CA2916970A1

CA2916970A1 - A smac mimetic compound for use in the treatment of proliferative diseases

Info

Publication number: CA2916970A1
Application number: CA2916970A
Authority: CA
Inventors: Stephen J. Morris
Original assignee: Pharmascience Inc
Current assignee: Pharmascience Inc
Priority date: 2016-01-08
Filing date: 2016-01-08
Publication date: 2017-07-08
Also published as: WO2017117684A1

Abstract

The present invention relates to a SMAC mimetic and pharmaceutical compositions thereof, more specifically the present invention is directed to a Compound with a following structure:
(see above formula) pharmaceutically acceptable salts and to pharmaceutical compositions comprising it, and to their use alone or in combination in the treatment of proliferative disorders, comprising cancers. Further the present invention relates to pharmaceutical composition in a dosage unit for intravenous infusion that comprises internally administering to the patient a SMAC mimetic, their regimens and dosing for treating a proliferative disorder in a patient. The invention further includes the stimulation of the concomitant degradation of of clAP1 and clAP2 in cancer cells.

Description

A SMAC MIMETIC COMPOUND FOR USE IN THE TREATMENT OF
PROLIFERATIVE DISEASES
Field of the Invention The present invention relates to a SMAC mimetics, more specific to a compound and to pharmaceutical compositions comprising them, and to their use in the treatment of proliferative disorders, comprising cancers BACKGROUND OF THE INVENTION
Apoptosis, or programmed cell death, typically occurs in the normal development and maintenance of healthy tissues in multicellular organisms. It is a complex process which results in the removal of damaged, diseased or developmentally redundant cells.
Intrinsic apoptotic pathways are known to be dysregulated, most particularly in cancer and lymphoproliferative syndromes, as well as autoimmune disorders such as multiple sclerosis, in neurodegenerative diseases and in inflammation. In cancer, failure to apoptose in response to anti-cancer therapies contributes to resistance of many cancer types to chemotherapy and biological agents.
The caspases are a family of proteolytic enzymes from the class of cysteine proteases which are known to initiate and execute apoptosis. In normal cells, the caspases are present as inactive zymogens, which are catalytically activated following external signals, for example those resulting from ligand driven Death Receptor activation, such as cytokines or immunological agents, or by release of mitochondrial factors, such as cytochrome C following genotoxic, chemotoxic, or radiation-induced cellular injury. The Inhibitors of Apoptosis Proteins (IAPs) constitute a family of proteins which are capable of binding to and inhibiting the caspases, thereby suppressing cellular apoptosis. Because of their central role in regulating Caspase activity, the IAPs are capable of inhibiting programmed cell death from a wide variety of triggers, which include loss of homeostatic, or endogenous cellular growth control mechanisms, as well as chemotherapeutic drugs and irradiation.
The IAPs contain one to three homologous structural domains known as baculovirus IAP repeat (BIR) domains. They may also contain a RING zinc finger domain at the C-terminus, with a capability of inducing ubiquitinylation of IAP-binding molecules via its E3 ligase function.
The human IAPs, XIAP, clAP1 and clAP2 each have three BIR domains, and a carboxy terminal RING zinc finger. Another IAP, NAIP, has three BIR domains (BIR1, BIR2 and BIR3), but no RING domain, whereas Livin, TslAP and MLIAP have a single BIR domain and a RING domain. The X chromosome-linked inhibitor of apoptosis (XIAP) is an example of an IAP which can inhibit the initiator caspase, known as caspase-9, and the effector caspases, Caspase-3 and Caspase-7, by direct binding. It can also induce the removal of caspases through the ubiquitylation-mediated proteasome pathway via the E3 ligase activity of a RING
zinc finger domain. It is via the BIR3 domain that XIAP binds to and inhibits caspase-9. The linker-BIR2 domain of XIAP inhibits the activity of caspases-3 and -7. The BIR domains have also been associated with the interactions of 1APs with tumor necrosis factor-receptor associated factor (TRAFs)-1 and -2, and to TAB1, as adaptor proteins effecting survival signaling through NFkB
activation. The IAPs thus function as a direct brake on the apoptosis cascade, by preventing the action of, or inhibiting active caspases and by re-directing cellular signaling to a pro-survival mode.
A growing body of data indicates that cancer cells may avoid apoptosis by the sustained over-expression of one or more members of the IAP family of proteins, as documented in many primary tumor biopsy samples, as well as most established cancer cell lines. Epidemiological studies have demonstrated that over-expression of the various IAPs is associated with poor clinical prognosis and survival. For XIAP this is shown in cancers as diverse as leukemia and ovarian cancer. Over expression of clAP1 and clAP2 resulting from the frequent chromosome amplification of the 11q21-q23 region, which encompasses both, has been observed in a variety of malignancies, including medulloblastomas, renal cell carcinomas, glioblastomas, and gastric carcinomas. (X)IAP negative regulatory molecules such as XAF, appear to be tumor suppressors, which are very frequently lost in clinical cancers. Thus, by their ability to suppress the activation and execution of the intrinsic mediators of apoptosis, the caspases, the 1APs may directly contribute to tumor progression and resistance to pharmaceutical intervention.
The IAPs are antagonized by SMAC, an intracellular protein, which binds to BIR
domains. SMAC binding can displace caspases from XIAP as well as activate the E3 ligase activities of clAP1 and clAP2.
Importantly, activation of the E3 ligase activity can lead to protein degradation. We and others have synthesized small molecule drugs that mimic the action of SMAC. When administered to patients or animals suffering proliferative disorders, the Smac mimetics antagonize IAPs, causing an increase in apoptosis among abnormally proliferating cells including cancer cells.
Examples of Smac peptidomimetics are those disclosed in, without limitation, US 7,517,906; US
7,419,975; US 7,589,118; US 7,932,382; US 7,345,081; US 7,244,851; US
7,674,787; US 7,772,177; US
7,989,441; US20100324083; US20100056467; US20090069294; US20110065726; U5201 10206690;
W02011098904.
SUMMARY OF THE INVENTION
The present invention relates to a compound for use in a method of treating a patient suffering a proliferative disorder that comprises administering a selected dose of N1,N4-bis((35,5S)-1-((S)-3,3-dimethy1-24(S)-2-(methylamino)propanimido)butanoy1)-5-((R)-1,2,3,4-tetrahydronaphthalen-1-ylcarbamoyl)pyrrolidin-3-ypterephthalamide and pharmaceutically acceptable salts thereof, as well as various forms of such compound and salts thereof as further described herein below. The treatment resulting in degradation of both clAP1 and clAP2. This compound is also referred to herein as Compound and it is also known as AEG40826 and HGS1029.

