. (A) free paclitaxel, (B) CLR1601 and (C) CLR1603. 2015/059382 Detailed Description of the Invention Definitions As used herein, the term ”treating" includes preventative as well as disorder remittent treatment including reducing, suppressing and inhibiting cancer progression or recurrence. As used herein, the terms ”reducing", "suppressing" and "inhibiting" have their commonly understood meaning of lessening or decreasing. As used herein, the term "progression" means increasing in scope or severity, advancing, growing or becoming worse. As used herein, the terms "recurrence” and “recurrent” refer to the return of a disease after a remission.
As used herein, the term "administering" refers to ng a patient, tissue, organ or cells in contact with an ancer compound of the present invention. As used herein, administration can be accomplished in vitro (i.e. in a test tube) or in viva, (i.e. in cells or tissues of living organisms, for example, humans). In certain embodiments, the present invention encompasses administering the compounds useful in the present invention to a patient or subject. A nt" or ct", used lently herein, refers to a mammal, preferably a human, that either: (1) has a disorder remediable or treatable by administration of the anti-cancer substance using a PLE compound or (2) is susceptible to a disorder that is preventable by administering the anti—cancer compound of the present invention.
As used herein, the term tive ” refers to an amount sufficient to affect a desired ical effect, such as a beneficial result, including, without limitation, prevention, diminution, amelioration or elimination of signs or symptoms of a disease or disorder. Thus, the total amount of each active component of the pharmaceutical composition or method is sufficient to show a meaningful subject benefit. Thus, an tive amount" will depend upon the t in which it is being administered. An effective amount may be administered in one or more prophylactic or therapeutic administrations.
As used herein the term “therapeutic nd” refers to any chemical compound capable of providing treatment for cancer.
As used herein the term “cancer” refers to any disease that results from the uncontrolled on of cells capable of metastasizing.
The terms “chemotherapy drug” “anti—cancer drug” and tumor drug” are used interchangeably throughout the specification.
The term “malignant tumor cell” and “cancer cell” are used interchangeably throughout the specification. The term “malignant tumor stem cell” and “cancer stem cell” are used interchangeably throughout the specification.
As used herein, the term “composition” is intended to encompass a product comprising the specified ingredients in the specified s, as well as any t which results, directly or ctly, from a combination of the specified ingredients in the specified amounts.
As used herein the term “A” refers to an phospholipid ether of the formula U 4/ at, w a...{cg.t,}fi;wQ-mgwwocchHgmrcagn Wm(CH2),,,WOCHQCHECHQ mo “é““GCHQCl'bNWHsh 33 {5) a if“) 01‘ " 01” ?”R i? a W“”{CH9),TT“QCHQCHCH; ””0”“? ”OCHQCH2N(CH3)3 As used herein the term “W” refers to an aryl, a C1-C6 alkyl, an alkenyl, an optionally substituted C3—C6 cycloalkyl and an optionally substituted C3~C6 heterocycloalkyl.
As used herein the term “aryl” refers to an aromatic ring including a phenyl group.
As used herein the term “alkyl” refers to a branched or ht—chain alkyl consisting of a saturated hydrocarbon group of 1 to 24 carbon atoms (Cl-€24) unless otherwise stated. The alkyl group can be cyclic or c.
As used herein the term “alkenyl” refers to a carbon-carbon double bond.
As used herein the term “cycloalkyl” refers to a cyclic alkyl group of 3 to 24 carbon atoms (C3—C24).
As used herein the term “heterocycloalkyl” refers to a cyclic group of 3 to 24 atoms (C3- C24) selected from , en, sulfur, phosphate and oxygen wherein at least one atom is carbon.
In general, the term “substituted,” whether preceded by the term nally” or not, means that one or more hydrogens of the ated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of -11_ substituents envisioned by this invention are ably those that result in the ion of stable or ally feasible compounds.
As used herein the term “R” refers to a en (H) or an alkyl.
As used herein the term “m” refers to an r of 12 to 24.
As used herein the term “n” refers to an integer of 0 to 6.
As used herein the term “B” refers to a linker compound. As used herein the term “linker nd” refers to any chemical compound or compounds capable of forming a chemical bond with two or more other distinct chemical compounds such that all nds form a single larger compound. In one ment, the linker compound is a bond. Multiple linker compounds may be used in the formation of the larger compound. In specific embodiments, the term linker nd is a bond or a compound of the formula Y—(CH2)n—Z.
As used herein the term “Y” refers to a bond, 0, NH, C=O, NHSOgO, or OC(=O)O.
As used herein the term “Z” refers to O, NH, C==O, C(=O)O, C(=O)NH, 802, OC(=O)OCH2, and —S—S-.
As used herein the term “D” refers to any anti-cancer drug currently known or in development.
As defined herein, the term "isomer" includes, but is not limited to optical isomers and analogs, structural isomers and analogs, conformational isomers and s, and the like. In one embodiment, this invention encompasses the use of different optical isomers of the present invention. It will be appreciated by those skilled in the art that the anti—cancer compounds useful in the present invention may contain at least one steriogenic . Accordingly, the compounds used in the methods of the present invention may exist in, and be isolated in, optically~active racemic forms. Some compounds may also t polymorphism.
It is to be understood that the present invention may encompass the use of any racemic, optically-active, polymorphic, or stereroisomeric form, or mixtures thereof, which form possesses properties useful in the treatment of cancer—related conditions described and claimed herein. In one embodiment, the anti—cancer compounds may include pure (R)—isomers. In another embodiment, the anti—tumor compounds may include pure (S)-isomers. In another embodiment, the nds may include a mixture of the (R) and the (S) isomers. In another embodiment, the compounds may include a racemic mixture comprising both (R) and (S) isomers. It is well known in the art how to prepare optically—active forms (for example, by resolution of the c form by recrystallization ques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral nary phase).
The invention includes the use of pharmaceutically acceptable salts of amino-substituted compounds with c and inorganic acids, for example, citric acid and hydrochloric acid. The invention also includes N—oxides of the amino substituents of the nds described herein.
Pharmaceutically acceptable salts can also he prepared from the phenolic compounds by treatment with inorganic bases, for example, sodium hydroxide. Also, esters of the phenolic compounds can be made with aliphatic and aromatic ylic acids, for e, acetic acid and benzoic acid esters. As used herein, the term "pharmaceutically acceptable salt" refers to a nd ated from a base compound which achieves substantially the same pharmaceutical effect as the base compound.
This invention further includes derivatives of the ancer compounds. The term atives" includes but is not d to ether derivatives, acid derivatives, amide derivatives, ester derivatives and the like. In addition, this invention further includes methods utilizing es of the anti—tumor compounds. The term "hydrate" includes but is not limited to hemihydrate, monchydrate, dihydrate, trihydrate and the like.
This invention further es metabolites of the anti-cancer compounds. The term "metabolite" means any substance produced from ancther substance by metabolism or a metabolic process.
Cancers that can be treated with compounds of the present invention include, but are not limited to: breast cancer including male breast cancer; digestive/gastrointestinal cancers including anal cancer, appendix cancer, epatic bile duct cancer, gastrointestinal carcinoid tumor, colon cancer, esophageal cancer, gallbladder cancer, gastric cancer, gastrointestinal stromal tumors (“gist”), Islet cell tumors, adult primary liver cancer, childhood liver cancer, pancreatic , rectal cancer, small intestine cancer, and stomach (gastric) cancer; endocrine and neuroendocrine cancers including pancreatic adenocarcinoma, adrenocortical carcinoma, pancreatic neuroendocrine tumors, Merkel cell carcinoma, non-small cell lung neurcendocrine tumor, small cell lung neuroendocrine tumor, parathyroid cancer, pheochromocytoma, pituitary tumor and thyroid cancer; eye s including intraocular melanoma and retinoblastoma; genitourinary cancer including bladder cancer, kidney (renal cell) cancer, penile cancer, prostate cancer, tional cell renal pelvis and ureter cancer, testicular , urethral cancer and Wilms tumor; germ cell cancers including childhood central nervous system cancer, childhood ranial germ cell tumor, extragonadal germ cell tumor, ovarian germ cell tumor and testicular cancer; gynecologic cancers including cervical cancer, endometrial cancer, gestational trophoblastic tumor, ovarian lial cancer, ovarian germ cell tumor, uterine sarcoma, vaginal cancer and vulvar cancer; head and neck s including aryngeal cancer, laryngeal cancer, lip and oral cavity cancer, metastatic squamous neck cancer with occult primary, mouth cancer, nasopharyngeal , oropharyngeal cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, pharyngeal cancer, salivary gland cancer and throat cancer; leukemias including adult acute lymphoblastic ia, childhood acute lymphoblastic leukemia, adult acute myeloid leukemia, childhood acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous ia and hairy cell leukemia; lymphomas including AIDS-related lymphoma, cutaneous t—cell lymphoma, adult Hodgkin ma, childhood Hodgkin lymphoma, Hodgkin lymphoma during pregnancy, mycosis fungoides, adult non-Hodgkin lymphoma, childhood non-Hodgkin lymphoma, dgkin lymphoma during pregnancy, primary central s system lymphoma, Se’zary syndrome and Waldenstrém macroglobulinemia; musculoskeletal cancers including Ewing sarcoma, osteosarcoma and malignant fibrous histocytoma of bone, childhood rhabdomyosarcoma and soft-tissue sarcoma; neurological cancers including adult brain tumor, childhood brain tumor, astrocytomas, brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal , craniopharyngioma, ependymoma, neuroblastoma, primary l nervous system (CNS) lymphoma; respiratory/thoracic s including all cell lung cancer, small cell lung cancer, malignant mesothelioma, thymoma and thymic carcinoma; and skin cancers including Kaposi sarcoma, melanoma and squamous cell carcinoma.
Compounds of formula (I) of the present invention have been demonstrated to be sequestered by cancer stem cells. See, rt J .P., et al. (2014) at 2, Figure 2 (demonstrating that CLR-lSOl, a CLR1404 fluorescent analog, has enhanced uptake by human glioblastoma stemlike cells and serum-cultured human glioblastoma cells as compared to normal human astrocytes and fetal human neural stem cells.) Cancer stem cells are associated with most, if not all, major cancer types. Tumor hypoxia stimulates cancer stem cell propagation, leading to increased resistance and metastatic potential. As such, cancer stem cells are associated with herapy resistance, tumor re—growth, and asis ing chemotherapy and radiation therapy. Thus, compounds of the present invention have the potential to treat various forms of cancer that have proven resistant to traditional therapy regimens.
