METHOD FOR PREDICTING DRUG RESPONSIVENESS IN MYELOID NEOPLASMS
BACKGROUND OF THE INVENTION FIELD OF THE INVENTION
[01] This invention relates generally to the analytical testing of tissue samples in vitro, and more particularly to methods for predicting the responsiveness of patients with myeloid neoplasms to chemotherapy agents. In particular, this invention relates to the use of apoptosis assays performed on peripheral blood and bone marrow samples to predict patient response to any cancer therapeutic agent that induces apoptosis as part of it's mechanism of action, including tyrosine kinase inhibitors, such as staurosporine derivatives including N-benzoylstaurosporine (midostaurin).
DESCRIPTION OF THE RELATED ART
[02] The myeloid neoplasms are primary malignant diseases of the blood-forming organs and are characterized by a predominance of immature myeloid precursors (blasts). The blasts progressively replace normal bone marrow, migrate and invade other tissues. There is diminished production of normal erythrocytes, granulocytes and platelets in patients with acute leukaemia, and this leads to the most important complications of this disease, i.e., anaemia, infection and haemorrhage.
[03] Three main categories of myeloid neoplasms are recognized: acute myeloid leukaemias, myelodysplastic syndromes and myeloproliferative disorders. See Harris, J. Clin. Oncol. 17(12): 3835-3849 (1999). The acute leukaemias are classified morphologically by reference to the predominant cell line involved. This classification broadly divides them into lymphoblastic (ALL) and acute myeloid leukaemia (AML) forms. If untreated, both forms are universally fatal within a period of months to one year. Therapy has markedly altered prognosis, and many patients with acute leukaemia remain free of disease for prolonged periods.
[04] Normal haematopoiesis requires tightly regulated proliferation and differentiation of pluripotent haematopoietic stem cells that become mature peripheral blood cells. Acute leukaemia is the result of a malignant event or events occurring in an early haematopoietic precursor. Instead of proliferating and differentiating normally, the affected cell gives rise to progeny that fail to differentiate and instead continue to proliferate in an uncontrolled fashion. As a result, immature myeloid cells in AML or lymphoid cells in ALL, (blasts), rapidly accumulate and progressively replace the bone marrow; causing diminished production of
normal red cells, white cells and platelets. This loss of normal marrow function in turn gives rise to the common clinical complications of leukaemia: anaemia, infection and bleeding. With time, the leukaemia blasts pour out into the blood stream and eventually occupy the lymph nodes, spleen and other vital organs. If untreated, acute leukaemia is rapidly fatal; most patients die within several months of diagnosis. With appropriate therapy, the natural history of acute leukaemia can be markedly altered, and many patients can be cured. [05] The aetiology of most cases of acute leukaemia is not well understood. However, sometimes a possible cause can be identified. This may include radiation; oncogenic viruses including retroviruses, such as HTLV-1 ; genetic and congenital factors; chemicals, such as benzene and kerosene; and drugs, such as epipodophyllotoxins. [06] Newer treatments for leukaemia. Recently, other kinds of chemotherapeutic agents have been found effective in treating various forms of myeloid neoplasms, including leukaemias, such as AML. These drugs may work, at least in part, by inhibiting cell growth signal transduction pathways and inducing apoptosis in malignant cells and are referred to herein as apoptosis inducing chemotherapy agents. These include, but are not limited to: Tyrosine Kinase Inhibitors (TKIs), including but not limited to, imatinib mesylate and staurosporine derivatives such as N-benzoylstaurosporine (midostaurin; PKC412); HDAC inhibitors; B-Raf inhibitors, including the biaryl isoquinoline group; FAK inhibitors; Edg-1 inhibitors; IAP inhibitors; Hepararanase inhibitors; Protein Tyrosine Phosphatese inhibitors; CXCR4 inhibitors; DNA methyl transferase inhibitors; PPARgamma and alpha agonists; epothilones; Edg-4 receptor antagonists and many others, see below. [07] However, the development of resistance toward these agents is frequently observed especially in advanced leukaemias. This can be a serious clinical problem since it may expose the patient to unnecessary side effects and/or delay the implementation of other more effective treatments. Thus, there is a need for a simple assay that can predict which patients are likely to respond to an apoptosis inducing chemotherapy agent including any of the above and which will not.
SUMMARY OF THE INVENTION
[08] This invention provides a solution for the above mentioned problem. Thus this invention provides a method to estimate the response of a patient with a myeloid neoplasm to treatment with an apoptosis inducing chemotherapy agent comprising: (a) measuring the extent of apoptosis in the blasts in a patient sample; and (b) determining from the measurement in Step (a) the likelihood that the patient will respond to treatment with an apoptosis inducing chemotherapy agent.
[09] In addition, this invention provides a method wherein the patient sample consists of a smear of blood or a smear of bone marrow aspirate or cytospin of aspirate.
[10] In other embodiments, this invention provides a method wherein the apoptosis inducing chemotherapy agent is a TKI including, but not limited to, N-benzoylstaurosporine.
In some embodiments, the method of the invention involves the determination step above consists of finding that the patient is a responder if the percentage of apoptosis is greater than 5%.
[11] In other embodiments, this invention provides a method to determine if a patient with a myeloid neoplasm should be included in a drug study of an apoptosis inducing chemotherapy agent comprising: (a) measuring the extent of apoptosis in the blasts in a patient sample; and (b) determining from the measurement in Step (a) if the patient will respond to treatment with an apoptosis inducing chemotherapy agent and should be included in the study.
[12] This method may make use of a patient sample consisting of a smear of blood or of a smear of bone marrow aspirate or cytospin of aspirate. In this method the patient will be considered to be a responder if the percentage of apoptosis is greater than 5%. [13] Another aspect of the invention relates to the use of N-benzoylstaurosporine in the manufacture of a medicament for the treatment of myeloid neoplasms in a selected patient population, wherein the patient population is selected on the basis of a measurement of the pre-treatment extent of apoptosis in the blasts in a patient sample.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[14] The present invention provides methods to estimate or predict which patients with a myeloid neoplasm will show a favourable response to an apoptosis inducing chemotherapy agent. These methods, to predict drug resistance and drug response can be used to guide
the choice of therapy for a particular patient, help determine drug dose and schedule and can be used to determine the correct patients to include in a drug study.
[15] These methods may make use of blood smears made from samples of a patient's peripheral blood or they may make use of bone marrow aspirates or cytospins of bone marrow.
[16] This invention is based, in part, on the discovery that the extent of apoptosis seen in blast cells in peripheral blood or bone marrow of patients with myeloid neoplasm, including
AML, prior to treatment with an apoptosis inducing therapeutic agent can predict the patients response to that agent.
[17] Incidence of AML. The annual new case incidence of all leukaemias is 8-10 per
100,000. This rate has remained static over the past three decades. The relative incidences for the four categories of leukaemia are as follows: 1) ALL (11 %); 2) chronic lymphocytic leukaemia (29%); 3) AML (46%); and 4) CML (14%).