2 This compound is disclosed in US patent 7579320, the entire disclosure of which is hereby incorporated by. reference as though fully set forth herein, and the compound has the following structure:
C) 41I
sccm:
(I¨JD/
Figure 1. The structure of Compound.
The invention, in related aspects, comprises a pharmaceutical composition in a dosage unit for intravenous infusion comprising such compound in a dose as hereinafter described and a method of treating a proliferative disorder in a human or non-human mammalian subject in need thereof that comprises internally administering to the subject an effective amount of said compound or a pharmaceutically acceptable salt thereof wherein the effective amount is a dose as defined more fully hereinafter. In additional illustrative embodiments, the invention comprises a method of potentiating apoptosis of abnormally proliferating cells in a human or non-human mammalian subject that comprises internally administering, e.g., by intravenous infusion, a hereinafter defined dose of Compound .
In addition, the invention comprises a method of stimulating the degradation of both clAP1 and clAP2 protein from the cells of a patient suffering from a condition that is caused or exacerbated by abnormal regulation of apoptosis, including, for example, cancer. In an embodiment of the present invention includes a method of stimulating the concomitant degradation of clAP1 and clAP2 by administering the Compound or a pharmaceutically acceptable salt thereof to a human or non-human mammalian subject.
Further, the invention comprises a method of treating an autoimmune disease, in which the condition is caused or exacerbated by abnormal regulation of apoptosis, in a patient in need thereof, including, for example, systemic lupus erythematosus, psoriasis, and immune thrombocytopenic purpura that comprises internally administering to the patient a hereinafter defined dose of Compound or a pharmaceutically acceptable salt thereof.