Compounds oft/1e Invention Drug delivery vehicles that are useful for the present invention include, but are not limited to, compounds of formula (I), G I“ 3: ti} W““~"’{CH3),.,§WOWl? "’“OCl’igCHszCH3)3 {573‘ (I) or formula (II), C) i Wm {CHELWocngcazcaz-o«wrfivwocuzcrizngcugg V (II), or formula (III) SW 9 63> W"“”’(CH2)5§WOCH2CHCH2“O’”$‘”QCHQCHQN(CH3)3 0‘33 (III), or a combination thereof, wherein W is selected from the group consisting of an aryl, a C1—C6 alkyl, an alkenyl, an optionally substituted C3—C6 cycloalkyl and an optionally substituted C3—C6 heterocycloalkyl, wherein R is H or an alkyl and wherein m is an integer from 12 to 24.
The basis for ive tumor targeting of compounds of the t invention lies in differences between the plasma membranes of cancer cells as compared to those of most normal cells. Specifically, cancer cell membranes are highly enriched in “lipid rafts”. Cancer cells have five to ten times more lipid rafts than healthy cells. Lipid rafts are lized regions of the membrane phospholipid bilayer that contain high, concentrations of cholesterol and olipids and serve to organize cell surface and intracellular signaling les (e.g., growth factor and cytokine receptors, the phosphatidylinositol 3-kinase (Pl3K)/Akt survival pathway). Data suggests that lipid rafts serve as portals of entry for PLEs. The marked ivity of these compounds for cancer cells versus non—cancer cells is attributed to the high affinity of PLES for cholesterol and the abundance of cholesterol-rich lipid rafts in cancer cells. The pivotal role played by lipid rafts is underscored by the fact that disruption of lipid raft architecture suppresses uptake of PLEs into cancer cells. It has been shown that the uptake of PLE’s is reduced by 60% when lipid rafts are blocked from forming. (See Example 2 and Fig. l).
Preliminary results obtained in over 55 aft and spontaneous tumor models have universally shown CLR1404 to undergo selective uptake and prolonged retention in tumors.
Because the agent is lized to some extent in the liver, the inventors avoided earlier compound evaluation in liver tumor models due to high liver background radioactivity levels.
CLR1404 is a PLE. Results obtained in a variety of tumor models indicate that CLR1404 is sequestered and ively retained by cancer cells and cancer stem cells. In fact, CLRI404 has been shown to remain in cancer cells for up to 20 days. See Fig. 3. 4 localizes in both primary and atic lesions regardless of anatomic location including those found in lymph nodes. See es 3~8. The high tumor to background avidity and tumor selectivity of CLR1404 suggests the core molecule is well—suited for use as an anti~cancer drug delivery vehicle.
Linker compounds that are useful for the present invention include any chemical linker capable of binding a drug delivery vehicle of the t invention to an anti—cancer drug of the present invention. Linker compounds that are useful for the present invention include both cleavable and non—cleavable s. In one embodiment, linker compounds that are useful for the present invention include, but are not limited to, aminobutyramide, amino acids, glutaramic acids, dicarboxylic acids, carbamic acids, a carbonyl, 9,10-anthracenedicarboxylic acid, biphenyl-3,3’,5,5’—tetracarboxylic acid, biphenyl-3,4’,5-tricarboxylic acid, 5—bromoisophthalic acid, 5—cyano-1,3—benzenedicarboxylic acid, 2,2’-diamino-4,4’—stilbenedicarboxylic acid, 2,5— diaminoterephthalic acid, 2,5—dihydroxyterephthalic acid, S-ethynyl-l,3-benzenedicarboxylic acid, 2—hydroxyterephthalic acid, imidazole, 2-methylimidazole, 2,6~naphthalenedicarboxylic acid, oxalic acid dehydrate, terephthalic acid, [l,l':4’,1”]terphenyl- 3,3”,5,5"—tetracarboxylic acid, 3,3’,5,5’—tetracarboxydiphenylmethane, l,2,4,5—tetrakis(4-carboxyphenyl)benzene, 4,4',4"- s-triazine-Z,4,6-triyl-tribenzoic acid, ic acid, tris(4’-carboxy[l,1’—biphenyl]—4- yl)benzene, 1,3,5-tri3(4—carb0xyphenyl)benzene, and l,3,5-triscarboxyphenylethynylbenzene.
In another embodiment, linker nds useful for the present invention also include, but are not limited to, lysosomal protease sensitive s with or Without an aniline—based self— immolative fragment. Non-limiting examples of lysosomal protease sensitive linkers are —16— W r: o a and \g/ which contain the valine— citrulline dipeptide linker designed to display an optimal balance between plasma stability and intracellular protease cleavage. See, Tranoy—Opalinski I., Design of self-immolative linkers for -activated prodrug therapy, ncer Agents Med Chem, 2008 Aug, 8(6):618-637, which is incorporated by reference herein in its entirety.
In another embodiment, linker compounds useful for the present invention also include, but are not limited to, self-immolative linkers that are cleaved by B-glucuronidase. [3~ glucuronidase is t in high concentration in necrotic area surrounding cancer cells. See, Tranoy-Opalinski 1., B-glucuronidase- responsive prodrugs for ive cancer chemotherapy: An update, Eur J Med Chem, 2014 Mar 3, 74, 302-313, which is incorporated by reference herein in its entirety. Non-limiting examples of B-glucuronidase-cleavable mmolative COOH )L “0%, H of” linkers are N02 and f—i _,_,.
OH Y“ , wherein X is NH; or N02 and wherein Y is O or NCH3. red linker nds of the present invention are a bond or a compound of formula (IV), Y—(CH2)n-Z (IV), wherein: Y is bound to A; Z is bound to D; Y is selected from the group consisting of a bond, 0, NH, C=O, NHSOgo, and OC(=O)O; and Z is selected from the group consisting of O, NH, C=O, C(=O)O, H, 302, OC(=O)OCH2, and ~S—S--; and n is an integer from O to 6.
More preferred linker nds of the present invention are a bond or a compound of a (IV), wherein n is an integer from O to 6, Y is bound to A, Z is bound to D, Y is selected from the group ting of a bond and C=O and Z is selected from the group consisting ofNH, C=O, H and C(=O)O.
Anti-cancer drugs that are useful for the present invention include, but are not limited to, paclitaxel, ecan, topotecan, abine, cisplatin, geldanamycin, mertansine, abiraterone, afatinib, aminolevulinic acid, aprepitant, axitinib, azacitidine, belinostat, bendamustine, bexarotene, bleomycin, bortezomib, bosutinib, busulfan, cabazitaxel, cabozantinib, capecitabine, latin, carfiizomib, carmustine, ceritinib, cetuximab, chlorambucil, abine, crizotinib, cyclophosphamide, cytarabine, enib, dacarbazine, dactinomycin, dasatinib, daunorubicin, decitabine, denosumab, dexrazoxane, docetaxel, dolastatins (e.g. monomethyl auristatin E), doxorubicin, enzalutamide, epirubicin, eribulin mesylate, nib, etoposide, everolimus, floxuridine, fludarabine phosphate, racil, ganetespib, gefitinib, gemtuzumab ozogamicin, hexamethylmelamine, hydroxyurea, ibritumomab an, ibrutinib, idelalisib, ifosfamide, imatinib, ipilimumab, ixabepilone, lapatinib, leucovorin calcium, lomustine, maytansinoids, mechlorethamine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin C, mitotane, mitoxantrone, nelarabine, nelfmavir, nilotinib, obinutuzumab, ofatumumab, axine mepesuccinate, latin, panitumumab, pazopanib, pegaspargase, pembrolizumab, pemetrexed, pentostatin, pertuzumab, plicanycin, pomalidomide, ponatinib hydrochloride, pralatrexate, procarbazine, radium 223 dichloride, ramucirumab, fenib, retaspimycin, ruxoiitinib, semustine, siltuximab, sorafenib, streptozocin, sunitinib malate, tanespimycin, temozolomide, temsirolimus, teniposide, thalidomide, thioguanine, thiotepa, toremifene, trametinib, trastuzumab, vandetanib, vemurafenib, vinblastine, vincristine, vinorelbine, vismodegib, vorinostat, and ziv-aflibercept. Any compounds that are currently known to or are capable of acting as anti-cancer drugs are also useful for the present invention.
PLE drug delivery vehicles of the present invention may attach singularly or in multiple to an anti-cancer drug in any number of possible stable attachment sites via a linker compound directly. 2015/059382 Compositions ofthe Invention In another aspect, the present invention provides a pharmaceutical composition containing a compound of the present invention in ation with one or more pharmaceutically acceptable carriers. In a preferred aspect the pharmaceutical composition is free of Kolliphor® EL (Kolliphor is a registered trademark of BASF SE). Kolliphor® BL is formerly known as Cremophor® EL (Cremophor is a registered trademark of BASF SE).
Actual dosage levels of active ingredients in the therapeutic compositions of this invention can be varied so as to obtain an amount of the active compound(s) which is effective to achieve the desired therapeutic response for a particular patient, compositions and mode of administration. The selected dosage level will depend upon the ty of the ular compound, the route of administration, the severity of the condition being treated and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound at levels lower than required to e the desired therapeutic effect and to gradually se the dosage until the desired effect is achieved.
The phrase peutically effective amount" of the compound of the invention means a sufficient amount of the compound to treat disorders, at a reasonable /risk ratio able to any medical treatment. It will be tood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being d and the severity of the disorder; activity of the specific compound employed; the specific ition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
The total daily dose of the nds of this invention administered to a human or lower animal may range from about 0.000] to about 1000 mg/kg/day. For purposes of oral administration, more preferable doses can be in the range of from about 0.001 to about 5 -19_ 2015/059382 mg/kg/day. If desired, the ive daily dose can be divided into multiple doses for purposes of administration; consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose.
The present invention also provides pharmaceutical compositions that comprise compounds of the t invention formulated together with one or more pharmaceutically acceptable carriers. The pharmaceutical compositions can be specially formulated for oral administration in solid or liquid form, for parenteral administration or for rectal administration.
The pharmaceutical compositions of this invention can be administered to humans and other mammals orally, rectally, parenterally, intracisternally, intravaginally, transdermally (e.g. using a patch), transmucosally, sublingually, pulmonary, intraperitoneally, topically (as by powders, cintments or drops), bucally or as an oral or nasal spray. The terms “parenteral” or ”parenterally," as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
In another aspect, the present invention provides a pharmaceutical composition comprising a ent of the t invention and a logically tolerable diluent. The present invention includes one or more compounds as described above formulated into itions together with one or more physiologically tolerable or acceptable diluents, carriers, adjuvants or es that are collectively referred to herein as diluents, for parenteral injection, for intranasal ry, for oral administration in solid or liquid form, for rectal or topical administration, among others.
