WO2011009523A1 - Eg5 as biomarker in rare thoracic cancer - Google Patents

Eg5 as biomarker in rare thoracic cancer Download PDF

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WO2011009523A1
WO2011009523A1 PCT/EP2010/003904 EP2010003904W WO2011009523A1 WO 2011009523 A1 WO2011009523 A1 WO 2011009523A1 EP 2010003904 W EP2010003904 W EP 2010003904W WO 2011009523 A1 WO2011009523 A1 WO 2011009523A1
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aryl
phenyl
heteroaryl
quinoline
treatment
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PCT/EP2010/003904
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French (fr)
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Frank Zenke
Lucia Trandafir
Sonja Kroesser
Benjamin Besse
Jean-Charles Soria
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Merck Patent Gmbh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Abstract

The invention relates to an in-vitro method for predicting the likelihood that a patient suffering from a thoracic tumor will respond to the treatment with an Eg5 inhibitory compound by determining the expression level of the eg5 gene or Eg5 protein as biomarker in a tissue sample from the patient, wherein a higher expression level of the eg5 gene or Eg5 protein indicates that the patient is likely to respond to the treatment compared to a reference value. Another object of the invention concerns a method for monitoring a thoracic tumor, which is associated with Eg5 expression, by administering an effective amount of at least a single Eg5 inhibitor to a mammal in need of such treatment and determining the Eg5 expression in a biological sample withdrawn from the mammal, and wherein a decrease in Eg5 expression indicates an increased likelihood that the mammal responds to the treatment with the inhibitor. The invention also relates to the use of Eg5 as biomarker for rare thoracic tumors.