3 In a further illustrative embodiment, the invention comprises a method of treating an autoimmune disease, in which the condition is caused or exacerbated by abnormal regulation of apoptosis, in a patient in need thereof, including, for example, systemic lupus erythematosus, psoriasis, and immune thrombocytopenic purpura that comprises internally administering to the patient a hereinafter defined dose of Compound or a pharmaceutically acceptable salt thereof.
In a further illustrative embodiment, the invention comprises a method of treating a viral infection in which the condition is caused or exacerbated by abnormal regulation of apoptosis, in a patient in need thereof, including, for example, hepatitis.
In additional illustrative embodiments, the invention comprises any one or more of the above methods that further comprises administering a second cancer-related therapy, such as, e.g., TRAIL agonists, anti-CD20, tumor vaccine, checkpoint inhibitor, radiation, chemotherapy, immunotherapy, photodynamic therapy, and combinations thereof.
Detailed Description of the Invention In many cancer and other diseases, an up-regulation of 1APs has been correlated to an increased resistance to apoptosis. Conversely, our results show that cells with decreased in IAP levels are more sensitive to chemotherapeutic agents and to death receptor agonists such as TRAIL. It is believed that a small molecule, which will antagonize IAP function, or cause a loss of IAPs from diseased cells, will be useful as a therapeutic agent. We report herein that Compound causes a down regulation of both clAP1 and clAP2 proteins in cells to sensitize them to cell death and provide methods of use for treatment of cancer and other diseases and disorders characterized by inappropriate apoptosis.
The compound administered in accordance with the present invention is a SMAC
mimetic that can be used in the treatment of proliferative disorders, e.g.: various benign tumors or malignant tumors (cancer), benign proliferative diseases (e.g., psoriasis, benign prostatic hypertrophy, and restenosis), or autoimmune diseases (e.g., autoimmune proliferative glomerulonephritis, lymphoproliferative autoimmune responses).
It has been found as an aspect of this invention that Compound administration to humans unexpectedly results in loss of its target clAP1 from cells and tissues for longer than 1 week after administration.
Additionally, it has been found as an aspect of this invention that Compound results in loss of both clAP1 and clAP2 in contrast to other SMAC mimetics in development that only cause loss of clAP1.
Compound can therefore be administered less frequently than other SMAC
mimetics, some of which are dosed daily. Additionally, loss of both clAP1 and clAP2 contributes to enhanced cellular potency of Compound relative to other compounds that affect only clAP1. In an embodiment of the present invention includes a method of stimulating the concomitant degradation of clAP1 and clAP2 by administering the Compound or a pharmaceutically acceptable salt thereof to a human or non-human mammalian subject.

4 The present invention discloses a method of treatment that includes the administration to a subject in need thereof of a therapeutically effective amount of Compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a pharmaceutical carrier and a therapeutically effective amount of Compound, or a pharmaceutically acceptable salt thereof.
In particular, Compound and compositions and methods of the present invention are useful for the treatment of any cancer negatively affected by the concomitant degradation of clAP1 and clAP2, including solid tumors such as skin, breast, brain, lung, testicular carcinomas, and the like. Cancers that may be treated by the compounds, compositions and methods of the invention include, but are not limited to the following:
Table 1 Tissue Example Adrenal gland neuroblastoma Bone osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors Cardiac sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma Gastrointestinal esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma) Genitourinary kidney (adenocarcinoma, Wilms tumor [nephroblastoma], tract lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma,

5 Table 1 Tissue Example choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma) Gynecological uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma) Hematologic blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]
Liver hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma Lung bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma Nervous system skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma) Skin malignant melanoma, basal cell carcinoma, squamous cell