Compositions suitable for eral ion may comprise physiologically acceptable, sterile aqueous or eous solutions, dispersions, suspensions or emulsions and e powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene , glycerol, and the like), vegetable oils (such as olive Oil), injectable organic esters such as ethyl oleate, and suitable mixtures f.
These compositions can also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be d by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for e sugars, sodium chloride and the like. ged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
Suspensions, in addition to the active compounds, may contain ding agents, as for e, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar—agar and anth, or es of these substances, and the like.
Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide—polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug e can be controlled. Examples of other biodegradable rs include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body s.
The injectable formulations can be sterilized, for example, by filtration through a ial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.
Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound may be mixed with at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and silicic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyn'olidone, e and acacia; c) humectants such as glycerol; d) egrating agents such as agar~agar, m carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) solution retarding agents such as paraffin; 1) absorption accelerators such as quaternary um compounds; g) wetting agents such as cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering .
Solid compositions of a similar type may also be ed as fillers in soft and hard- filled gelatin capsules using such ents as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
The solid dosage forms of tablets, dragees, es, pills and granules can be prepared with coatings and shells such as enteric gs and other coatings nown in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a composition such that they release the active ingredient(s) only, or entially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding itions which can be used include ric substances and waxes.
The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above—mentioned excipients.
Liquid dosage forms for oral administration e pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl e, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3— ne glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofin‘furyl alcohol, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof.
Besides inert diluents, the oral compositions may also e nts such as wetting agents, fying and suspending agents, sweetening, flavoring and perfuming agents.
Compositions for rectal or vaginal administration are preferably suppositories which can be ed by mixing the compounds of this invention with suitable non-irritating excipients carriers such as cocoa butter, polyethylene glycol or a itory wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
Compounds of the present invention can also be administered in the form of liposomes.
As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono~ or lamellar hydrated liquid crystals which dispersed in an aqueous medium. Any, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a compound of the present invention, stabilizers, preservatives, excipients and the like. The preferred lipids are natural and synthetic phospholipids and phosphatidyl cholines (lecithins) used separately or together. Methods to form liposomes are known in the art.
See, for example, Prescott, Ed, Methods in Cell BiologyLVolume XIV, Academic Press, New York, NY. (1976), p. 33 et seq. Such compositions will influence the physical state, solubility, stability, rate of in viva release, and rate of in viva clearance.
In one method of the present invention, a ceutical composition can be delivered in a controlled release system. For example, the agent may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump may be used (see Langer, supra; , CRC Crit.
Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N.
Engl. J. Med. 321:574 (1989). In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity to the therapeutic target, for example liver, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 . Other controlled release systems are discussed in the review by Langer ce 27—1533 (1990).
In another aspect, the invention is directed to a method of treating a disease or condition in a subject comprising administering to the subject an effective amount of a compound of the present ion.
In general, the invention is not limited to ent of any specific disease or condition but asses the treatment of any disease or condition Whose mechanism may be affected by the nds of the present invention.
Representative Embodiments Paclitaxel—CLR1404 Conjugates In one embodiment of the t invention the eutic compound is paclitaxel linked to an CLRI404 core compound by a dicarboxylic acid linker, n the dicarboxylic acid linker is attached to the CLR1404 core compound via an amide bond and to paclitaxel via an ester bond at the 2’~OH group, é, n feie3i/éCH33fi50 :QMECH) ("it In a preferred embodiment of the present invention the therapeutic compound is axel linked to the CLR1404 core compound by a glutaramic acid linker, wherein the glutaramic acid linker is attached to the CLR1404 core nd via an amide bond and to paclitaxel via an ester bound at the 2’—OH group, O «J? rm“ 0 33.7 \t }t*"€CH2)zaO§OCH3ci—12Nuie3 W“ o C»): CLR1601 conjugate.
In another embodiment of the present invention the therapeutic compound is paclitaxel linked to the CLR1404 core compound by a dicarboxylic acid linker, wherein the dicarboxylic acid linker is attached to the 4 core nd via an amide bond and to paclitaxel via an ester bond at the 7—OH group, ‘i "'5 ‘ «xv-«3‘ aminezgzaoaaeafimgrtaegA t, a a.
In another embodiment of the t invention the therapeutic compound is paclitaxel linked to the CLR1404 core compound by a carbamic acid linker, n the carbamic acid linker is attached to the CLR1404 core compound via an amide bond and to paclitaxel via an ester bond at the 7-OH group, W;“x (g 0 it to m M e of‘rN-r we a“NW5: (cagitgopocageagweg “j“ [I the“? “L H 0 Got x t“, , .1 5 0% o7 L x 4’} OH gym\‘x O fl Erw 0 CLR1602 conjugate.
In another embodiment of the present invention the therapeutic compound is paclitaxel linked to the 4 core compound by a carbonic—carboxylic acid linker, wherein the carbonic-carboxylic acid linker is attached to the 4 core compound via an amide bond and to paclitaxel via an ester bond at the 7—OH group, E Jtar/‘1?“ O ‘}>~O O OH r a.” r gain x O 41%;" EJH Q \ \l If "e i/Trbfifgxmfl-fi».mox \ : «V/ {a Xx) H i 0H6 0t W 0 We“; 57-.
Omf “Kay ‘50 O » 2“ 0:91:71; [fw‘x ? E} HNWKZK. fig—“(CHQMBOEQCHQCHENMBg \‘WMJ s CLR1603 conjugate.
In another embodiment of the present invention the therapeutic compound is paclitaxel linked to two 4 core compounds by dicarboxylic acid s, wherein the dicarboxylic acid linkers are attached to the CLR1404 core compounds Via amide bonds and to paclitaxel Via ester bonds at both the 2’-OH group and the 7—OH group, as $3 triegtéfifigfi H2353???) “it:, ‘12:) In another embodiment of the present invention the therapeutic compound is axel linked to the CLR1404 core compound by a dicarboxylic acid , wherein the dicarboxylic acid linker is attached to the CLR1404 core compound Via an amide bond and to paclitaxel Via carbonate or a carbamate bond at the 2’-OH group, WO 81203 2015/059382 32 i3; 3333333eH333H3333333 33:33-33 In another embodiment of the present invention the therapeutic compound is paclitaxel linked to an CLR1404 core compound by a dicarboxylic acid linker, wherein the dicarboxylic acid linker is attached to the CLR1404 core compound via an amide bond and to axel via carbonate or a carbamate bond at the 7—OH group, ffixff33H3333%333‘3—33333333333{*9 ’33?3.33eHH3 3:353 l3» 3.“3:: In another embodiment of the present invention the therapeutic compound is paclitaxel linked to two CLR1404 core compounds by oxylic acid linkers, wherein the dicarboxylic acid linkers are attached to the two CLRl404 core molecules via amide bonds and to paclitaxel via a carbonate or a carbamate bond at both the 2’—OH group and the 7—OH group, 23‘! Ci it Mm :3} “11* 93%er f teas}tgeéeeezeegxraag % ifitiréfiiizis {:3 {if} r,x > «a r: «w a {Ci—{Sin a Ema aegiscegeagoéawreafigggflmaaw; In one embodiment of the present invention the therapeutic compound is paclitaxel linked to a C18 alkyl phosphocholine nd via a carboxylic , wherein the carboxylic linker is attached to the C18 alkyl phosphocholine compound Via an amide or ate bond and to paclitaxel Via a carbonate or a carbamate bond at the 2’—OH group 69 9 ko<;>—< 0%— M€3NCHZCH20?O“(CH2)18“X Irinol’ecan-CLRI404 Conjugate In one embodiment of the present invention the therapeutic compound is irinotecan linked to the CLR1404 compound by a dicarboxylic acid linker, wherein the dicarboxylic acid linker is attached to the CLR1404 core compound Via an a carbonate or a ate bond and to irinotecan Via an ester bond, —28- — 5&3 HR / (CH2)gfllééfJCE-LCHQNi’lefig ecan-CI8 alkyl phosphocholine ate In one embodiment of the present invention the therapeutic compound is irinotecan linked to a C18 alkyl phosphocholine compound by a carbonyl linker, n the carbonyl linker is attached to the C18 alkyl phosphocholine compound via a carbon—carbon bond and to irinotecan via an ester bond, Gems-120w [2:363 Topotecan-CLR1404 Conjugates In one embodiment of the present invention the therapeutic compound is topotecan linked to the 4 core compound by a non-hydrolyzable phenyl ether, MeghCHECHZOPO{CHa)ng/—>«O\C} f! U,\ a I“ ”\O1WD In another embodiment of the present invention the therapeutic compound is topotecan linked to an CLR1404 core compound by a dicarboxylic acid linker, wherein the dicarboxylic acid linker is ed to the CLR1404 compound via a carbonate or a carbamate bond and to topotecan via an ester bond, {"1 ‘5, t w\V‘\ A“: ‘x O {‘3/ \Mnt“%\ (/71 {3‘0 H'&2')?! X-Q—MHQOPQCHgCHg‘éitie Gemcitabine-C18 alkylphosphocholine ate In one embodiment of the present invention the therapeutic compound is gemcitabine linked to two C18 alkyl phosphocholine compounds by carbonyl linkers, wherein the carbonyl linkers are attached to the C18 alkyl phosphocholine compounds via carbon-carbon bonds and to gemcitabine via ester bonds, if“? if ““N \Nirh at} 61:!) 1 ”A E: MagiJCHQCHZQPQ”WW (”NewA ,0x e i.
C3 0 iié’gi‘iCHECHEOi‘éG V”A "‘u/‘N r\ “x ”a. ”C F V“ m MMx \n % Q In another embodiment of the present invention the therapeutic compound is abine linked to a C18 alkyl phosphocholine nd by a carbonyl linker, wherein the carbonyl linker is attached to the C18 alkyl phosphocholine compound via a carbon-carbon bond and to gemcitabine via an ester bond, (‘9 9 o MeaNCHQCHZOEO 0 F 06 o OH F Cisplatin~CLR1404 Care Conjugate In one embodiment of the present invention the therapeutic compound is cisplatin linked directly to the CLR1404 core compound, ’t ,H Clk at r: 9 a (3] fat} (Gazhaogocmcwma H H O Geldanamycin—CLRZ404 Conjugates In one embodiment of the present invention the therapeutic nd is geldanamycin linked directly: the CLR1404 core nd, In another embodiment of the present invention the therapeutic nd is amycin linked to the CLR1404 core compound by a short amino acid linker, wherein the amino acid linker is connected to the CLR1404 core compound via a carbonate or carbamate bond and to the geldanamycin via an amide bond, ‘3) ’33? U , P isifigi‘lCHgCHgCt?G{CH2}~§g r’ \ in ASH-23 r: \rzt/ [r i? exit ”is i 1g Q Q ' “M - at a l taéflJ is, :3 In a preferred ment of the present invention the therapeutic compound is geldanamycin linked to the CLR1404 core compound by an aminobutyramide linker, wherein the aminobutyramide linker is connected to the CLR1404 core compound Via a carbamate bond and to the geldanamycin Via an amide bond: Mewmgmgnmgcam«waif:2 v Q?) a q, CLR1606 ate and o i o ’33 ('3 5Wi3 A, x Q ,lL hflegNCHgCHgo“PmOtCHglggwxf awn V v «if ‘ 0 Cl) \rxz/ H ii lL )LL J" (3) if g” a O I? \x; “av?! 1Men KN. * xx. 3/ Men 1 \X‘ Z Q M453 NH?i , CLR1607 conjugate.