[18] The leukaemias account for about 3% of all cancers in the U.S. The impact of leukaemia is heightened because of the young age of some patients. For example, ALL is the most common cancer and the second leading cause of death in children younger than 15 years. ALL has a maximal incidence between 2 and 10 years of age, with a second, more gradual rise in frequency later in life. The incidence of AML gradually increases with age, without an early peak. Approximately half of AML cases occur in patients younger than 50 years.
[19] Pathophysiology of AML. The precise molecular event or events that cause leukaemic transformation are unknown; the end result, however, is relentless proliferation of immature haematopoietic cells that have lost their capacity to differentiate normally. The development of leukaemia may be a multi-step process, as demonstrated by the fact that in many cases acute leukaemia develops in patients with a pre-existing myelodysplastic disorder. The disease is monoclonal, i.e., the final leukaemic event occurs in a single cell. The level of differentiation at which malignancy becomes evident is variable. In some cases of AML it appears that malignancy occurs in a very undifferentiated cell, similar to the normal haematopoietic stem cell, in this case, cells, platelets and myeloid precursors are all products of the malignant clone. In other cases of AML, the malignant event may occur in a more differentiated cell; therefore granulocyte and monocyte precursors will develop from
the malignant cell, but red cells and platelet precursors will not. In almost all cases of ALL, the myeloid lineage is not malignant, which suggests that in ALL the malignant event occurs in a cell that is at least partially differentiated. Although the majority of leukaemic cells are relatively undifferentiated, some mature circulating cells may be products of the malignant clone.
[20] As the malignant clone expands, it does so at the expense of normal haematopoiesis. The mechanism of normal marrow suppression in leukaemia is complex; in many patients with hypercellular marrow, the pancytopaenia is probably the result, at least in part, of physical replacement of normal marrow precursors by leukaemic cells. In some patients with acute leukaemia, however, a pancytopaenia with hypocellular marrow develops, thus suggesting that marrow failure is not simply due to physical replacement of the marrow space but may also be due to substances released by the malignant cells. [21] Classification. The acute leukaemias can be classified in a variety of ways, including morphology, cytochemistry, cell-surface markers, cytoplasmic markers, cytogenetics and oncogene expression. The most important distinction is between AML and ALL because these two diseases differ considerably in their clinical behaviour, prognosis, and response to therapy. The various subgroups of AML and ALL also have some important differences. [22] Morphology. Leukaemic cells in AML are typically 12-20 mm in diameter, with discrete nuclear chromatin, multiple nucleoli and cytoplasm that usually contains azurophilic granules. Auer rods, which are slender, fusiform cytoplasmic inclusions that stain red with Wright-Giemsa stain, are virtually pathognomonic of AML. The French-American-British (FAB) collaborative group has subdivided AML into eight subtypes based on morphology and histochemistry: • M0, M1 , M2 and M3 - reflect increasing degrees of differentiation of myeloid leukaemic cells; • M4 and M5 - features of the monocytic lineage; • M6 - features of the erythroid cell lineage; and • M7 - acute megakaryocytic leukaemia.
[23] Cell-surface markers. Monoclonal antibodies reactive with cell-surface antigens have been used to classify acute leukaemias. Antibodies that react with antigens found on normal immature myeloid cells, including CD13, CD14, CD33 and CD34, also react with blast cells from most patients with AML. Exceptions are the M6 and M7 variants, which have antigens restricted to the red cell and platelet lineage, respectively. Myeloid leukaemia blasts also express HLA-DR antigens but usually lack T-cell, B-cell and other lymphoid
antigens. In 10-20% of patients, however, AML blasts will express antigens usually restricted to B- or T-cell lineage. Expression of lymphoid antigens by AML cells does not change either the natural history or the therapeutic response of these leukaemias. [24] Cytogenetics and molecular biology. In most cases of acute leukaemia, an abnormality in chromosome number or structure is found. These abnormalities are clonal, essentially involving all of the malignant cells in a given patient, are acquired and not found in the normal cells of the patient, and are referred to as "non-random" because specific abnormalities are found in multiple cases of AML and are associated with distinct morphologic or clinical subtypes of the disease. These abnormalities may be simply the gain or loss of whole chromosomes, but more often they include chromosomal translocations, deletions or inversions. When patients with acute leukaemia and a chromosomal abnormality are treated and enter into complete remission, the chromosomal abnormality disappears; when relapse occurs, the abnormality reappears.
[25] In AML, the most frequent changes are a gain of chromosome 8 or loss of part or all of chromosome 7 or 5. These abnormalities are each seen in anywhere from 7-12% of cases and are not associated with a particular morphologic subtype of AML. They are, however, more frequently seen in patients with secondary leukaemia and are associated with an unfavourable prognosis. Whether these abnormalities cause leukaemia or occur secondarily is uncertain, but the presence of tumour suppressor genes on chromosomes 5 and 7 could explain the association.
[26] The signs and symptoms of acute leukaemia result from decreased normal marrow function and invasion of normal organs by leukaemic blasts. Anaemia is present at diagnosis in most patients and causes fatigue, pallor, and headache and, in predisposed patients, angina or heart failure. Thrombocytopenia is usually present, and approximately one-third of patients have clinically evident bleeding at diagnosis, usually in the form of petechiae, ecchymoses, bleeding gums, epistaxis or haemorrhage. Most patients with acute leukaemia are significantly granulocytopenic at diagnosis. As a result, approximately one- third of patients with AML and slightly fewer patients with ALL have significant or life- threatening infections when initially seen, most of which are bacterial in origin. [27] With the development of effective programs of combination chemotherapy and advances in marrow transplantation, many patients with acute leukaemia can be cured. These therapeutic measures are complex and are therefore best carried out at centres with appropriate support services and experience in treating leukaemia. Because leukaemia is a
rapidly progressive disease, specific anti-leukaemic therapy should be started as soon after diagnosis as possible, usually within 48 hours.
[28] Remission induction. Treatment with a combination of an anthracycline (daunomycin or idarubicin) and cytarabine leads to complete remission in 60-80% of patients with AML. Profound myelosuppression always follows when these agents are used at doses capable of achieving complete remission. Failure to achieve complete remission is usually due either to drug resistance or to fatal complications of myelosuppression. [29] Post-remission therapy. Intensive consolidation chemotherapy with repeated courses of daunomycin and cytarabine at conventional doses, high-dose cytarabine or other agents prolongs the average remission duration and improves the chances for long-term, disease-free survival. The best results reported to date with chemotherapy have generally used repeated cycles of high-dose cytarabine. Unlike the situation in ALL, low-dose maintenance therapy is of limited benefit after intensive consolidation treatment. In AML, leukaemic recurrence occurs less often in the CNS, being seen in only approximately 10% of cases, most commonly in patients with the M4 or M5 variants. There is no evidence that CNS prophylaxis improves overall disease-free survival in AML.