6 Table 1 Tissue Example carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids The methods of the invention can include administration of Compound alone or in combination with one or more additional chemotherapeutic agents. Administration of multiple agents can be simultaneous or sequential. Useful chemotherapeutic agents include, but are not limited to, alkylating agents (e.g., cyclophosphamide, mechlorethamine, chlorambucil, melp. halan), anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin), cytoskeletal disruptors (e.g., paclitaxel, docetaxel), epothilones (e.g., epothilone A, epothilone B, epothilone D), inhibitors of topoisomerase I and II (e.g., irinotecan, topotecan, etoposide, teniposide, tafluposide), nucleotide analogs precursor analogs (e.g., azacytidine, azathioprine, capecitabine, cytarabine, doxifluridine, fluorouracil, gemcitabine, mercaptopurine, methotrexate, tioguanine), peptide antibiotics (e.g., bleomycin), platinum-based agents (e.g., carboplatin, cisplatin, oxaliplatin), retinoids (e.g., all-trans retinoic acid), and vinca alkaloids and derivatives (e.g., vinblastine, vincristine, vindesine, vinorelbine). In some embodiments, chemotherapeutic agents include fludarabine, doxorubicin, paclitaxel, docetaxel, camptothecin, etoposide, topotecan, irinotecan, cisplatin, carboplatin, oxaliplatin, amsacrine, mitoxantrone, 5-fluoro-uracil, or gemcitabine. In addition Compound may be used in combination with TRAIL receptor antagonists such as DS-8273a, Mapatumumab, Lexatumumab, Conatumumab, or APG880. Compound may also be used in combination with anti-CD20 antibodies such as: Rituximab, Obinutuzumab, IGN-002, Tositumomab, Ocrelizumab, Veltuzumab, AME-133, PR0131921, and GA101.
Compound, alone or in combination with one or more other active pharmaceutical ingredients, are administered to a human or veterinary subject. The pharmaceutical compositions typically comprise at least one pharmaceutically acceptable excipient, e.g., a carrier or diluent, and can be administered in the conventional manner by routes including systemic, topical, or oral routes.
Administration is normally by intravenous injection, either as a bolus or infusion, but other routes of administration are not precluded including, e.g., subcutaneous, intramuscular, intraperitoneal, intrapleural, intrathecal, intraorbital, or intraarterial injection. An embodiment of the present invention includes de use of a combination of Compound and rituximab in the treatment of cancer by the stimulation of the concomitant degradation of clAP1 and clAP2.
The pharmaceutical composition of the invention is a composition in which the active pharmaceutical ingredient, i.e., Compound, is pure enough, and the composition is otherwise suitable, for internal administration to a human or other mammal. It can be prepared in unit dose form, i.e., a form suitable for single administration to a subject such as by infusion. So, e.g., a pharmaceutical composition in intravenous unit dose form may comprise a vial or pre-filled syringe, or an infusion bag or device, each comprising a sufficient amount of Compound to supply the desired dose (or a convenient fraction of such

7 dose), as described hereinafter, such that the contents of one vial or syringe (or a small number of multiple vials, depending upon the fraction of dose in each) are administered at a time.
Compound can be formulated as a 5 mg/mL solution in sterile water for injection for further dilution prior to infusion. Compound in sterile water for injection, USP (5 mg/mL) will be further diluted with dextrose 5% water (D5W) to achieve the appropriate concentration of compound for IV
infusion. After dilution in D5W to final concentrations range from 0.001 to 1 mg/mL or 0.006 to 0.768 mg/mL.
Typically, Compound is administered by intravenous infusion, including, e.g., by infusion over an infusion period of about 1 to about 120 minutes, or 1 to about 60 minutes, e.g., about 15 minutes.
A dosing regimen can be, once or repeated daily, twice- weekly, or three times weekly (i.e., thrice weekly) intravenous injections, or, e.g., once weekly injections in cycles of three weeks on and one week off, every week, or less frequently, for as long as the treatment is effective, e.g., until disease progresses or the drug is not tolerated. The effective dose administered in each injection is an amount that is effective and tolerated. An effective dose is one that over the course of therapy, which may be, e.g., 1 or more weeks results in treatment of the proliferative disorder, i.e., a decrease in the rate of disease progression, termination of disease progression, or regression or remission.
Doses employed in the practice of this invention can be effective in potentiating apoptosis of abnormally proliferating cells in a patient suffering a proliferative disorder such as cancer or autoimmune disease or certain other disorders, e.g., viral infection. For example, Compound can be administered intravenously, e.g., by infusion, at a dose of 0. 01 to 5 mg/m2 of patient body surface area (BSA) per day of treatment, e.g., 0.01 to 3, 0.01 to 1, 0.05 to 5, 0.05 to 3, 0.05 to 1, 0.1 to 5, 0.1 to 3, 0.1 to 1, 0.2 to 1.4 mg/m2, administered, e.g., by infusion over about 1 to about 120 minutes, e.g., about 15 minutes. The dose in most cases will be more than 0.05 mg/m2. Current clinical studies employ about 0.1 mg/m2 to about 5 mg/m2, specifically, 0.1 to 4.8 mg/m2.
Pharmaceutical compositions of the instant invention refer to compositions suitable for administration in a medical use, i.e., internal administration to a patient. Compositions suitable for infusion in accordance with the method of this invention conveniently comprise a sterile aqueous preparation of Compound, which is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using suitable carriers or diluents which may include a buffer. Thus, in one illustrative aspect, this invention comprises a pharmaceutical dosage unit comprising Compound and one or more pharmaceutically acceptable excipients in an aqueous solvent for use in intravenous or subcutaneous administration for the treatment of a cancer or an autoimmune disorder. The administration of Compound can occur simultaneous with, subsequent to, or prior to the combination therapy, such as chemotherapy or radiation, so long as the chemotherapeutic agent or radiation sensitizes the system to the method and compositions of the present invention.