Mertansine—CLR1404 Conjugates In another embodiment of the present invention the therapeutic compound is sine linked to the CLR1404 core compound by a maleimide linker, wherein the maleimide linker is attached to the CLR1404 core compound Via an amide bond and to mertansine Via a — sulfur bond, II’O O l O OJH/N\g/\/ \<N‘©'(CH2)1BO§OCH2CH2NM83S O 6-) o 09 , CLR1608 conjugate.
For all representative embodiments n is an integer from 2 to 6 and X is O or NH.
Examples Example 1Syntheses ofConjugates 2015/059382 9 ® NaNg, /N\/\N/ I—<j>—(CHZ)1got—TOCHZCHENMe3 9 9 H N3—Q—(CH2)1BOEOCHZCH2NMe3 09 Cut, Na aseorbate 0 09 ElOH'HZO’BO 035% CLR1401 CLR1401azide H2, Pd/C l? HZN‘Q‘(CH2)1BOE’OCH2CH2NMe3<9 MeOH, 87% 06 CLR1401 amine @WO>—OO 0 0 OH 0 9 f0 0 OH ‘ 2 .
HOH0)];OFl i O O + ‘ O“ 5R5 _ HzN ; (CH2)180?OCH2CH2NM93 Py CHCl3 99%' O 0O ’ ©‘i 0);“ 09 paclitaxel O paclitaer-2'-hemiglutarate ONH O COMU, Et3N CHCl3-iPrOH CLR1601 O 9 HN—Q—(CH2)150$OCH2CH2NMe3e 1. Synthesis of CLR1601 A. sis of CLR1401 azide 18-(p-Iodophenyl)octadecyl phosphocholine (4.01 g, 6.3 mmol), sodium azide (818 mg, 12.6 mmol) and sodium ascorbate (140 mg, 0.71 mmol) were dissolved in the mixture of degassed l (28 ml) and water (12 ml) in the reaction vessel. Copper (1) iodide (120 mg, 0.63 mmol) and N,N’-dimethyl—ethylenediamine (0.1 ml, 0.94 mmol) were added to the reaction mixture. Reaction vessel was tightly closed and the mixture was stirred at 80 °C for 45 min.
Reaction mixture was cooled to the room temperature, water (60 ml) was added, and the mixture was stirred for 30 min open to the air. The mixture was transferred to the separatory funnel, chloroform (80 ml) and methanol (52 ml) were added, and tion was performed by shaking. form layer was removed, and extraction was repeated (2 X 80 m1 of chloroform). ed chloroform extracts were washed with 0.01 N HCl, dried over Na2804, filtered and evaporated to dryness. Residue was dissolved in chloroform (4 ml) and acetone (170 ml) was slowly added with stirring. The mixture was stirred for 30 min and filtered. The product was rinsed on the filter with e, and dried under high vacuum to give 3.31 g (95%) of 18—(p— azidopheny1)octadecyl phosphocholine.
B. Synthesis of CLR1401 amine l8-(p—Azidopheny1)octadecy1 phosphocholine (3.116 g) was placed in a Parr pressure bottle, methanol (30 ml) and catalyst 10% I’d/C (100 mg) were added. The hydrogenation reaction was performed under hydrogen pressure (55 psi) with shaking for 24 h. The bottle was depressurized, form and methanol were added to dissolve some precipitated reaction t, and the mixture was filtered to remove the catalyst. Filtrate was evaporated to dryness and residue was dissolved in warm chloroform-methanol (1:1) mixture (10 ml). Hot acetone (150 ml) was added slowly with stirring, the mixture was cooled to the ambient temperature with stirring and filtered. Product was rinsed on the filter with acetone and dried under high vacuum.
Yield of aminophenyl)octadecyl phosphocholine: 2.597 g (87%).
C. Synthesis of paclitaxel—Z'—hemiglutarate Paclitaxel (404 mg, 0.437 mmol) and glutaric anhydride (67 mg, 0.588 mmol) were dissolved in chloroform (8 ml) and pyridine (0.5 ml) was added. Reaction mixture was stirred at room ature for 24 h and ated to dryness. Residue was kept under high vacuum for 1.5 h to remove the residual ne. Crude product was purified by silica gel chromatography in chloroform—methanol ent from 98:2 to 95:5) to yield 452 mg (99%) of paelitaxel-2'— hemiglutarate.
D. Synthesis of CLR1601 Paelitaxel-Z'-hemig1utarate (947 mg, 0.978 mmol) and 18—(p—aminopheny1)octadecyl phosphocholine (492 mg, 0.934 mmol) were suspended in chloroform (40 ml) and isopropanol (1.2 ml) mixture. To this suspension, trimethylamine (0.27 ml, 1.957 mmol) and COMU (419 mg, 0.978 mmol) were added. Reaction mixture was d at room ature for 20 h by which time it became clear and homogeneous. Reaction mixture was transferred to a separation funnel and mixed with chloroform (40 ml), methanol (80 ml) and cold water (72 ml).
Chloroform layer was removed, and extraction was ed (2 X 80 m1 of chloroform).
Combined chloroform extracts were dried over Na2S04, filtered and evaporated to dryness. The WO 81203 ing residue was purified by chromatography on silica gel with chloroform-methanol (gradient from 9:1 to 5:5) followed by final elution with chloroform—methanol~water (65:25z4).
After evaporation of the t, the product was dried under high vacuum to give 1.167 g (85%) of CLRl 601. 11. Synthesis of CLR1602 H N—Q-(CH 215) OSOCH CH NMe 5‘3” COM” 2 2 2 a + , BOCHNN3 . BOCHNNiN—Q—(CHZ)1BOPOCHZCH2NMe39 a Cb CHCig'leOH’ H (1% CLR1401 amine 18~[p-(4-N-BOC-aminobutyramido) o O phenyuoctadecyl phosphocholine HO! «1 (9 CHCI3-MeOH H2NMN—Q—(CthgoPOCl-JZCHZNMQH 18-[p~(4-amlnobutyramido)phenyl]octadecyl phosphocholine bis-2',7-(p-nitrophenyl ate) paclitaxel m““06szc ; o _ J o 3 P CHCI 0 ‘5‘) . o‘ : H ~ HZNNL H y’ 3 : N CH <:> )— ( 2)18OPOCH CH NM w2 2 es <0 0 H ‘ 40°C 18-[(4~aminobutyramido)pheny|]octadecyl phosphocholine E; O}_O JLNA/fig@MCH2)180POCHZCH2NMe3 0 gm 0 We . \‘ 0H5HO: 5 O CLR1602 0“ CH t” A. Synthesis of l8—[p-(4-N—BOC~aminobutyramido)phenyl]octadecyl phosphocholine 18—(p—Aminophenyl)octadecyl phosphocholine (76 mg, 0.144 mmol) and 4—N-BOC— aminobutyric acid (38 mg, 0.188 mmol) were suspended in chloroform (5 ml) and isopropanol (0.15 ml), then triethylamine (0.05 ml, 0.38 mmol) was added followed by COMU (80 mg, 0.188 mmol). Reaction mixture was stirred at room temperature for 24 h and quenched with 2 ml of saturated aqueous NaHC03 solution. Quenched reaction mixture was transferred into to a separation funnel and mixed with chloroform (35 ml), methanol (40 m1) and cold water (36 ml).
Chloroform layer was removed, and extraction was repeated (2 X 40 ml of chloroform).
Combined chloroform extracts were dried over NagSO4, filtered and evaporated to dryness.
Residue was purified by chromatography on silica gel with chloroform~methanol (gradient from 9:1 to 5:5) followed by final elution with form—methanol-water (65:25:4). After evaporation of the solvent, the product was dissolved in warm chloroform—methanol mixture (1.5 ml) and itated with acetone. Product was collected by filtration and drying under high vacuum to give a white powder (100 mg, 97%).
B. sis of l8-[p-(4-aminobutyramido)phenyl]octadecyl phosphocholine 18-[(4-N-BOC-aminobutyramido) phenyl]octadecy1 phosphocholine (98 mg, 0.138 mmol) was dissolved in a mixture of chloroform (4 ml), methanol (2ml) and concentrated HCl (0.2 ml). The reaction mixture was stirred ght at ambient temperature and then was quenched by slow on of the saturated aqueous NaHC03 solution (3 ml). Quenched reaction mixture was transferred into a separation funnel and mixed with chloroform (40 ml), methanol (40 ml) and cold water (36 ml). Chloroform layer was removed, and extraction was repeated (2 X 40 ml of chloroform). Combined chloroform extracts were dried over NaZSO4, d and evaporated to dryness. Product was purified by chromatography on silica gel with chloroform- methanol (100:65) followed by final elution with form-methanol-conc. NH4OH(aq) (100:65:15). After evaporation of the solvent, the product was dried under high vacuum to afford 50 mg (60%) of l8-[p—(4—aminobutyramido) ]octadecyl phosphocholine.
C. Synthesis of 7-(p—nitrophenyl carbonate) paclitaxel Paclitaxel (100 mg, 0.117 mmol) was dissolved in chloroform (4.5 ml), 8 drops of pyridine were added and the solution was cooled in an ice bath. Solid p—nitrophenyl chloroformate (200 mg, 1 mmol) was added in one portion. Reaction mixture was allowed to warm to t and was d for 24 h, then quenched with water (1 ml) and stirred for 15 min. The mixture was ted with chloroform, the extract was washed with water, dried over NaZSO4, filtered and evaporated to dryness. Crude bis—2',7—(p-nitrophenyl carbonate) paclitaxel was dissolved in chloroform and loaded on the silica gel column. The crude product was left in the column for 72 h to te hydrolysis ofp—nitrophenyl carbonate at 2’—position. The column was eluted with dichloromethane — ethyl acetate (gradient from 98:2 to 90:10). After evaporation of the solvent, —36- the product was precipitated with hexane and dried under high vacuum to provide 63 mg (53%) of 7-(p-nitr0phenyl carbonate) paclitaxel. See. Arpicco 8., et al., In! J Pharm, 2013, 454, 653- 659.