[30] Prognosis after initial chemotherapy. Among patients with AML in whom complete remission is achieved, 20-40% remain alive in continuous complete remission for more than 5 years, thus suggesting probable cure. As with ALL, younger patients and those with a low white cell count at diagnosis have a more favourable outcome. Patients whose disease is characterized by certain chromosomal abnormalities, particularly t(8;21), t(15;17) and inv(16), do somewhat better, whereas those with 5q- -7, 11q23, inv(3) or t(6;9) do worse. Patients who have a pre-leukaemic phase before their condition evolves into acute leukaemia and those whose leukaemia is secondary to prior exposure to alkylating agents or radiation respond poorly to chemotherapy. Expression of the multi-drug resistance gene 1 (MDR1 ) is also associated with a worse outcome.
[31] Treatment of recurrent AML. Patients whose AML recurs after initial chemotherapy can achieve second remission in about 50% of cases following re-treatment with daunomycin-cytarabine or high-dose cytarabine. Unfortunately, these remissions tend to be short-lived, and few patients in whom relapse occurs after first-line chemotherapy are cured by salvage chemotherapy.
[32] Apoptosis. Apoptosis is programmed cell death. During development and later, excess numbers of neurons, lymphocytes and many other kinds of cells die through a
genetically programmed sequence of changes called apoptosis. This phenomena is of vital importance in determining the malignancy and resistance to treatment of neoplastic cells. [33] The term apoptosis was coined by in 1972 as a means of distinguishing a morphologically distinctive form of cell death which was associated with normal physiology. See Kerr, Wyllie & Currie, BrJ Cancer 26(24): 239-257 (1972). Apoptosis was distinguished from necrosis, which was associated with acute injury to cells. Apoptosis is characterized by nuclear chromatin condensation, cytoplasmic shrinking, dilated endoplasmic reticulum and membrane blebbing, mitochondria remain unchanged morphologically. [34] This type of cell death is often hard to observe in vivo because the dying cells are rapidly phagocytosed by tissue macrophages, and this phagocytosis is clearly different from that seen in inflammation, when activated macrophages are recruited from outside the immediate area of death. One way to observe this phenomenon in vitro is to use a cell permeant DNA-staining fluorescent dye, such as Hoechst 33342, which allows a striking visualization of the chromatin condensation.
[35] Apoptotic death can be triggered by a wide variety of stimuli, and not all cells necessarily will die in response to the same stimulus. Among the more studied death stimuli is DNA damage (by irradiation or drugs used for cancer chemotherapy), which in many cells leads to apoptotic death via a pathway dependent on p53. Some hormones, such as corticosteroids, lead to death in particular cells, e.g., thymocytes, although other cell types may be stimulated. Some cells types express Fas, a surface protein which initiates an intracellular death signal in response to cross-linking. In other cases cells appear to have a default death pathway which must be actively blocked by a survival factor in order to allow cell survival. When the survival factor is removed, the default apoptotic death program is triggered.
[36] Biochemical correlates of these morphological features have emerged during the subsequent years of study of this phenomenon. The first and most dramatic is DNA fragmentation. When DNA from apoptotically dying cells was subjected to agarose gel electrophoresis, ladders with -200 bp repeats were observed, corresponding to histone protection in the nucleosomes of native chromatin. Subsequent pulsed field gel techniques have revealed earlier DNA cleavage patterns into larger fragments. Since even a few double-stranded DNA breaks will render the cell unable to undergo mitosis successfully, such DNA fragmentation can be regarded as a biochemical definition of death. However, in some apoptotic systems, e.g., Fas killing of tumour cells, artificially enucleated cells lacking a nucleus still die, showing that the nucleus is not always necessary for apoptotic cell death.
[37] The changes in the apoptotic cell trigger phagocytosis by non-activated macrophages. Macrophages appear to recognize apoptotic cells via several different recognition systems. For example, there is good evidence that apoptotic cells lose the normal phospholipid asymmetry in their plasma membrane, as manifested by the exposure of normally inward-facing phosphatidyl serine on the external face of the bilayer. Macrophages can recognize this exposed lipid headgroup via an unknown receptor, triggering phagocytosis. Exposure of phosphatidyl serine on the surface of apoptotic cells is depicted in the right blow-up at the bottom of the diagram above.
[38] Another biochemical hallmark of apoptotic death which increasingly appears general is the activation of caspases, which are cysteine proteases related to ced-3, the "death gene" of the nematode Caenorhabditis elegans. Caspases seem to be widely-expressed in an inactive proenzyme form in most cells. Their proteolytic activity is characterized by their unusual ability to cleave proteins at aspartic acid residues, although different caspases have different fine specificities involving recognition of neighbouring amino acids. Active caspases can often activate other pro-caspases, allowing initiation of a protease cascade. Persuasive evidence that these proteases are involved in most examples of apoptotic cell death has come from the ability of specific caspase inhibitors to block cell death, as well as the demonstration that knockout mice lacking caspase 3, 8 and 9 fail to complete normal embryonic development.
[39] Definitions. As used herein, the term "apoptosis inducing chemotherapy agent" (AICA) shall mean any compound, means or agent for use in treating a neoplastic disease that directly or indirectly induces apoptosis as part of the anti-tumour effect. This is intended to include all types of pharmacological therapeutics or other means or agents for treating a neoplastic disease except for immunotherapies and vaccines. This group of therapeutic agents is a broad one covering many chemotherapeutic agents having different mechanisms of action. Generally, chemotherapeutic agents are classified according to the mechanism of action. Many of the available agents are anti-metabolites of development pathways of various tumours, or react with the DNA of the tumour cells. There are also agents which inhibit enzymes, such as topoisomerase I and topoisomerase II, or which are antimiotic agents.
[40] By the term "apoptosis inducing chemotherapy agent" is meant, in addition, any chemotherapeutic agent including, but not limited to: i. an aromatase inhibitor;
ii. an antioestrogen, an anti-androgen (especially in the case of prostate cancer) or a gonadorelin agonist; iii. a topoisomerase I inhibitor or a topoisomerase II inhibitor; iv. a microtubule active agent, an alkylating agent, an anti-neoplastic anti- metabolite or a platin compound; v. a compound targeting/decreasing a protein or lipid kinase activity or a protein or lipid phosphatase activity, a further anti-angiogenic compound or a compound which induces cell differentiation processes; vi. a bradykinin 1 receptor or an angiotensin II antagonist; vii. a cyclooxygenase inhibitor, a bisphosphonate, a rapamycin derivative, such as everolimus, a heparanase inhibitor (prevents heparan sulphate degradation), e.g., PI-88, a biological response modifier, preferably a lymphokine or interferons, e.g. interferon γ, an ubiquitination inhibitor or an inhibitor which blocks anti-apoptotic pathways; viii. an inhibitor of Ras oncogenic isoforms or a famesyl transferase inhibitor; ix. a telomerase inhibitor, e.g., telomestatin; x. a protease inhibitor, a matrix metalloproteinase inhibitor, a methionine aminopeptidase inhibitor, e.g., bengamide or a derivative thereof; or a proteosome inhibitor, e.g., PS-341 ; xi. agents used in the treatment of haematologic malignancies or FMS-like tyrosine kinase inhibitors; xii. an HSP90 inhibitors; xiii. histone deacetylase (HDAC) inhibitors; xiv. serine/theorine mTOR inhibitors; xv. somatostatin receptor antagonists; xvi. ocvβ 3/5 integrin antagonists; xvii. anti-leukaemic compounds; xviii. tumour cell damaging approaches, such as ionizing radiation; xix. EDG binders; xx. antranilamide class of compounds; xxi. ribonucleotide reductase inhibitors; xxii. S-adenosylmethionine decarboxylase inhibitors; xxiii. monoclonal antibodies of VEGF or VEGFR; or a VEGF inhibitor or a derivative thereof.