8 Compound, or pharmaceutically acceptable salts thereof, may also be administered simultaneously with, prior to, or after administration of one or more of the therapeutic agents described below. Such combination therapy may include administration of a single pharmaceutical dosage formulation which contains a compound of the present invention and one or more additional agents given below, as well as administration of the compound of the present invention and each of additional agent in its own separate pharmaceutical dosage formulation. For example, Compound and a chemotherapeutic agent, such as taxol (paclitaxel), docetaxel, etoposide, cisplatin, vincristine, vinblastine, and the like, can be administered to the patient either together in a single oral dosage composition such as a tablet or capsule, or each agent administered in separate oral dosage formulations or via intravenous injection. Additionally, Compound may be co-administered with TRAIL receptor agonists or with anti-cancer antibodies such as anti-CD20. Where separate dosage formulations are used, the compounds of the present invention and one or more additional agents can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially; combination therapy is understood to include all these regimens.
EXAMPLES
Data from various experiments with Compound ( also known as AEG40826 0rHGS1029) are provided in the following Examples.
Example 1 ¨ Efficacy of Compound in combination with Rituximab (Anti-CD20) treatment in a mouse model of lymphoma ( see Figure 2).
The effect of combining Compound with Rituximab was examined in a mouse model of Burkitt's lymphoma. Ramos cells were injected into the flank of female SCID mice and allowed to grow to an average size of 330mm3. Compound (AEG40826) was administered once weekly at a dose of 5 mg/kg by intravenous bolus tail vein injection. Rituximab (Rituxan) ) was administered once weekly at a dose of 10 mg/kg by intravenous bolus tail vein injection. Tumor sizes were measured by caliper. Data indicate that Compound has some single agent activity against the lymphoma cancer cells and that the combination of Compound and Rituximab resulted in strong anti-cancer efficacy.
Figure 2. Efficacy of Compound in combination with Rituximab.

9 Borkitt's Lymphoma (RAMOS) Model in Female SOD mice X
U: 2500- = T
rel : I-1 t ."-ts- ICinIkb 'I Orngthg 2000- -co- AEG40826 5rogikg E e *
= ;>== Rituxan 1 Otnglkg g .1 -4- AEG40626 . Rituxan 2 '1500-7.
s., t .. ISEG40826: 5 mpg IV

= 1000-p gmr= i . Rituxon: 10 mgAtg 0/
r. T
t.-. ..... ,,e . = 4 Mc* FD bet& ae f 160 sne1Cf.7; beet ;wens e= ¨
e> 500- = 0 tad r.. obssii 1 se appesetd . ,Airoprow ,.. 1 . .
15 20 A rik's ''''''''''''''''''''' ' `4.5= ' ' = a " t t t It I
5 ri bVd25 D291732 I r39 S 47 Example 2. Efficacy of SMAC mimetics in combination with TRAIL.
Human HCT-1116 colorectal tumor cells were treated with SMAC mimetics for 72 hours in the presence of lOng/mL recombinant human TRAIL (Figure 3). Cellular viability was measured by Cell Titre Glo 5 (Promega). Data indicate that Compound is approximately 4 fold more potent than Birinapant.
Figure 3. Combinational efficacy of SMAC mimetics with TRAIL.
Skimp= curve +/- hTra 0 on H CT-116 eons AEG00401121.6 CUM 44- Mad on a CT-116 sells 12e = Ditinapant EC50 = >100nAl s 40820A EC50 = >100reA _ Adnapant =
1500/n1 . ire2"+"=0 tenõ ^ (6.- ¨ -*1-'87"--rir4,4, " hTraA BM
= 0 57344 ....
An r¨,_ . ¨ , . i i k. ea F 4, 'n o ee e ..
3 '11\1 4tithe .