D. Synthesis of CLR1602 7~(p-Nitrophenyl carbonate) axel (53 mg, 0.052 mmol) and l8-[p-(4-aminobutyramido) phenyl]octadecyl phosphocholine (47 mg, 0.077 mmol) were suspended in chloroform (2 ml) and pyridine (0.5 ml) and stirred at 40 °C for 5 h. The reaction mixture was evaporated to dryness, and residue was purified by chromatography on silica gel with chloroform-methanol (gradient from 9:1 to 5:5) followed by final elution with form-methanol~water (6522524).
After evaporation of the solvent, compound was dried under high vacuum to give 67 mg (86%) of solid CLR1602. 111. Synthesis of CLR1603 u <9 0 0 H N-G CH2 ( 2)”OPOCH CH NMe2 2 “ + BnOWOH ~_,Et N COMU BnOWLfi‘Q”(CH2)1sO ll 6) (59 CHCI3 H2NM€3 CLR1401 amine 18—[p-(5-benzyloxy-valeramido) phenylloctadecyl phosphocholine 0 0 NO H2. Pd/C W n (’3 O O 2 fi—le‘izhaOEOCHzCHzNMea FY _.....___. HO + —__» MeOH Clio CHCI3 18-[p-(5-hydroxy-valeramido)phenyl]octadecyl phosphocholine o o u©~(CH2)1EOEOCHZCH2NMeg 18~[p-(5-(p-nitro-phenoxycarbonyloxy)valeramido)phenyl]octadecyl phosphocholine )\—o 0 OH DMAP ©/‘VLL . .~ /\/iLNNQ—(CH2)1SOPOCHZCHZNMe3 CHC|3_Py 6H ®O O r.t to 60 °C 18---[p(5-”(p-nitro-~phenoxycarbonyloxy)valeramido)phenyt] octadecy! phosphocholine paclitaxel CLR1603 O 9 HN@‘(CH2)130E’OCH2CH2NM83e A. Synthesis of (5-benzyloxy-valeramid0)phenyl]octadecyl phosphocholine 18—(p—Aminophenyl)octadecyl phosphocholine (760 mg, 1.443 mmol) and 5— oxyvaleric acid (361 mg, 1.732 mmol; synthesized according to Can J Chem, 1992, 70, 1472-1445 and Org Lett', 2014, 16, 516-519) were suspended in chloroform (25 m1) and triethylamine (0.3 m1, 2.164 mmol) was added followed by solid COMU (741 mg, 1.732 mmol).
Reaction mixture was stirred at room ature for 24 h and after completion, it was transferred into a separation funnel and mixed with chloroform (55 ml), methanol (80 ml) and cold water (72 m1). Chloroform layer was removed, and tion was repeated (2 X 80 ml of chloroform). Combined chloroform extracts were dried over NaZSO4, filtered and evaporated to dryness. Residue was purified by cinematography on silica gel with chloroform-methanol (gradient from 9:1 to 5:5) followed by final elution with chloroform—methanol-water (6522523).
After evaporation of the solvent and drying under high vacuum, the product was dissolved in warm chloroform—methanol mixture (3 m1) and hot acetone (75 ml) was slowly added with stirring. The mixture was cooled to the ambient temperature with ng and filtered. Collected product was dried under high vacuum to give 18—DJ—(S~benzyloxy—valeramid0)pheny1]octadecyl ocholine (887 mg, 86%) as a white powder.
B. Synthesis of 18-[p~(hydroxy-valeramido)phenyl]octadecyl phosphocholine 18—[p-(5-Benzyloxy—valeramido)phenyl]octadecyl phosphocholine (868 g) was dissolved in methanol (15 ml), transferred into a Parr pressure bottle, and 10% Pd/C (75 mg) catalyst was added. The hydrogenation reaction was performed under hydrogen pressure (55 psi) with shaking for 24 h. The bottle was depressurized, and the mixture was filtered to remove the catalyst. te was ated to s and residue was dissolved in warm chloroform— methanol mixture (3—4 ml). Hot acetone (75 ml) was slowly added with stirring. The mixture was cooled to the ambient temperature with stirring and filtered. Collected t was dried under high vacuum to yield l8—[p-(5-hydroxy-valeramido)phenyl]octadecyl phosphocholine (718 mg, 95%) as a white powder.
C. Synthesis of 18-[p—(5-(p-nitro-phenoxycarbonyloxy)valeramido)phenyl]octadecyl phosphocholine 18—[p-(5-Hydroxy~valeramido)pheny1]octadecyl phosphocholine (40 mg, 0.064 mmol) and p-nitrophenyl chloroformate (25 mg, 0.124 mmol) were ded in chloroform (3 ml) and pyridine (0.2 ml) was added. The on mixture was stirred for 24 h at room ature. An additional portion ofp-nitrophenyl chloroformate (15 mg) was added, and ng was continued for another 1.5 h. Reaction was te by TLC analysis. Reaction mixture was quenched with 1 ml of 1N HCl and transferred into the separation funnel with chloroform (20 ml), ol (20 m1) and cold water (15 ml). Extraction was repeated (3 X 20 ml of chloroform). Combined chloroform extracts were dried over NazSO4, filtered and evaporated to dryness. Residue was purified by chromatography on silica gel with chloroform-methanol (gradient from 9:1 to 5:5) followed by final elution with chloroform-methanol-water (65:25z4). After evaporation of solvent and precipitation with acetone, the residue was dried under high vacuum to give 48 (95%) of solid material.
D. Synthesis of CLR] 603 Paclitaxel (46 mg, 0.054 mmol) and 18~[p-(5-(p—nitro- phenoxycarbonyloxy)valeramido)phenyl]octadecyl phosphocholine (43 mg, 0.054 mmol) were suspended in chloroform (2 ml) and ne (0.5 ml) in a reaction vial. DMAP (8 mg, 0.065 mmol) was added, the vial was tightly closed and the contents were stirred at 60 °C for 48 h. An additional quantity of paclitaxel (20 mg) was added, and the reaction was continued at 60 °C for another 48 h. Reaction mixture was concentrated, and residue was purified by silica gel chromatography with chloroform-methanol (gradient from 9:1 to 5:5) ed by final elution with chloroform-methanol—water (65252) and (65:25z4). Evaporation of solvent and drying under high vacuum provided 3 (50 mg, 62%).
IV. sis of CLR1607 CHCIa-MeOH + HZNMfi-Q—(CH2)180FI>OCHZCH2NM83n 18-[p-(4-aminobutyramido)phenyl] octadecyl phosphocholine geldanamycin MS3NCHZCH20éO(CH2)18©—fi@ t JJ\/\/ CLR1607 Geldanamycin (111 mg, 0.198 mmol) and l8—[p~(4—aminobutyramido) phenyl]octadecyl phosphocholine (110 mg, 0.18 mmol) were dissolved in chloroform (3.5 ml) and methanol (1 ml). One drop of triethylamine was added, and the reaction mixture was stirred at room temperature for 24 h. TLC showed about 80% tion of reaction. Additional amycin (10 mg) was added, and stirring was continued for another 24 h. Reaction mixture was concentrated and residue d by silica gel chromatography with chloroform—methanol (gradient from 9:1 to 5:5) followed by final elution with chloroform-methanol-water (651252), (652513) and (6522524). After evaporation of the solvent and drying under high vacuum, acetone was added and the mixture was evaporated. CLR1607 was obtained as a purple solid (174 mg, 85%).
Identity for each isolated product was confirmed by lH—nmr and mass spectral analysis.
Examples 2 through 8 exhibit the ability of 4 and d molecules to be sequestered and retained by various cancer types while simultaneously being eliminated from healthy tissue.
V. Synthesis of CLR1608 0 HO I o 0 \ .
HzN‘Q‘1CHzlta‘3WCHZCH2NMe3 o COMU. Eth H a O HN—Q—(CthsoifOCHZCHZNMm@ ——-—————————————.> CHCi3 Oe DMAC, 90 °C 09 CLR‘MM amine CLR1401 maleamic acid N—Q—(CHZMOEOCHZCHzNMeg" e I E: N3 0 Co CHClg-MeOH 37 °c CLR1401 maleimide O / mertansine o/Kr \g/v \qN—Q—(cuzmoifOCHZCHzNMeafit O 3 o 0 e A. Synthesis of CLR1401 maleamic acid CLR1401 amine (300 mg, 0.57 mmol) was dissolved in N,N—dimethylacetamide (12 ml) at 90°C and maleic anhydride (61 mg, 0.627 mmol) was added in one portion. Reaction mixture was stirred at 90°C for 1 h, cooled to the room temperature and stirred for 24 h. Acetone (25 ml) was slowly added with ng, and the mixture was stirred at room temperature for 1 h.
Precipitated t was filtered and rinsed on the filter with acetone, then dried under high vacuum. Yield: 327 mg (92%).
B. Synthesis of CLR1401 maleimide CLR1401 maleamic acid (100 mg, 0.16 mmol) was suspended in ethanol—free form (5 ml), then triethylamine (0.05 ml, 0.352 mmol) and COMU (75 mg, 0.176 mmol) were added.
The reaction mixture was stirred for 24 h, then transferred to a separation funnel and mixed with chloroform (40 m1), methanol (40 m1) and cold water (36 ml). Chloroform layer was removed, and extraction was ed (2 X 40 ml of chloroform). Combined chloroform extracts were dried over Na2804, d and evaporated to dryness. The remaining residue was purified by chromatography on silica gel with chloroform—methanol ent from 9:1 to 5:5) followed by final elution with ch]oroform—methanol—water (65:2524). After evaporation of the solvent, the product was precipitated with acetone, collected and dried under high vacuum to give 87 mg (90%) of CLR1401 maleimide.
C. Synthesis of CLR1608 CLR1401 maleimide (40 mg, 0.066 mmol) and mertansine (53 mg, 0.072 mmol) were dissolved in the mixture of chloroform (1.7 ml) and methanol (03 ml). Triethylamine (0.08 ml) was added, and the mixture was stirred at 37°C for 24 h. The reaction mixture was concentrated, and residue was purified by chromatography on silica gel with chloroform-methanol (gradient from 9:1 to 5:5) followed by final n with chloroform-methanol—water (65:25:3). After evaporation of the t, the product was dried under high vacuum to give 62 mg (70%) of CLR1608.