xxix. photodynamic therapy; xx. angiostatic steroids; xxi. implants containing corticosteroids; xxii. AT1 receptor antagonists; and xxiii. ACE inhibitors. [41] The term "aromatase inhibitor", as used herein, relates to a compound which inhibits the oestrogen production, i.e., the conversion of the substrates androstenedione and testosterone to oestrone and oestradiol, respectively. The term includes, but is not limited to, steroids, especially atamestane, exemestane and formestane; and, in particular, non- steroids, especially aminoglutethimide, roglethimide, pyridoglutethimide, trilostane, testolactone, ketokonazole, vorozole, fadrozole, anastrozole and letrozole. Exemestane is marketed as AROMASIN®; formestane as LENTARON®; fadrozole as AFEMA®; anastrozole as ARIMIDEX®; letrozole as FEMARA® or FEMAR®; and aminoglutethimide as ORIMETEN®. A combination of the invention comprising a chemotherapeutic agent which is an aromatase inhibitor is particularly useful for the treatment of hormone receptor positive tumours, e.g., breast tumours.
[42] The term "anti-oestrogen", as used herein, relates to a compound which antagonizes the effect of estrogens at the oestrogen receptor level. The term includes, but is not limited to, tamoxifen, fulvestrant, raloxifene and raloxifene hydrochloride. Tamoxifen can be administered in the form as it is marketed, e.g., NOLVADEX®; and raloxifene hydrochloride is marketed as EVISTA®. Fulvestrant can be formulated as disclosed in U.S. Pat. No. 4,659,516 and is marketed as FASLODEX®. A combination of the invention comprising a chemotherapeutic agent which is an anti-oestrogen is particularly useful for the treatment of oestrogen receptor positive tumours, e.g., breast tumours. [43] The term "anti-androgen", as used herein, relates to any substance which is capable of inhibiting the biological effects of androgenic hormones and includes, but is not limited to, bicalutamide (CASODEX®), which can be formulated, e.g., as disclosed in U.S. Pat. No. 4,636,505.
[44] The term "gonadorelin agonist", as used herein, includes, but is not limited to, abarelix, goserelin and goserelin acetate. Goserelin is disclosed in U.S. Pat. No. 4,100,274 and is marketed as ZOLADEX®. Abarelix can be formulated, e.g., as disclosed in U.S. Patent No. 5,843,901.
[45] The term "topoisomerase I inhibitor", as used herein, includes, but is not limited to, topotecan, irinotecan, camptothecian and its analogues, 9-nitrocamptothecin and the
macromolecular camptothecin conjugate PNU-166148 (compound A1 in WO 99/17804). Irinotecan can be administered, e.g., in the form as it is marketed, e.g., under the trademark CAMPTOSAR®. Topotecan can be administered, e.g., in the form as it is marketed, e.g., under the trademark HYCAMTIN®.
[46] The term "topoisomerase II inhibitor", as used herein, includes, but is not limited to, the anthracyclines, such as doxorubicin, including liposomal formulation, e.g., CAELYX®, daunorubicin, epirubicin, idarubicin and nemorubicin; the anthraquinones mitoxantrone and losoxantrone; and the podophillotoxines etoposide and teniposide. Etoposide is marketed as ETOPOPHOS®; teniposide as VM 26-BRISTOL®; doxorubicin as ADRIBLASTIN® or ADRIAMYCIN®; epirubicin as FARMORUBICIN®; idarubicin as ZAVEDOS®; and mitoxantrone as NOVANTRON®.
[47] The term "microtubule active agent" relates to microtubule stabilizing, microtubule destabilizing agents and microtublin polymerization inhibitors including, but not limited to, taxanes, e.g., paclitaxel and docetaxel; vinca alkaloids, e.g., vinblastine, especially vinblastine sulfate; vincristine, especially vincristine sulfate and vinorelbine; discodermolides; cochicine and epothilones; and derivatives thereof, e.g., epothilone B or a derivative thereof. Paclitaxel may be administered, e.g., TAXOL®; docetaxel as TAXOTERE®; vinblastine sulfate as VINBLASTIN R.P®; and vincristine sulfate as FARMISTIN®. Discodermolide can be obtained, e.g., as disclosed in U.S. Pat. No. 5,010,099. Also included are Epotholine derivatives which are disclosed in U.S. Pat. No. 6,194,181 , WO 98/10121 , WO 98/25929, WO 98/08849, WO 99/43653, WO 98/22461 and WO 00/31247. Especially preferred are Epotholine A and/or B.
[48] The term "alkylating agent", as used herein, includes, but is not limited to, cyclophosphamide, ifosfamide, melphalan or nitrosourea (BCNU or Gliadel®). Cyclophosphamide can be administered, e.g., in the form as it is marketed, e.g., under the trademark CYCLOSTIN®; and ifosfamide as HOLOXAN®. [49] The term "anti-neoplastic anti-metabolite" includes, but is not limited to, 5- fluorouracil (5-FU); capecitabine; gemcitabine; DNA de-methylating agents, such as 5- azacytidine and decitabine; methotrexate; and edatrexate. Capecitabine can be administered, e.g., in the form as it is marketed, e.g., under the trademark XELODA®; and gemcitabine as GEMZAR®. Also included is the monoclonal antibody trastuzumab which can be administered, e.g., in the form as it is marketed, e.g., HERCEPTIN®.