2* \I \ AO
, 0 _______________________________________ = IIMW4 4 i i i ; WI A ii 4 i i Lop AEG004082GA inii) Log Birinaperi (r01) Example 3. Compound but not other SMAC mimetic in development causes loss of clAP1 and clAP2

10 from cancer cells (Figure 3).

Hey ovarian cancer cells were treated with the following Smac mimetics:
Compound (AEG40286), Birinapant and LCL-161. Cell proteins were harvested after 3 or 18 hours of treatment. clAP1 levels were detected and quantified in cell lysates by clAP1 specific EL1SA. clAP2 levels were detected by western blot and quantified using a Licor Odyssey scanner. Data show that Compound more potently causes reduction of clAP1 than either LCL-161 or Birinapant at either 3 or 18 hours of treatment.
Compound results in loss of clAP2 at 3 hours. Unexpectedly, Compound does not cause an increase in clAP2 levels at 18 hours in contrast to LCL-161 and Birinapant which increase clAP2 levels to approximately 140 and 180 percent of control, respectively. These results are consistent with enhanced efficacy of Compound..
Figure 3. Effects of SMAC mimetics on clAP1 and clAP2 levels in ovarian cancer cells.
CIAP-1 protein level in Hey cells treated with MP antagonists for either 3h or 18h o k o 100=
E g 80 o ¨
$.... 0 . a -ia ris 40 li e. .-Ijili u n ri, __ Ilt_ < 0 _ __________________________ f Vi cte I ....I.1 - ..I . '6 ,,- 4-.5. r ap = ?ALI
( sunm .. I AE jim , j t a 13 .................................................................. I
.3 hours la hours clAP-2 protein level in Hey cells treated with IAP antagonists for either 3h or 18h Cellular :t 180 -160.1 E 1,7), 140 1 120 .
4: 100 4 , .
^ iL) ' ______________________________________________ 1 ill El o <
Vehod= U 61 loht AEt F I Nu i ..,,inio i - Alm v,iiii..i 361,0 i .4141 1 31A4 I 31301 I3 nom lb boom Example 4. Administration of Compound and pharmacokinetics in cancer patients.
Compound in water at a concentration of 5 mg/mL was diluted into dextrose 5%
water and administered via 15 minute intravenous infusion. Plasma samples were isolated at the indicated timepoints and Compound concentration in plasma was determined by LC-MS/MS. Data indicate that Compound exhibited dose-linear pharmacokinetics following adminstration at doses between 0.1 and 4.8 mg/m2.

11 Figure 4. Plasma pharmacokinetics of Compound in cancer patients.
Cmax :1400 o Dose (mg/m2) AIJC
30o Dose (mgin12) Example 5. Peripheral blood mononuclear cell levels of clAP1 and clAP2 following administration of Compound.
Peripheral blood samples were obtained from patients immediately prior to administration of Compound and 2 hours post-administration. Cells were lysed and clAP1 and clAP2 levels were detected by quantitative ELISA. Samples were collected on day 1 pre and post infusion as well as one week later pre and post infusion and on subsequent weeks. Data indicate that administration of Compound resulted in profound loss of clAP1 which was maintained for at least one week at doses of 0.2 mg/m2 and higher.
Consistent with the unexpected ability of Compound to reduce clAP2 acutely and to prevent SMAC
mediated up-regulation of clAP2 (see Example 2) levels of clAP2 were acutely decreased in peripheral

12 blood mononuclear cells and remained suppressed one week later when assayed prior to the next weekly infusion.
Figure 5. Pharmacodynamic effect of Compound on clAP1 and clAP2 levels in peripheral blood mononuclear cells. Samples were collected pre and post administration of Compound on week 1 and week 2. clAP2 levels were below the lower limit of detection post administration of 2.1 or 4.8 mg/m2 Compound on day 8. No sample was available for detection of clAP2 post adminstration of 3.2mg/m2 Compound on day 8.