Example 2—CLR1501 is Preferentially Sequestered by Cancer Cells via Lipid Rafts Materials and s: PC—3 cells were pretreated with either 2 pig/ml filipin III or vehicle for 15 min, then washed and incubated with 2 uCi of 125I-CLR1404 for 1 h. The media was removed and the cells were washed with ate buffered saline containing 0.1% bovine serum albumin, trypsinized, then split into two samples for determination of cell number by DNA content (A280 compared to a cell line specific standard curve) and counts per minute using a Gamma Counter (Perkin Elmer).
Pretreatment of PC?) cells with filipin 111, an agent that sequesters cholesterol and disrupts lipid rafts, resulted in nearly 40% less uptake of 12SI—CLR1404 compared to untreated control cells (Fig. 1). This supports the hypothesis that CLR1404 uses lipid rafts as portals of entry into cancer cells. Notably, higher filipin III trations are cytotoxic, and therefore, complete lipid raft ablation (and ably complete inhibition of CLR1404 analog ) could not be demonstrated.
Example 3-1’refermial uptake 0fCLR«1501 by Cancer Cells over Healthy Cells Materials and Methods: Human cancer cell lines were sed from the American Type Culture Collection (ATCC). They included the following: Caki-2 (renal; clear cell carcinoma), HCT—l 16 (colorectal carcinoma); MES—SA/DXS (uterine sarcoma) [all ined in s 5a medium supplemented with 10% fetal bovine serum (FBS)], Ovcar—3 (ovarian adenocarcinoma) [maintained in RPMI medium supplemented with 20% FBS], U87—MG (glioma) [maintained in minimum essential medium supplemented with 10% FBS], Mia Paca—2 (pancreatic carcinoma) (maintained in co’s modified Eagle’s medium supplemented with 10% FBS), PC-3 (prostate carcinoma) (maintained in F—12K medium supplemented with 10% FBS), MDA-MB- 231 (triple—negative mammary gland adenocarcinoma) (maintained in Leibovitz’s medium supplemented with 10% FBS), and A549 (non—small cell lung carcinoma) (maintained in F-12 medium supplemented with 10% FBS). Normal human skin fibroblasts were purchased from ATCC and grown in last Basal Medium PCS-201—030 supplemented with serum-free kit (Fibroblast Growth Kit—Serum-Free PCS-20l-040). All media (except for MDA—MB- 231 cell line) also contained llin (100 U/ml) and streptomycin (100 ug/ml) and were maintained at 37°C with 5% C02 in air.
All cells were ined at 37°C in riate medium supplemented with 10% FBS and 5% C02. Before imaging, the cells were removed from flasks with 0.25% trypsin and were allowed to grow overnight on the microslides VI ). The next day, the cells were washed with phosphate—buffered saline (PBS) and were incubated with either 5 or 7.5 uM (as indicated) of CLRlSOl in appropriate serum-free medium for 24 hours. CLRlSOl is a fluorescently labeled CLR1404 analog. CLR1501 was formulated with 0.4% of Polysorbate 20, 2% of ethanol, and . After washing thoroughly with PBS, the cells were imaged using Bio—Rad Radiance 2100 MP Rainbow laser scanning/multiphoton confocal microscope using a l-s re time.
Alternatively, cells were visualized using a Nikon AIR confocal microscope (Keck Laboratory, University of Wisconsin-Madison). The emission signal of CLRlSOl was detected using Alexa Fluor 488 filters (ex/em 480/520 nm).
Results: CLRlSOl was administered to five different cancer cell lines (renal, ovarian, pancreatic, melanoma, and prostate) and a normal human skin fibroblast line in vitro. Twenty-four hours later, CLRlSOl exhibited from five to nine-fold ential uptake in these cancer cell lines in vitro ed to normal fibroblasts (Fig. 2). Retained CLRlSOl was associated with plasma and organelle membranes.
Example 4-Rar Glioma Model Materials and Methods: All animals were housed and handled in accordance with the University of Wisconsin Research Animal Resources Center guidelines. Rat C6 glioma cells were propagated in DMEM medium (Life Technologies, Gaithersburg, MD) supplemented with % heat—inactivated FBS (BioWhittaker, Walkersville, MD), 100 U/ml penicillin G, ml streptomycin, and 0.0.1 M HEPES (Life Technologies, Gaithersburg, MD). Intracranial tumor implantation was performed as bed previously. Cohen JD, et a1., Intracranial C6 glioma model in adult Wistar-Furth rats. J Neuro Oncol 1990 5-6. Briefly, 1X106 C6 cells were resuspended in 5 ml 1.2 0/0 methylcellulose and injected into the frontal lobes of anesthetized female Wistar rats (Harlan, Indianapolis, IN). Sham—operated animals ed intracranial injections of an equal volume of methylcellulose without tumor cells.
Imaging Studies: Ten days after implantation, the presence of intracranial tumors was confirmed with MRI. Briefly, anesthetized rats (6) received 2 ml of Gadodiamide (Gd, Omniscan 287 mg/ml, Nycomed, ton, NJ) intraperitoneally and imaged 10 min later using a 1.5 Tesla clinical MR system (GE Signa LX) and a GE phased array ity coil. The Tl-weighted (TR=500 ms, S ms) multislice sequences covering the entire brain of each rat were inspected to select tumor-bearing rats with varying tumor sizes, and perated rats for NM404 injections.
NM404 [l8-(4-iodophenyl)—octadecylphosphocholine] (100mg) was radioiodinated with 1251 via isotope exchange with Naml in a melt of pivalic acid. rt, et al. Int J Appl Rad Isotopes. 1986; 37:907—913. NM404 has the same chemical structure as CLR1404 except that it is radioiodinated with 12SI instead of 1241 or 13‘1. Following HPLC purification NM404 was dissolved in an aqueous 2% Polysorbate 20 on prior to tail vein injection (5-20uCi/200g rat) into four tumor-bearing and three sham—operated rats. At 1 (n=l), 2 (n=l), and 4 (11:2) days after NM404 injection, s were euthanized (C02) and brains were excised and imaged on a modified Bioscan AR2000 radio—TLC r (1mm increments at 2 min acquisition/lane and 1 mm high—resolution collimator). In addition, normal brain, blood, kidney, liver, spleen, thyroid, and tumor tissues were weighed, and radioactivity counted in a gamma r. The tissue distribution of radioactivity was then correlated to brain histology.
Results and Discussion: Initial imaging results with NM404 indicated striking uptake and prolonged retention in all gliomas ranging from 3—5 mm in diameter. Radioactivity in normal brain tissue was minimal in sham operated control s (Fig. 4A and 4B), whereas NM404 cmwmmmeMwmQMMfifigMbU)fimmmmmnmm0Mmmwmmflfim% glioma—bearing rats were 10.5, 12.2, and 6.7 at 24, 48, and 96h, respectively. As has been observed in previous cell culture and in vivo animal model studies, NM404 is apparently metabolized and eliminated from normal cells but s metabolically trapped in tumor cell membranes. Previous autoradiography experiments in other tumor models have ted that only viable tumor cells, and not normal tissue or necrotic s, are capable of accumulating NM404. Interestingly, even small tumors measuring a few mm in diameter, were also detected after NM404 administration. These inary findings suggest that CLR1404 may also be useful for visualization of small invasive tumor foci.
Conclusion: As has been the case in all tumor models ed previously, NM404 displayed selective and ged retention by rat C6—gliomas evaluated in this study.
Example —CLR1404 Uptake in Various Malignant Tumors Materials and Methods: All described animal studies were performed ing to animal protocols approved by the Institutional Animal Care and Use Committee. Female athymic nude mice (Hsd:Athymic Nude—Foxnlnu or CrlzNU—Foxnlnu, Charles River Laboratories) about 4 to 5 weeks of age, 16 to 18 g (n = 6), were used for human tumor xenograft studies. Mice were anesthetized with isoflurane and injected aneously with viable tumor cells in 100 ul of Dulbecco’s PBS (or, for glioma cells, 50 ml of PBS) into the right flank. Inoculum sizes were 1 X 106 (for renal, ovarian, glioma, pancreatic, prostate, and NSCLC models), 2 X 106 (for colorectal and uterine models), or 3 X 106 (breast).
Results: Radioiodinated 124I—CLR1404 was tested in subcutaneous and orthotopic xenografts of 60 different spontaneous, transgenic, human, and rodent malignant cell lines and tumor types. After intravenous administration, 124l-CLR1404 localized in almost all primary and metastatic malignant tumors regardless of ic location. entative examples are of both human (Fig. SA-SI) and rodent (Fig. SJ—M) tumors.
Table l. Uptake of 124I-CLR1404 in a Broad Range of Cancer Types Tumor model Species Prostate PC-3 SCID mouse Adenocarcinoma Lung A-549 (NSCLC) SCID mouse Adenocarcinoma \OOOQO‘iU‘a-bw Lung NCI H-69 (Oat 551137 SClD mouse Small cell carcinoma Yes Adrenal H-295 SClD mouse Adenocarcinoma Yes Adrenal RL-251 SClD mouse Adenocarcinoma Yes Colon—5 l SClD mouse Adenocarcinoma Yes Colon LSl 80 SClD mouse arcinoma Yes Colon DLDl SClD mouse Adenocarcinoma Yes Colon PIT—29 SClD mouse Adenocarcinoma Yes Colon LS—180 Nude mouse Adenocarcinoma Yes Nude mouse and NOD- ll Glioblastoma U87 Glioma Yes SClD 12 Melanoma A—3 75 Nude mouse Adenocarcinoma Yes 13 Multiple myeloma Nude mouse Myeloma Yes MM. 1 S l4 Neuroblastoma SK—N—AS Nude mouse Neuroblastoma Yes l5 lastoma NB]691 Nude mouse Neuroblastoma Yes l6 lastoma CHLA-ZO Nude mouse Neuroblastoma Yes l7 Neuroblastoma LanS Nude mouse Neuroblastoma Yes l8 Ovarian RTE-77 Nude mouse Adenocarcinoma Yes 19 n Ovcar—3 Nude mouse Adenocarcinoma Yes Pancreatic BXPC3 Nude mouse arcinoma Yes 21 Pancreatic Mia Pacer-2 Nude mouse Carcinoma Yes 22 Pancreatic Capan-l Nude mouse Adenocarcinoma Yes 23 Renal cell Caki—2 Nude mouse (orthotopic) Clear cell carcinoma Yes 24 Renal cell ACHN Nude mouse (orthotopic) Adenocarcinoma Yes Sarcoma A) Nude mouse Fibrosarcoma Yes 26 Head and neck SCCl Nude mouse Squamous cell carcinoma 27 Head and neck SCC6 Nude mouse Squamous cell carcinoma 28 Prostate LNCap Mouse Adenocarcinoma Yes 29 Prostate LuCap Mouse arcinoma Yes Breast MCF—7 Rat Adenocarcinoma Yes 31 Triple negative breast Nude mouse Adenocareinoma MDA—MB23 l Yes 32 Uterine MES SA/Dx5 Nude mouse Sarcoma Yes NOD-SClD mouse 33 Glioblastoma 22 GSC Glioma Yes (orthotopic) NOD—SClD mouse 34 Glioblastoma 105 GSC Glioma Yes (orthotopic) Breast 4T1 Endogenous mouse Adenocarcinoma Yes (orthotopic) 36 Bladder SV40 Mouse (orthotopic) Adenocarcinoma Yes 37 Prostate MatLyLu Rat Adenocarcinoma Yes 38 Walker256 Rat osarcoma Yes 39 TRAMP prostate Endogenous mouse Adenocarcinoma Yes —46- r710 “T Colon CT26 SClD mouse Adenocarcinoma Yes 41 Colon Pirc Autochthonous Pirc rat Adenocarcinoma Yes 42 Min mouse intestinal Endogenous mouse Adenocarcinoma Yes 43 Melanoma Mouse Adenocarcinoma Yes 44 Mammary SCC + mouse 82:33:36“ Yes 45 Mammary AC ApeMin/+ mouse Adenocarcinoma Yes 46 Hepatocellular carcinoma Endogenous mouse Adenocarcinoma Yes 47 Glioma L9 Rat xenograft Glioma Yes 48 Glioma C6 Rat xenograft Glioma Yes 49 Glioma CNS} Rat xenogralt Glioma Yes 50 Glioma RG2 Rat xenograft Glioma Yes 51 Retinoblastoma Endogenous mouse Blastoma Yes 52 Pancreatic c—myc Endogenous mouse Adenocarcinoma Yes 53 Pancreatic Kras Endogenous mouse Adenocarcinoma Yes 54 Cervical—HPV Endogenous mouse Adenocarcinoma Yes 55 Esophageal Endogenous Mouse Adenocarcinoma Yes 56 Intestinal polyp nous mouse a (benign) No 57 Maggi/13:31:3:012“. Endogenous mouse Hyperplasia (benign) No 58 Hepatoma Hep-3B Nude mouse oma No 59 Hepatoma Hep-G2 Nude mouse Carcinoma No 60 Fire rat colon adenoma Pirc rat Adenoma No .._I_ .J *Tumor uptake was considered positive if tumor to muscle ratio was greater than 3.