[50] The term "platin compound", as used herein, includes, but is not limited to, carboplatin, cis-platin, cisplatinum and oxaliplatin. Carboplatin can be administered, e.g., in the form as it is marketed, e.g., CARBOPLAT®; and oxaliplatin as ELOXATIN®. [51] The term "compounds targeting/decreasing a protein or lipid kinase activity; or a protein or lipid phosphatase activity; or further anti-angiogenic compounds", as used herein, includes, but is not limited to, protein tyrosine kinase and/or serine and/or threonine kinase inhibitors or lipid kinase inhibitors, e.g., i) compounds targeting, decreasing or inhibiting the activity of the platelet-derived growth factor-receptors (PDGFR), such as compounds which target, decrease or inhibit the activity of PDGFR, especially compounds which inhibit the PDGF receptor, e.g., a /V-phenyl-2-pyrimidine-amine derivative, e.g., imatinib, SU101 , SU6668 and GFB-111 ; ii) compounds targeting, decreasing or inhibiting the activity of the fibroblast growth factor-receptors (FGFR); iii) compounds targeting, decreasing or inhibiting the activity of the insulin-like growth factor receptor 1 (IGF-1 R), such as compounds which target, decrease or inhibit the activity of IGF-IR, especially compounds which inhibit the IGF-1 R receptor, such as those compounds disclosed in WO 02/092599; iv) compounds targeting, decreasing or inhibiting the activity of the Trk receptor tyrosine kinase family; v) compounds targeting, decreasing or inhibiting the activity of the Axl receptor tyrosine kinase family; vi) compounds targeting, decreasing or inhibiting the activity of the Ret receptor tyrosine kinase; vii) compounds targeting, decreasing or inhibiting the activity of the Kit/SCFR receptor tyrosine kinase; viii) compounds targeting, decreasing or inhibiting the activity of the C-kit receptor tyrosine kinases (part of the PDGFR family), such as compounds which target, decrease or inhibit the activity of the c-Kit receptor tyrosine kinase family, especially compounds which inhibit the c-Kit receptor, e.g., imatinib
ix) compounds targeting, decreasing or inhibiting the activity of members of the c-Abl family and their gene-fusion products, e.g., BCR-Abl kinase, such as compounds which target decrease or inhibit the activity of c-Abl family members and their gene fusion products, e.g., a Λ/-phenyl-2-pyrimidine-amine derivative, e.g., imatinib, PD180970, AG957, NSC 680410 or PD173955 from ParkeDavis; x) compounds targeting, decreasing or inhibiting the activity of members of the protein kinase C (PKC) and Raf family of serine/threonine kinases, members of the MEK, SRC, JAK, FAK, PDK and Ras/MAPK family members, or PI (3) kinase family, or of the Pl(3)-kinase-related kinase family, and/or members of the cyclin-dependent kinase family (CDK) and are especially those staurosporine derivatives disclosed in U.S. Pat. No. 5,093,330, e.g., N- benzoylstaurosporine (midostaurin); examples of further compounds include, e.g., UCN-01 ; safingol; BAY 43-9006; Bryostatin 1 ; Perifosine; llmofosine; RO 318220 and RO 320432; GO 6976; Isis 3521 ; LY333531/LY379196; isochinoline compounds, such as those disclosed in WO 00/09495; FTIs; PD184352 or QAN697, a P13K inhibitor; xi) compounds targeting, decreasing or inhibiting the activity of protein-tyrosine kinase inhibitors, such as compounds which target, decrease or inhibit the activity of protein-tyrosine kinase inhibitors include imatinib mesylate (GLEEVEC®) or tyrphostin. A tyrphostin is preferably a low molecular weight (Mr <1500) compound, or a pharmaceutically acceptable salt thereof, especially a compound selected from the benzylidenemalonitrile class or the S-arylbenzenemalonirile or bisubstrate quinoline class of compounds, more especially any compound selected from the group consisting of Tyrphostin A23/RG-50810, AG 99, Tyrphostin AG 213, Tyrphostin AG 1748, Tyrphostin AG 490, Tyrphostin B44, Tyrphostin B44 (+) enantiomer, Tyrphostin AG 555, AG 494, Tyrphostin AG 556 and AG957 and adaphostin (4-{[(2,5- dihydroxyphenyl)methyl]amino}-benzoic acid adamantyl ester, NSC 680410, adaphostin); and
xii) compounds targeting, decreasing or inhibiting the activity of the epidermal growth factor family of receptor tyrosine kinases (EGFR, ErbB2, ErbB3, ErbB4 as homo- or heterodimers), such as compounds which target, decrease or inhibit the activity of the epidermal growth factor receptor family are especially compounds, proteins or antibodies which inhibit members of the EGF receptor tyrosine kinase family, e.g., EGF receptor, ErbB2, ErbB3 and ErbB4 or bind to EGF or EGF-related ligands, and are in particular those compounds, proteins or monoclonal antibodies generically and specifically disclosed in WO 97/02266, e.g., the compound of Example 39, or in EP 0 564 409, WO 99/03854, EP 0520722, EP 0 566 226, EP 0 787 722, EP 0 837 063, U.S. Patent No. 5,747,498, WO 98/10767, WO 97/30034, WO 97/49688, WO 97/38983 and, especially, WO 96/30347, e.g., compound known as CP 358774, WO 96/33980, e.g., compound ZD 1839; and WO 95/03283, e.g., compound ZM105180, e.g., trastuzumab (Herpetin®), cetuximab, Iressa, OSI-774, CI-1033, EKB-569, GW-2016, E1.1 , E2.4, E2.5, E6.2, E6.4, E2.11 , E6.3 or E7.6.3, and 7H-pyrrolo-[2,3-c]pyrimidine derivatives which are disclosed in WO 03/013541. [52] By antibody is meant intact monoclonal antibodies, polyclonal antibodies, multi- specific antibodies formed from at least 2 intact antibodies, and antibodies fragments so long as they exhibit the desired biological activity.
[53] Compounds which target, decrease or inhibit the activity of a protein or lipid phosphatase are, e.g., inhibitors of phosphatase 1 , phosphatase 2A, PTEN or CDC25, e.g., okadaic acid or a derivative thereof.
[54] Further anti-angiogenic compounds include compounds having another mechanism for their activity, e.g., unrelated to protein or lipid kinase inhibition, e.g., thalidomide (THALOMID) and TNP-470.
[55] Compounds which induce cell differentiation processes are e.g. retinoic acid, α-, γ- or δ-tocopherol or α- γ- or δ-tocotrienol.
[56] The term cyclooxygenase inhibitor as used herein includes, but is not limited to, e.g., Cox-2 inhibitors, 5-alkyl substituted 2-arylaminophenylacetic acid and derivatives, such as celecoxib (CELEBREX®), rofecoxib (VIOXX®), etoricoxib, valdecoxib or a 5-alkyl-2- arylaminophenylacetic acid, e.g., 5-methyl-2-(2'-chloro-6'-fluoroanilino)phenyl acetic acid or lumiracoxib.
[57] The term "bisphosphonates", as used herein, includes, but is not limited to, etridonic, clodronic, tiludronic, pamidronic, alendronic, ibandronic, risedronic and zoledronic
acid. Etridonic acid can be administered, e.g., in the form as it is marketed, e.g.,
DIDRONEL®; clodronic acid as BONEFOS®; tiludronic acid as SKELID®; pamidronic acid as
AREDIA®; alendronic acid as FOSAMAX®; ibandronic acid as BONDRANAT®; risedronic acid as ACTONEL®; and zoledronic acid as ZOMETA®.
[58] The term "heparanase inhibitor", as used herein, refers to compounds which target, decrease or inhibit heparin sulphate degradation. The term includes, but is not limited to,
PI-88.
[59] The term "biological response modifier", as used herein, refers to a lymphokine or interferons, e.g., interferon γ.