cilAP-1 levels in patient PBMCs prior and 2 h post infusion o -0 .01 DI Pte-dose v tr. k 11101 01 Post-dOse m [101 OS Pre-dose 0 i 4:: I OCI DB Post-dose ei 60 ..rg 40 - l u .
:
el ' I
o.
I 1 !
I :
i I I HI 1 i 1 0 ¨
0 DI 02 C4 0.6 09 14 11 3.2 Dose HGS1029 (AEG40826 mg/m2) 100 1 Effect of AEG40826 on ciAP2 in PBNIC

a Cl D1 Post-dose -0-, 80 1 0C108 Pre-dose i 70 1 e 1 60 0 el DaPose-closer c6 1 .;.¶ 1 7 12, 50 -I , i >, 1 it 1-4s1 20 1 'H i ' la. T T i 0.9 1.4 2.1 3.2 4.8

13

Claims

1. Use of a SMAC mimetic for treating a proliferative disorder in a patient comprising internally administering of a SMAC mimetic to patient.

2. The use according to claim 1, wherein internally administering to the patient causes the loss of clAP1 from patient cells.

3. The use according to claim 1, wherein internally administering to the patient causes the loss of clAP2 protein from patient cells.

4. The use according to claim 1, wherein the SMAC mimetic does not result in an upregulation of clAP2 levels.

5. The use according to claim 1, wherein internally administering to the patient causes the concomitant loss of clAP1 and of clAP2 protein from patient cells.

6. The use according to claim 1, wherein the SMAC mimetic is or pharmaceutical acceptable salts thereof.

7. The use according to claim 1, wherein the patient is a human or non-human mammalian subject.

8. A SMAC mimetic compound of formula a pharmaceutically acceptable salts for use in the treatment of a proliferative disorder in a human patient , comprises internally administering to the patient in an amount ranging from 0.01 to 5 mg/rn2 of patient body surface area (BSA) by intravenous infusion over a period from 1 to 120 minutes on a weekly schedule.

9. The SMAC mimetic of claim 8, wherein the amount of the compound is administered per dose ranging from 0.1 to 5 mg/m2 and the period of infusion is from 1 to 60 minutes.

10. The SMAC mimetic of claim 8, wherein the amount of compound is administered per dose ranging 0.1 to 4.8 mg/m2 and the period of infusion is 15 minutes.

11. A use of a SMAC mimetic compound of formula or a pharmaceutically acceptable salts for the treatment of a proliferative disorder in a patient that comprises internally administering to the patient in combination with one or more additional chemotherapeutic agents .

12. The use according to claim 11, wherein the treatment of a proliferative disorder in a patient comprises internally administering to the patient of a combination of the SMAC
mimetic with TRAIL receptor agonists.

13. The use according to claim 11, wherein the treatment of a proliferative disorder in a patient comprises internally administering to the patient of a combination of the SMAC
mimetic with Rituximab.

14. The use according to claim 11, wherein the treatment of a proliferative disorder in a patient comprises internally administering to the patient a combination of the SMAC
mimetic with an anti-CD20 antibody.

15. The use according to any one of claims 1 to 14, wherein the proliferative disorder is selected from the group of: lung adenocarcinoma, pancreatic cancer, colon cancer, ovarian cancer, breast cancer, mesothelioma, peripheral neuroma, bladder cancer, glioblastoma, melanoma, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, bladder cancer, meningioma, glioma, astrocytoma, breast cancer, cervical cancer, chronic myeloproliferative disorders (e.g., chronic myelogenous leukemia), chronic lymphocytic leukemia, colon cancer, endocrine cancers, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gestational trophoblastic tumors, hairy cell leukemia, Hodgkin lymphoma, non- Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi sarcoma, laryngeal cancer, leukemia, acute lymphoblastic leukemia (ALL) , acute myeloid leukemia (AML), lip cancer, oral cavity cancer, liver cancer, male breast cancer, malignant mesothelioma, meduUoblastoma, melanoma, Merkel cell carcinoma, metastatic squamous neck cancer, multiple myeloma and other plasma cell neoplasms, mycosis fungoides and the Sezary syndrome, myelodysplastic syndromes, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, small cell lung cancer, oropharyngeal cancer, bone cancers, including osteosarcoma and malignant fibrous histiocytoma of bone, ovarian epithelial cancer, ovarian germ cell tumors, ovarian low malignant potential tumors, pancreatic cancer, paranasal sinus cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumors, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, small intestine cancer, soft tissue sarcoma, supratentorial primitive neuroectodermal tumors, pineoblastoma, testicular cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilm's tumor and other childhood kidney tumors.