Tumor:muscle ratio less than or equal to 2 was considered negative.
Example 6-Clim'cal Trial Evaluating ts with Non—Small Cell Lung Carcinoma C ”) using CLR1404 Although CLR1404 has displayed selective and prolonged tumor retention in 55/60 xenograft and spontaneous rodent models, a physician red IND initiated clinical evaluation of the agent in Stage 4 human NSCLC patients in order to determine whether or not it would exhibit r tumor uptake and retention properties in humans. To date, two patients with advanced NSCLC were imaged after an injection of <1 mCi of ‘3 1I—CLR1404. Blood and urine samples were collected at predetermined times, and gamma imaging performed at several time points following administration. In both patients, significant tumor uptake and retention of CLRl404 was demonstrated in the y lung tumor, as seen in Fig. 6. Relative to the high liver uptake values seen previously with its first tion predecessor, NM324, liver and abdominal activity are much lower with 4, suggesting the feasibility of evaluating this agent in other abdominal cancers including atic, colon, and prostate.
Materials and Methods: ing intravenous injection of iodine—131 labeled CLRl404 (l mCi/20 pg), patients with advanced NSCLC where scanned at 3, 6, 24, 48, 96 h and at 7 and ll days on a GE Maxxus dual Head SPECT scanner. Blood and urine s were collected for pharmacokinetic analysis as well as clinical hematologic, renal, and hepatic bioanalysis.
Results: Initial qualitative imaging results indicate that iodine-131 labeled CLR1404 y localizes in bilateral pulmonary masses as early as 24 h after injection and is selectively retained in these tumors in excess of 11 days. Moreover, background radioactivity in the liver and lower abdominal region ing urinary bladder, kidneys, and intestines was significantly less than was observed previously with its predecessor, NM324. No adverse reactions were observed in any of the patients.
Conclusions: These preliminary findings t that CLR1404 exhibits similar tumor uptake and retention properties in human NSCLC as was seen usly in rodent models.
Although based on only two patients at this point, it appears that CLR1404 does indeed localize in and undergo selective and prolonged tumor retention in human non—small cell lung cancer.
Patient 1: 55 year old male with bilateral 3 cm left lobe and infiltrative right lobe NSCLC and a brain metastasis and a small right adrenal mass. He has participated in numerous standard and experimental treatment ns. Images are shown in Fig. 6A-C.
Patient 2: 70 year old male recently sed with 6 cm upper lobe all cell lung carcinoma, a 5 cm liver mass, an iliac bone metastasis and a very small brain metastasis. He had recently completed low dose carboplatin/taxol chemotherapy and tive radiotherapy to the iliac and brain metastases the week prior to initiating the 4 trial. Images are shown in Fig. 6D—G.
Example 7-Detectz'0n of 3 usly unknown brain tumor metastases in NSCLC patient using ’241.CLR1404 Materials and Methods: Human PET brain scans were acquired on a 64—slice PET/CT scanner (Discovery VCT, General Electric) at multiple time points after the injection of about 5 mCi of ‘24I-CLR1404 using a 90~min dynamic acquisition sequence (2D, nine frames at 10 min each, VIP list mode on) and reconstructed [Advantage Workstation version AW4.4, General Electric, 30 cm DFOV WO 81203 (display field of view), 128 X 128, OSEM VUE Point, 10 subsets with two iterations, standard 2 axis, attenuation correction and dead time, r, and decay correction].
Results: Preliminary s were obtained in an NSCLC patient t neurological symptoms using 124l-CLRl404 . Imaging revealed three previously unknown brain lesions highly suspicious for metastases that were subsequently confirmed with gadolinium—enhanced MRI (Fig. 7).
Example 8—Detecti0n of tumor recurrence of a right frontal falcine metastasis using 124]- CLR1404 Recurrent brain asis in 60-year-old woman with malignant melanoma. Magnetic resonance (“MR”) (Fig. 8A) and LR1404 PET images (Fig. SB) and images 8 months after stereotactic radiosurgery for tumor recurrence of a right frontal falcine metastasis (Fig. 8C) shows a focus of abnormal activity with CLRl404 (arrow). Corresponding enhancing focus on initial MR imaging was interpreted as radiation is versus possible recurrence. Subsequent MR imaging showed further increase in size of the nonspecific enhancing lesion, coupled with increased perilesional edema indicating a recurrence of the ant tumor. These results indicate that 124I—CLR1404 was sequestered by cancer cells that were resistant to the radiosurgery and eventually established a recurrent tumor. nds of the present invention include anti-cancer drugs linked to the CLR1404 core molecule. These compounds are capable of targeting cancer cells and cancer stem cells including brain cancer cells such that the anti—cancer drug is sequestered and retained by the cancer cell. These compounds provide the first targeted treatment of cancer capable of being adapted to specifically administer a range of anti—cancer drugs to cancer cells to both treat the cancer and prevent metastasis and ence.
Example 9—Paclitaxel~C0njugates and 1C50for Various Cancer Cell Lines Method Cancer cell lines including, MDA—MB—468 (Breast), NCl-HIZ99 (Lung), NCI-H460 , Capan—2 (Pancreas), MiaPaCa—l (Pancreas), HT29 (Colorectal), HCTll6 (Colorectal) and P03 (Prostate) were treated with serial concentrations of paclitaxel and CLRl404-paclitaxel conjugates (i.e. CLRléOl, CLR1602 and CLR1603). The cell lines were then measured for cell viability and reported as ICSO for each treatment.
Results CLR1601 and CLR1603 were capable of reducing cell viability for each of MDA—MB— 468 (Breast), 299 (Lung), NCI—H460 (Lung) Capan-2 (Pancreas), MiaPaCa-l (Pancreas), HT29 (Colorectal), HCT116 (Colorectal) and PC-3 (Prostate) cancer cell lines. See Figures 9- 16, respectively. ICSO for each axel-1404 conjugate (i.e. CLR1601 and CLR1603) and paclitaxel are reported in Table 2. ICSO for CLR1602 is not shown, r CLR1602 was not capable of significantly reducing cancer cell line viability because CLR1602 is non— hydrolyzable. In vivo, free paclitaxel is taken up by cancerous tumor cells at a much lower rate due to the non-specific nature of paclitaxel uptake. Thus, in vivo, the amount of PLE—paclitaxel conjugate necessary for cancer cell death should be on par or less than that for paclitaxel and may result in a greatly reduced toxicity to non—cancer cells.
Table 2. ICSO for CLR1601, CLR1603 and Paclitaxel CLR1601 CLR1603 Paclitaxel MDA-MB—468 t) 3.77 nM 3.42 nM 1.9 nM NCI—H11299 (Lung) 60.3 nM 108 nM 1.82 nM NCI—H46O (Lung) 29.5 nM 171.1 nM 1.66 nM Capan—2 (Pancreas) 56.7 nM 83.6 nM 7.91 nM MiaPaCa-l (Pancreas) 37.3 nM 38.9 nM 1.32 nM HT29 (Colorectal) 92.2 nM 70.6 nM 1.07 nM HCT116 (Colorectal) 7.6 nM 11 nM 0.87 11M PC-3 (Prostate) 34.4 11M 29.5 nM 0.9 nM Example 10—Flow lry Assays Methods n V and PI hatidylinositide) staining by flow cytometry was utilized to determine percentages of live, early apoptotic, late apoptotic, and necrotic cells. In short, cells were treated with xic agents and stained with an Annexin V031 labeling kit (Life Technologies). Cells were analyzed on an LSRII flow cytometer (BD Biosciences). As shown in Tables 3—9, cells were classified as: Live (Annexin V negative, P1 negative), Early apoptotic (Annexin V positive, PI negative), Late apoptotic (Annexin V positive, PI positive), and ic (Annexin V negative, PI positive). A representative scatter plot is shown in Figure 17 for MBA— MB-468 cells treated with 5 uM of 1 for 72 hours. Annexin V (attached to AlexaFluor 488) is shown on the x—axis, while PI is shown on the y—axis. The lower left quadrant tes live cells, the upper left quadrant indicates necrotic cells, the upper right quadrant indicates late apoptotic cells and the lower right quadrant tes early apoptotic cells. Debris was eliminated from this analysis.
Results MDA~MB~468 cells, a triple negative breast cancer cell line, were d with CLR conjugates (CLR1601 and CLR1603) for 72 hours and with axel (“PTX”) for 24 hours.