[60] The term "inhibitor of Ras oncogenic isoforms", e.g., H-Ras, K-Ras or N-Ras, as used herein, refers to compounds which target, decrease or inhibit the oncogenic activity of
Ras, e.g., a farnesyl transferase inhibitor (FTI), e.g., L-744832 or DK8G557.
[61] The term "telomerase inhibitor", as used herein, refers to compounds which target, decrease or inhibit the activity of telomerase. Compounds which target, decrease or inhibit the activity of telomerase are especially compounds which inhibit the telomerase receptor, e.g., telomestatin.
[62] The term "methionine aminopeptidase inhibitor" or (MMP inhibitor), as used herein, refers to compounds which target, decrease or inhibit the activity of methionine aminopeptidase. Compounds which target, decrease or inhibit the activity of methionine aminopeptidase are, e.g., bengamide or a derivative thereof.
[63] The term "proteosome inhibitor", as used herein, refers to compounds which target, decrease or inhibit the activity of the proteosome. Compounds which target, decrease or inhibit the activity of the proteosome include, e.g., PS-341 and MLN 341.
[64] The term "matrix metalloproteinase inhibitor", as used herein, includes, but is not limited to, collagen peptidomimetic and non-peptidomimetic inhibitors; tetracycline derivatives, e.g., hydroxamate peptidomimetic inhibitor batimastat; and its orally-bioavailable analogue marimastat (BB-2516), prinomastat (AG3340), metastat (NSC 683551)
BMS-279251 , BAY 12-9566, TAA211 , MMI270B or AAJ996.
[65] The term "agents used in the treatment of haematologic malignancies", as used herein, includes, but is not limited to, FMS-like tyrosine kinase inhibitors, e.g., compounds targeting, decreasing or inhibiting the activity of FMS-like tyrosine kinase receptors (Flt-3R); interferon, 1-b-D-arabinofuransylcytosine (ara-c) and bisulfan; and ALK inhibitors, e.g., compounds which target, decrease or inhibit anaplastic lymphoma kinase.
[66] Compounds which target, decrease or inhibit the activity of FMS-like tyrosine kinase receptors (Flt-3R) are especially compounds, proteins or antibodies which inhibit members of the Flt-3R receptor kinase family, e.g., N -benzoylstaurosporine (PKC412, midostaurin, a staurosporine derivative) and MLN518.
[67] The term "HSP90 inhibitors", as used herein, includes, but is not limited to, compounds targeting, decreasing or inhibiting the intrinsic ATPase activity of HSP90; degrading, targeting, decreasing or inhibiting the HSP90 client proteins via the ubiquitin proteosome pathway. Compounds targeting, decreasing or inhibiting the intrinsic ATPase activity of HSP90 are especially compounds, proteins or antibodies which inhibit the ATPase activity of HSP90, e.g., 17-allylamino,17-demethoxygeldanamycin (17AAG), a geldanamycin derivative; other geldanamycin-related compounds; radicicol and HDAC inhibitors. [68] Compounds which target, decrease or inhibit activity of histone deacetylase (HDAC) inhibitors, such as sodium butyrate and suberoylanilide hydroxamic acid (SAHA) inhibit the activity of the enzymes known as histone deacetylases. Specific HDAC inhibitors include MS275, SAHA, FK228 (formerly FR901228), Trichostatin A and compounds disclosed in U.S. Patent No. 6,552,065, in particular, Λ/-hydroxy-3-[4-[[[2-(2-methyl-7H-indol-3-yl)-ethyl]- amino]methyl]phenyl]-2E-2-propenamide, or a pharmaceutically acceptable salt thereof and Λ/-hydroxy-3-[4-[(2-hydroxyethyl){2-(W-indol-3-yl)ethyI]-amino]methyl]phenyl]-2E-2- propenamide, or a pharmaceutically acceptable salt thereof, especially the lactate salt. [69] Compounds which target, decrease or inhibit the activity/function of serine/theronine mTOR kinase are especially compounds, proteins or antibodies which target/inhibit members of the mTOR kinase family, e.g., RAD, RAD001 , CCI-779, ABT578, SAR543, rapamycin and derivatives/analogs thereof, AP23573 and AP23841 from Ariad, everolimus (CERTICAN®) and sirolimus.
[70] "Somatostatin receptor antagonists", as used herein, refers to agents which target, treat or inhibit the somatostatin receptor, such as octreoride and SOM230. [71] "Tumour cell damaging approaches" refers to approaches, such as ionizing radiation. The term "ionizing radiation", referred to above and hereinafter, means ionizing radiation that occurs as either electromagnetic rays, such as X-rays and gamma rays; or particles, such as alpha and beta particles. Ionizing radiation is provided in, but not limited to, radiation therapy and is known in the art. See Hellman, Cancer, 4th Edition, Vol. 1, Devita et al., Eds., pp. 248-275 (1993).
[72] The term "anti-leukaemic compounds" includes, e.g., Ara-C, a pyrimidine analog, which is the 2'-α-hydroxy ribose (arabinoside) derivative of deoxycytidine. Also included is the purine analogue of hypoxanthine, 6-mercaptopurine (6-MP) and fludarabine phosphate. [73] The term EDG binders as used herein refers a class of immunosuppressants that modulates lymphocyte recirculation, such as FTY720.
[74] CERTICAN® (everolimus, FRAD) an investigational novel proliferation signal inhibitor that prevents proliferation of T-cells and vascular smooth muscle cells. [75] The term "ribonucleotide reductase inhibitors" refers to pyrimidine or purine nucleoside analogues including, but not limited to, fludarabine and/or ara-C; 6-thioguanine; 5-FU; cladribine; 6-mercaptopurine, especially in combination with ara-C against ALL; and/or pentostatin. Ribonucleotide reductase inhibitors are especially hydroxyurea or 2-hydroxy- 7H-isoindole-1 ,3-dione derivatives, such as PL-1, PL-2, PL-3, PL-4, PL-5, PL-6, PL-7 or PL-8. See Nandy et al., Ada Oncologica, Vol. 33, No. 8, pp. 953-961 (1994). [76] The term "S-adenosylmethionine decarboxylase inhibitors", as used herein, includes, but is not limited to, the compounds disclosed in U.S. Patent No. 5,461 ,076. [77] Also included are in particular those compounds, proteins or monoclonal antibodies of VEGF disclosed in WO 98/35958, e.g., 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine or a pharmaceutically acceptable salt thereof, e.g., the succinate, or in WO 00/09495, WO 00/27820, WO 00/59509, WO 98/11223, WO 00/27819 and EP 0 769 947; those as described by Prewett et al., Cancer Res, Vol. 59, pp. 5209-5218 (1999); Yuan et al., Proc NatlAcad Sci USA, Vol. 93, pp. 14765-14770 (1996); Zhu et al., Cancer Res, Vol. 58, pp. 3209-3214 (1998); and Mordenti et al., Toxicol Pathol, Vol. 27, No. 1 , pp. 14-21 (1999) in WO 00/37502 and WO 94/10202; ANGIOSTATIN®, described by O'Reilly et al., Cell, Vol. 79, pp. 315-328 (1994); ENDOSTATIN®, described by O'Reilly et al., Cell, Vol. 88, pp. 277-285 (1997); anthranilic acid amides; ZD4190; ZD6474; SU5416; SU6668; bevacizumab; or anti- VEGF antibodies or anti-VEGF receptor antibodies, e.g., rhuMAb and RHUFab; VEGF aptamer, e.g., Macugon; FLT-4 inhibitors; FLT-3 inhibitors; VEGFR-2 lgG1 antibody; Angiozyme (RPI 4610); and Avastan®. In summary, VEGF inhibitor compounds are compounds which target, decrease or inhibit the activity of VEGFR and are may be compounds, proteins or antibodies which inhibit or interact with at least one VEGF receptor tyrosine kinase, inhibit a VEGF receptor or bind to VEGF.