16. A pharmaceutical composition in a dosage unit for intravenous infusion that comprises internally administering to the patient a SMAC mimetic of formula - a pharmaceutically acceptable salts for treating a proliferative disorder in a patient.

17. The pharmaceutical composition according to claim 16, wherein the patient is a human or non-human mammalian subject.

18. A method of treating a proliferative disorder in a patient that comprises internally administering to the patient a SMAC mimetic of formula or a pharmaceutically acceptable salts .

19. The method of claim 18, wherein the SMAC mimetic causes loss of clAP1 from patient cells.

20. The method of claim 18, wherein the SMAC mimetic causes loss of clAP2 protein from patient cell.

21. The method of claim 18, wherein the SMAC mimetic does not result in an upregulation of clAP2 levels.

22. The method according to claim 18, wherein internally administering the SMAC mimetic to the patient causes the concomitant loss of clAP1 and clAP2 protein from patient cells.

23. The method according to claim 22, wherein internally administering the SMAC mimetic to the patient stimulates the concomitant degradation of clAP1 and clAP2 protein from patient cells.

24. The method according to claim 18, wherein the patient is a human or non-human mammalian subject.

25. A method of claim 18 that comprises internally administering to the patient an amount of the SMAC mimetic ranging from 0.01 to 5 mg/m2 of patient body surface area (BSA) by intravenous infusion over a period from about 1 to about 120 minutes on a weekly schedule.

26. The method of claim 25, wherein the amount of the SMAC mimetic administered per dose ranges from about 0.1 to about 5 mg/m2 and the period of infusion is from 1 to 60 minutes.

27. The method of claim 25, wherein the amount of the SMAC mimetic administered per dose is ranging from about 0.1 to about 4.8 mg/m2 and the period of infusion is 15 minutes.

28. The method of treating according to claim 25, wherein further comprises internally administering to the patient of the SMAC mimetic in a combination one or more additional chemotherapeutic agents.

29. The method of treating according to claim 25, wherein the combination includes Rituximab.

30. The method of treating according to claim 26, wherein the combination includes an anti-CD20 antibody.

31. The method of treating according to claim 26, wherein the combination includes a TRAIL
receptor agonists

32. The method of any of claims 18 to 31, wherein the proliferative disorder is selected from the group of: lung adenocarcinoma, pancreatic cancer, colon cancer, ovarian cancer, breast cancer, mesothelioma, peripheral neuroma, bladder cancer, glioblastoma, melanoma, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, bladder cancer, meningioma, glioma, astrocytoma, breast cancer, cervical cancer, chronic myeloproliferative disorders (e.g., chronic myelogenous leukemia), chronic lyrnphocytic leukemia, colon cancer, endocrine cancers, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gestational trophoblastic tumors, hairy cell leukemia, Hodgkin lymphoma, non- Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi sarcoma, laryngeal cancer, leukemia, acute lymphoblastic leukemia (ALL) , acute myeloid leukemia (AML), lip cancer, oral cavity cancer, liver cancer, male breast cancer, malignant mesothelioma, meduUoblastoma, melanoma, Merkel cell carcinoma, metastatic squamous neck cancer, multiple myeloma and other plasma cell neoplasms, mycosis fungoides and the Sezary syndrome, myelodysplastic syndromes, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, small cell lung cancer, oropharyngeal cancer, bone cancers, including osteosarcoma and malignant fibrous histiocytoma of bone, ovarian epithelial cancer, ovarian germ cell tumors, ovarian low malignant potential tumors, pancreatic cancer, paranasal sinus cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumors, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, small intestine cancer, soft tissue sarcoma, supratentorial primitive neuroectodermal tumors, pineoblastoma, testicular cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilm's tumor and other childhood kidney tumors.