See Table 3. MDA-MB-468 cells, were also treated with CLR conjugates (CLR1606 and CLR1607) for 72 hours and with geldanamycin (“GEL”) for 48 hours. See Table 4. For PTX conjugates, cell viability was reduced from 61.1% (no drug treatment) to 17.0%, 17.8%, 22.2%, 19.7%, 53.8%, and 48.9% after treatment with 1 uM CLR1601, 5 uM CLR1601, 1 11M CLR1603, 5 uM 3, 100 nM PTX, and 1 uM PTX, respectively. See Table 3. For GEL conjugates, cell viability was reduced from 61.1% to 58.8%, 42.7%, 52.7%, 56.9%, and 26.2% after treatment with 1 uM CLR1606, 10 11M CLR1606, 1 uM CLR1607, 10 nM CLR1607, and l 1.1M GEL, respectively. See Table 4.
Table 3. MDA-MB—468 Treated with Paclitaxel Conjugates for 72 Hours MDA-MB-468 72 hrs CLR1601 CLR1601 CLR1603 CLR1603 PTX PTX (% of total cells) No Drug (luM) (511M) (luM) (511M) (100 nM) (luM) Live 61.1 17 17.8 22.2 19.7 53.8 48.9 Necrotic 2.83 15.8 21.6 22 22.5 8.89 10.8 Late Apoptotic 20.5 57.5 57.9 49.1 55.2 31.4 34.8 Early Apoptotic 15.5 9.83 2.71 6.74 2.65 5.9 5.5 Table 4. MDA-MB—468 Cells Treated with Geldanamycin Conjugates for 72 Hours MBA-M13468 72 hrs CLR1606 CLR1606 CLR1607 CLR1607 GEL (% of total cells) No Drug (luM) (lOuM) (luM) (lOuM) (luM) Live 61.1 58.8 42.7 52.7 56.9 26.2 Necrotic 2.83 2.31 19.4 1.62 1.63 16.3 Late Apoptotic 20.5 25.7 33.8 25.1, 26.4 37.9 Early Apoptotic 15.5 13.2 4.13 20.6 15.1 19.6 COLO 829 cells, a ma cell line, were treated with GEL conjugates (CLR1606 and CLR1607) for 72 hrs and GEL for 48 hours. See Table 5. Cell ity was reduced from 80.8% (no drug ent) to 70.4%, 21.1%, 67.9%, 54.3%, 32.4%, and 18.6% after treatment with 1 nM CLR1606, 10 uM CLR1606, 1 uM CLR1607, 1.0 uM CLR1607, 100 nM GEL, and 1 11M GEL, respectively. See Table 5.
Table 5. COLO 829 Cells Treated with amycin Conjugates for 72 Hours COLO 829 72 hrs CLR1606 CLR1606 CLR1607 CLR1607 GEL GEL (% oftotal cells) No Drug (luM) (10uM) (luM) (10uM) (100 nM) (luM) Live 80.8 70.4 21.1 67.9 54.3 32.4 18.6 Necrotic 1.74 1.59 28.6 2.36 16.1 4.9 6.42 Late Apoptotic 4.18 6.7 40.1 9.05 20.9 40.8 53.7 Early Apoptotic 13.3 21.3 10.2 20.7 8.69 21.9 21.4 PANC—l cells, a pancreatic cancer cell line, were treated with GEL conjugates (CLR1606 and CLR1607) for 72 hrs and GEL for 48 hours. See Table 6. Cell viability was reduced from 44.2% (no drug treatment) to 42.0%, 21.5%, 44.0%, 33.0%, 23.3%, and 18.9% after treatment with 1 uM CLR1606, 10 pM CLR1606, 1 pM CLR1607, 10 uM CLR1607, 100 nM GEL, and 1 uM GEL, respectively. See Table 6.
Table 6. PANC-l Cells Treated with Geldanarnycin Conjugates for 72 Hours PANC-l 72 hrs 6 CLR1606 CLR1607 CLR1607 GEL GEL (% of total cells) No Drug (luM) (10uM) (luM) (10uM) (100 nM) (luM) Live 44.2 42 21.5 44 33 23 .3 18.9 Necrotic 47.9 46.8 65.3 48.1 54.7 59.4 66.4 Late Apoptctic 5.65 8.63 12.6 4.83 9.81 12.6 12.8 Early Apoptotic 2.23 2.59 0.63 3.1 2.43 4.68 1.93 22RV1 cells, a prostate cancer cell line, were treated with GEL conjugates (CLR1606 and CLR1607) for 72 hrs and GEL for 48 hours. See Table 7. Cell viability was 20.3%, 21.3%, 16.0%, 21.9%, 15.7%, 19.4%, and 28.1% after treatment with no drug, 1 uM CLR1606, 10 uM CLR1606, 1 uM CLR1607, 10 uM CLR1607, 100 nM GEL, and 1 uM GEL, respectively. See Table 7. Basal cell death was high with this cell line; the cells did not respond well to the method of cell collection.
Table 7. 22RV1 Cells Treated with Geldanamycin for 72 Hours 22RV1 72 hrs CLR1606 6 CLR1607 7 GEL GEL (% of total cells) No Drug (1uM) (10uM) (luM) (10uM) (100 nM) (luM) Live 20.3 21.3 16 21.9 15.7 19.4 28.1 Necrotic 49.5 51.7 57.7 55.2 59.1 47.1 46.6 Late Apoptotic 26.2 23.4 22.9 20.7 23.1 28.4 19.1 Early Apoptotic 4.1 3.63 3.44 2.27 2.19 5.09 6.22 CLR conjugates appear to require a longer treatment period with cells to induce cell death. ent of —468 cells with 1 [.LM CLR1601 and 1 uM CLR1603 for 48 hours resulted in a reduction of cell viability from 86.2% (no drug ent) to 79.4% and 81.4%, respectively. See Table 8. Treatment of COLO 829 cells with 1 [.LM CLR1606 and 1 pM CLR1607 for 48 hours resulted in a ion of cell, viability from 91.7% (no drug treatment) to 90.1% and 82.7%, respectively. See Table 9.
Table 8. MDA—MB-468 Treated with axel Conjugates for 48 Hours MDA—MB-468 48 hrs CLR1601 CLRl 603 PTX PTX (% of total cells) No Drug (luM) (luM) (100 nM) (luM) Live 86.2 79.4 81.4 77.9 73.3 Necrotic 2.87 13.1 12.4 14.1 15.6 Late Apoptotic 8.33 6 4.78 5.54 9.18 Early Apoptotic 2.56 1.49 1.4 2.47 1.97 Table 9. COLO 829 Cells Treated with Geldanamycin Conjugates for 48 Hours COLO 829 48 hrs CLR1606 CLR1607 GEL GEL (% of total cells) No Drug (luM) (luM) (100 nM) (luM) Live 91.7 90.1 82.7 36.1 24.6 Necrotic 5.96 6.91 8.94 22.7 22.7 Late Apoptotic 1.67 2.2 5.54 29 38.3 Early Apoptotic 0.72 0.79 2.77 12.2 14.4 Overall, PLE—paclitaxel and PLE—geldanamycin conjugates were shown to be capable of ng tumor cell Viability including inducing cell death for a variety of tumor types.

Claims (13)

WHAT IS CLAIMED IS:
1. A therapeutic compound comprising the formula A-B-D wherein: A is at least one compound of a (I), (I), at least one nd of formula (II), (II), at least one compound of formula (III) (III), or a combination thereof, wherein W is ed from the group ting of an aryl, a C1-C6 alkyl, an alkenyl, a C3-C6 cycloalkyl, and a C3-C6 heterocycloalkyl, wherein R is H or an alkyl and wherein m is an integer from 12 to 24; B is a linker compound; and D is a chemotherapy drug, wherein the ratio of A to D is from 1:2 to 2:1.
2. The therapeutic compound of claim 1 wherein the linker compound is a bond or a compound of formula (IV), Y-(CH2)n-Z (IV), wherein: Y is bound to A; Z is bound to D; Y is selected from the group consisting of a bond, O, NH, C=O, NHSO2O, and OC(=O)O; Z is selected from the group consisting of O, NH, C=O, C(=O)O, C(=O)NH, SO2, OC(=O)OCH2, and –S-S-; and 1003091162 n is an integer from 0 to 6.
3. The therapeutic compound of claim 1 wherein: A is compound of formula (I), wherein W is selected from the group consisting of a C1 alkyl, , , and and wherein m is 18; B is a linker compound selected from a bond and a compound of formula (IV), Y-(CH2)n- Z (IV), wherein n is an r from 0 to 6, Y is bound to A, Z is bound to D, Y is selected from the group consisting of a bond and C=O and Z is selected from the group consisting of NH, C=O, C(=O)NH and C(=O)O; and D is selected from the group consisting of paclitaxel, irinotecan, topotecan, gemcitabine, cisplatin, geldanamycin and mertansine.
4. The therapeutic compound of claim 3 wherein: A is a compound of formula (I), wherein W is and m is 18; B is a nd of a (IV), wherein Y is C=O and Z is C=O, C(=O)NH or C(=O)O and n is 3 or 4; and D is paclitaxel, wherein the ratio of A to D is 1:1.
5. The eutic compound of claim 3 wherein: A is a compound of formula (I), wherein W is and m is 18; B is a bond or a compound of a (IV), wherein Y is C=O, Z is NH and n is 1 or 3; D is geldanamycin, 1003091162 wherein the ratio of A to D is 1:1.
6. The therapeutic compound of claim 3 wherein: A is a compound of formula (I), wherein W is and m is 18; B is a bond; and D is mertansine, wherein the ratio of A to D is 1:1.
7. The therapeutic nd of claim 1, selected from the group consisting of: 1003091162 1162 O O OH O NH O O H O OH O O O O O 2CH2OPO (CH2)18 X O O ; 1003091162 1162 N O O O F 2CH2OPO O O OH F ; 1003091162 O H Me3NCH2CH2OPOCH2CH2O O O H HO O NH2; and wherein n is an integer from 0 to 6 and X is O or NH.
8. The therapeutic compound of claim 1, which is a nd of Formula (V), 1003091162 (V).
9. A pharmaceutical composition comprising a eutic compound of claims 1, 7, 8, or 13 and one or more pharmaceutically acceptable carriers.
10. Use of a therapeutic nd of claims 1, 7, 8, or 13 for the manufacture of a medicament for treatment of cancer.
11. The use of claim 10 wherein the cancer comprises cancer stem cells.
12. The use of claim 10 wherein the cancer is recurrent.
13. A therapeutic compound of claim 2, wherein D is selected from the group consisting of paclitaxel, irinotecan, topotecan, gemcitabine, cisplatin, geldanamycin, and sine. 1003091162
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