[78] "Photodynamic therapy", as used herein, refers to therapy which uses certain chemicals known as photosensitizing agents to treat or prevent cancers. Examples of
photodynamic therapy includes treatment with agents, such as, e.g., VISUDYNE® and porfimer sodium.
[79] "Angiostatic steroids", as used herein, refers to agents which block or inhibit angiogenesis, such as, e.g., anecortave, triamcinolone, hydrocortisone, 11-α- epihydrocotisol, cortexolone, 17α-hydroxyprogesterone, corticosterone, desoxycorticosterone, testosterone, estrone and dexamethasone.
[80] Implants containing corticosteroids refers to agents, such as, e.g., fluocinolone and dexamethasone.
[81] AT1 receptor antagonists include agents, such as DIOVAN®.
ACE inhibitors include CIBACEN®, benazepril, enazepril (LOTENSIN®), captopril, enalapril, fosinopril, lisinopril, moexipril, quinapril, ramipril, perindopril and trandolapril.
[82] Other chemotherapeutic agents include, but are not limited to, plant alkaloids, hormonal agents and antagonists, biological response modifiers, preferably lymphokines or interferons, antisense oligonucleotides or oligonucleotide derivatives; or miscellaneous agents or agents with other or unknown mechanism of action.
[83] In each case where citations of patent applications or scientific publications are given, in particular with regard to the respective compound claims and the final products of the working examples therein, the subject matter of the final products, the pharmaceutical preparations and the claims is hereby incorporated into the present application by reference to these publications. Comprised are likewise the corresponding stereoisomers, as well as the corresponding crystal modifications, e.g., solvates and polymorphs, which are disclosed therein. The compounds used as active ingredients in the combinations disclosed herein can be prepared and administered as described in the cited documents, respectively.
[84] The structure of the active agents identified by code numbers, generic or trade names may be taken from the actual edition of the standard compendium "The Merck Index" or from databases, e.g., Patents International, e.g., IMS World Publications, or the publications mentioned above and below. The corresponding content thereof is hereby incorporated by reference.
[85] It will be understood that references to the components (a) and (b) are meant to also include the pharmaceutically acceptable salts of any of the active substances. If active substances comprised by components (a) and/or (b) have, for example, at least one basic centre, they can form acid addition salts. Corresponding acid addition salts can also be formed having, if desired, an additionally present basic centre. Active substances having an acid group, e.g., COOH, can form salts with bases. The active substances comprised in
components (a) and/or (b) or a pharmaceutically acceptable salts thereof may also be used in form of a hydrate or include other solvents used for crystallization. 2-[(Pyridin-6(7rV)-on-3- yl)methyl]amino-Λ/-[3-(trifIuoromethyl)phenyl]-3-pyridine-carboxamide, is the most preferred combination partner (a).
[86] Thus, one aspect of this invention is a method to predict response of a patient with a myeloid neoplasm, such as AML, to treatment with a therapeutic agent including an agent that can induce apoptosis or a tyrosine kinase inhibitor, i.e., an apoptosis inducing chemotherapy agent. In one embodiment, before and at several time points during treatment, a sample of blood or bone marrow aspirate is obtained from the patient. The sample of blood or bone marrow is then applied as a thin layer on a slide and dried and fixed, e.g., with methanol, neutral-buffered formalin or para-formaldehyde. Techniques for preparing such slide or smears are well known to those of skill in the art. [87] The degree of apoptosis of the blasts seen in the blood or bone marrow can then be determined by any method known in the art. The likelihood of response of a patient with a myeloid neoplasm to an apoptosis inducing chemotherapy agent can be estimated from the pre treatment degree of apoptosis seen in the blasts in the peripheral blood or bone marrow sample.
[88] One of skill in the art will recognize that there is no absolute cut-off value for the relationship between the percentage of apoptosis seen in the pre-treatment blasts and the responsiveness of the patient to an apoptosis inducing chemotherapy agent. In one embodiment the percentage of apoptosis seen in the pre-treatment blasts can be used as a relative indicator of the expected responsiveness of the patient when given the medication. [89] In other embodiments of the invention, a specific cut-off value can be adopted in order to produce a responder/non-responder result for the purpose, e.g., of including or excluding a patient from a particular study or making a determination of which agent or combination of agents to use when treating a patient or dosing or other clinical decisions. [90] Thus, in this aspect of the present invention, if the percentage of apoptosis in blast cells, prior to treatment is greater than about 5%, the patient will be considered to be a responder to an apoptosis inducing chemotherapy agent. In a more preferred embodiment, if the percentage of apoptosis in blast cells prior to treatment is greater than about 10%, the patient will be considered to be a responder to an apoptosis inducing chemotherapy agent. In a still more preferred embodiment, if the percentage of apoptosis in blast cells prior to treatment is greater than about 20%, the patient will be considered to be a responder to an apoptosis inducing chemotherapy agent, and in most preferred embodiments, if the
percentage of apoptosis in blast cells prior to treatment is greater than about 30% or greater than 40%), the patient will be considered to be a responder to an apoptosis inducing chemotherapy agent.
[91] As used herein, the term "myeloid neoplasm" shall mean any neoplastic disorder of the myeloid lineage and includes but is not limited to AMLs, myelodysplastic syndromes and myelo-proliferative disorders. See Harris et al. (1999), supra; and Bennet et al., Hematol Oncol, Vol. 1 , pp. 187-192 (1982), both incorporated herein by reference in their entirety. [92] Apoptosis Assays. One of skill in the art will know that apoptosis can be measured in many ways and, it is intended that any of these assays can be used in the methods of this invention. For general information on available apoptosis assays, see Apoptosis and Cell Proliferation, 2nd Edition (Boehringer Mannheim GmbH, Biochemie 1998), incorporated by reference herein in its entirety.
[93] In one preferred embodiment, the degree or extend of apoptosis is determined by means of a TUNEL assay. In one embodiment of this invention, the degree of apoptosis in the blasts in the blood or bone marrow from a patient with a myeloid neoplasm is determined prior to treatment with an apoptosis inducing chemotherapy agent. If the degree of apoptosis is less than about 5% the patient could be predicted to be a non-responder and an alternative form of treatment would be considered, e.g., a different type of chemotherapy agent or combination therapy with multiple agents. If the degree of apoptosis is found to be about 20%) or greater, such as for example greater than 30% or greater than 40%, the patient could be predicted to be a responder and treatment with the apoptosis inducing chemotherapy agent would proceed. If the degree of apoptosis is found to be between 5% and 20%, the patient could be predicted to be a partial responder and treatment may proceed, but other treatment including, but not limited to, combination therapy, will be considered.
[94] In another embodiment of this invention, the degree of apoptosis in the blasts in the blood or bone marrow from a patient with a myeloid neoplasm is determined prior to the patients inclusion in a study of an apoptosis inducing chemotherapy agent. If the degree of apoptosis is less than about 5%, the patient could be predicted to be a non-responder and will not be included in the study. If the degree of apoptosis is found to be about 20% or greater, such as for example greater than 30% or greater than 40%, the patient could be predicted to be a responder and would be included in the study with the apoptosis inducing chemotherapy agent. If the degree of apoptosis is found to be between 5% and 20%, the
patient could be considered to be a partial responder and might or might not be included in the study depending on the individual circumstances.
[95] Another aspect of the invention relates to the use of N-benzoylstaurosporine in the manufacture of a medicament for the treatment of myeloid neoplasms in a selected patient population, wherein the patient population is selected on the basis of a measurement of the pre-treatment extent of apoptosis in the blasts in a patient sample. According to a preferred embodiment of the invention, the patient population is selected if the degree of apoptosis is found to be about 20% or greater, such as for example greater than 30% or greater than 40%. According to another embodiment, the patient population may be selected if the degree of apoptosis is found to be between 5% and 20%. Preferably, the patient sample comprises of a smear of blood, a smear of bone marrow aspirate or a cytospin of an aspirate isolated from said patient.
[96] Any method known in the art to measure the extent of apoptosis in the blasts or other cells of a myeloid neoplasm may be used in the methods of the invention. These methods may involve the use of various characteristics of the apoptosis process, as described below, and often make use of light microscopy to identify marked or tagged cells in the blood or bone marrow smear or slide. In order to establish a reliable percentage at least 10 cells should be counted, in a preferred embodiment at least 100 cells are counted. See Apoptosis and Cell Proliferation 2nd Edition (1998), supra, incorporated by reference herein, in its entirety.
[97] These methods include methods to study apoptosis in cell populations, as well as methods to measure apoptosis in individual cells. Methods for use in cell populations include assays for DNA fragments, such as the apoptotic DNA Ladder Kit [see Gavrieli, Sherman and Ben-Sasson, J Cell Biol, Vol. 119, No. 3, pp. 493-501 (1992)] and quantification of histone complex with DNA fragments with an ELISA. In addition, assay for caspase activation can be used, including the detection of the cleavage of a substrate, such as poly ADP ribose polymerase (PARP).
[98] Methods for use in individual cells include staining of chromosomal DNA after permeabilization, active labelling of cells by nick translation, the detection of translocated membrane components or the detection of damage or leakage of plasma membrane, such as by trypan blue or propidium iodide exclusion assays.
[99] In one preferred embodiment of the invention, the apoptosis assay used is the TUNEL enzymatic labelling assay. See White et al., J Virol 52: 410 (1984). The TUNEL assay makes use of the fact that extensive DNA degradation often occurs in the early stages
of apoptosis. Cleavage of the DNA may yield double-stranded, LMW DNA fragments (mono- and olgionucleosomes), as well as single-strand breaks "nicks" in HMW-DNA. These DNA strand breaks can be detected by enzymatic labelling of the free 3'-OH termini with modified nucleotides, such as X-dUTP, X-brotin, DIG or fluorescein. The TUNEL assay makes use of terminal dioxynucleotidyl transferase (TdT) to label the blunt ends of double- stranded DNA breaks independent of a template. The term TUNEL refers to TdT-mediated X-dUTP nick end labelling.
[100] Kits for performing any of the above assays can be obtained from Boehringer Mannheim, Indianapolis, IN.
EXAMPLE
[101] Patients with AML were enrolled in a Phase II clinical study of N- benzoylstaurosporine (midostaurin). The study drug was midostaurin (chemical name is N- (9S, 10R, 11 R, 13R)-2,3, 10,11,12,13-Hexahydro-10-methoxy-9-methyI-1 -oxo-9, 13-epoxy- 1H,9ry-diindolo[1,2,3-gtj:3',2M'-/m]pyrrolo[3,4-i][1,7]benzodiazonin-11-yl]-/V- methylbenazmide).
[102] Midostaurin is a staurosporine derivative, a tyrosine kinase inhibitor of a variety of targets, including but not limited to PKC, alpha, beta and gamma, VEGFR-2, c-kit, PDGFR- alpha, PDGFR-beta and FLT3. Midostaurin has also been described as an apoptosis inducing chemotherapy agent able to induce apoptosis in malignant cells. (See, Fabbo, D Anticancer Drug Des. 15(1): 17-28 (February 2000). Before initiation of drug treatment and at several time points during drug treatment, blood samples are collected into an anticoagulant, e.g., EDTA. The blood sample is then applied as a thin layer on a glass slide, dried and fixed with methanol, neutral-buffered formalin or para-formaldehyde. [103] The cells fixed on the slide are then assayed with a TUNEL apoptosis assay via the In Situ Cell Death Detection Kit (Boehringer Mannheim, Indianapolis, IN, USA) to determine the percentage of blast cells undergoing apoptosis. This result is correlated with the drug response data obtained by determining the percentage of blasts in the sample at the initiation of treatment and at the several time points after starting drug treatment. A substantial reduction in the percentage of blasts in the blood sample after initiation of the treatment is an indication of a positive clinical response to the study drug. [104] Results. Patient No. 1 showed a positive response to the study drug as shown by a reduction of blasts from 80% prior to initiation of treatment to 40% on day 8 and to 0-5% on
day 15 and this patient had a high rate of 40% of apoptosis in blast cells in the TUNEL assay prior to initiation of drug.
[105] Patient No. 2 showed minimal response to the study drug, Midostaurin, as shown by no reduction in percentage of blasts during treatment. The differential for this patient showed 90% blasts prior to the drug and 90% blasts at day 21 of treatment. This patient showed a low rate of apoptosis of 0-5% prior to treatment and at day 15 and at day 21. [106] The conclusion was that a low percentage of apoptosis seen in blasts prior to drug treatment predicts lack of response or resistance to the effects of the study drug while a relatively high rate of apoptosis prior to treatment predicts a good response to this apoptosis inducing chemotherapy agent
REFERENCES CITED
[107] All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. The discussion of references herein is intended merely to summarize the assertions made by their authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited references.
[108] The present invention is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the invention. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatus within the scope of the invention, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications and variations are intended to fall within the scope of the appended claims. The present invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.