Description

Eg5 as biomarker in rare thoracic cancers
The invention relates to an in-vitro method for predicting the likelihood that a patient suffering from a thoracic tumor will respond to the treatment with an Eg5 inhibitory compound by determining the expression level of the eg5 gene or Eg5 protein as biomarker in a tissue sample from the patient, wherein a higher expression level of the eg5 gene or Eg5 protein indicates that the patient is likely to respond to the treatment compared to a reference value. Another object of the invention concerns a method for monitoring a thoracic tumor, which is associated with Eg5 expression, by administering an effective amount of at least a single Eg5 inhibitor to a mammal in need of such treatment and determining the Eg5 expression in a biological sample withdrawn from the mammal, and wherein a decrease in Eg5 expression indicates an increased likelihood that the mammal responds to the treatment with the inhibitor. The invention also relates to the use of Eg5 as biomarker for rare thoracic tumors.
During mitosis, various kinesins regulate the formation and dynamics of the spindle apparatus, which is responsible for correct and coordinated alignment and separation of the chromosomes. It has been observed that specific inhibition of a mitotic motor protein - Eg5 - results in collapse of the spindle fibers. The result of this is that the chromosomes can no longer be distributed correctly over the daughter cells. This results in mitotic arrest and can thus cause cell death. Up-regulation of the motor protein Eg5 has been described, for example, in tissue from breast lung and colon tumors. Since Eg5 takes on a mitosis- specific function, it is principally rapidly dividing cells and not fully differentiated cells that are affected by Eg5 inhibition. In addition, Eg5 regulates exclusively the movement of mitotic microtubuli (spindle apparatus) and not that of the cytoskeleton. This is crucial for the side-effect profile of the compounds according to the invention, since, for example, neuropathies, as observed in the case of Taxol, do not occur or only do so to a weakened extent. The inhibition of Eg5 by the compounds according to the invention is therefore a relevant therapy concept for the treatment of malignant tumors.
In general, all solid and non-solid tumors can be treated with the compounds of the formula (I), such as, for example, monocytic leukemia, brain, urogenital, lymphatic system, stomach, laryngeal and lung carcinoma, including lung adenocarcinoma and small-cell lung carcinoma. Further examples include prostate, pancreatic and breast carcinoma.
Expression data on Eg5 in different tumor samples have been addressed in US 6,414, 121 B1 describing methods that can be used for diagnosis and prognosis of cellular proliferation and to screen candidate bioactive agents for the ability to modulate cellular proliferation. Additionally, methods and the molecular target Eg5 for therapeutic intervention in cancers from breast, lung and colon are disclosed. However, thymoma data have not been generated so far.
Epithelial tumors of the thymus, known as thymoma, thymic carcinoma, thymic carcinoid or thymic epithelial tumor (TET), are the most commonly encountered mediastinal tumors. Surgical resection is considered the primary treatment choice, if possible, and these neoplasms have been the target of intensive surgical research for many years. Adjuvant radiation therapy is indicated when the integrity of the dish is uncertain. A loco regional treatment is indicated if the tumor is inextricable. Patients with type A, AB, and B1 thymomas have demonstrated better postoperative survival than those with type B2 or B3 thymoma. A systematic treatment is proposed for mediastinal stage, which offers an objective response about 50 %. A feature of these tumors is their variable morphological appearance, and studies regarding the correlation between morphological characteristics and clinical behavior have been conducted. Another biological feature of these neoplasms, especially thymomas, is their frequent association with various autoimmune diseases, as represented by myasthenia gravis. Neither the biological functions of thymomas nor the genetic characteristics of TETs in relation to the WHO histological classification system has been fully elucidated.
Therefore, the technical problem forming the basis of the present invention is to provide a method for predicting the likelihood that a patient suffering from a thoracic tumor will effectively respond to the treatment with compounds, which are suggested as presumable cancer drugs. It is another problem to provide a method for monitoring a thoracic tumor, which makes a simple and fast evaluation of tumor stage and susceptibility possible. It is still another problem of the invention to provide a biomarker, which allows the identification and characterization of rare thoracic tumors properties in-vitro and in-vivo.
The present invention solves the first problem by providing an in-vitro method for predicting the likelihood that a patient suffering from a thoracic tumor, who is a candidate for treatment with an Eg5 inhibitory compound, will respond to the treatment with the compound, which comprises the step (a) of determining an expression level of a prognostic gene or a gene expression product thereof (biomarker), which is eg5 gene or Eg5 protein, in one or more tissue samples taken from the patient, wherein (i) a higher expression level of the eg5 gene or Eg5 protein indicates that the patient is likely to respond to the treatment compared to a reference value of at least one healthy reference person and/or (ii) a similar expression level of the eg5 gene or Eg5 protein indicates that the patient is likely to respond to the treatment compared to a reference value of at least one reference patient suffering from the thoracic tumor in the same stage and susceptible to the treatment.
It has been surprisingly demonstrated by the inventors that the eg5 gene and particularly the Eg5 protein do not only act as therapeutic target, but represent a powerful biomarker with high predictive value in rare thoracic cancers. Consequently, the aforementioned Eg5 protein represents a novel biomarker that is well suited for predicting the response likelihood to Eg5 inhibitors. The underlying biomarker Eg5 of the invention has been unexpectedly found to be positively stained in all types of rare thoracic tumors, such as TET, after histological based studies using Eg5 inhibitors. The aforementioned eg5 gene and Eg5 protein may be named in another way, but are easily assigned by the accession number, which is generally accepted and fixed in numerous data bases, such as the GenBank, SwissProt and the like. Both, the eg5 gene and Eg5 protein have already been described in the state of the art by sequence and other features; however, a link to rare thoracic tumors has not been identified previously. That means the inventive principle underlying the present method comprises prospecting for the eg5 gene or the gene product Eg5 thereof in thoracic tumor samples, which can be either detected on the genetic level or on the protein level, wherein the protein level is preferred.
The inhibitory compounds according to the invention effect specific inhibition of mitotic motor proteins, in particular Eg5. The compounds according to the invention preferably exhibit an advantageous biological activity which can easily be detected. The efficacy of the compounds can be determined, for example, via the Eg5 ATPase activity, which is measured via an enzymatic regeneration of the product ADP to ATP by means of pyruvate kinase (PK) and subsequent coupling to an NADH-dependent lactate dehydrogenase (LDH) reaction. The reaction can be monitored via the change in absorbance at 340 nm by coupling to the NADH-dependent LDH. The regeneration of the ATP simultaneously ensures that the substrate concentration remains constant. The change in absorbance per time unit is analyzed graphically and a linear regression carried out in the visually linear region of the reaction. In such assays, the compounds according to the invention preferably exhibit and cause an inhibiting effect, which is usually documented by IC50 values in a suitable range, preferably in the micromolar range and more preferably in the nanomolar range. It can also be shown that the compounds according to the invention have an advantageous effect in a xenotransplant tumor model. As discussed herein, effects of the compound according to the invention are relevant to rare thoracic diseases. Accordingly, the compounds according to the invention are useful in the prophylaxis and/or treatment of these tumor diseases that are influenced by inhibition of one or more mitotic motor proteins, in particular Eg5. The cancer as concerned by the invention are thoracic cancerous diseases, preferably rare thoracic cancers. Cancers can also be designated as tumors in the meaning of the invention. In a preferred embodiment of the invention, the patient suffers from a thymic epithelial tumor (TET). TETs are rare thoracic tumors that develop in the anterior mediastinum, but have shown modest responses to standard chemotherapy. Although the inventive method provides a reliable indicator in any genetic type of TET according to the genetic characterization, it is preferably applied in C types, AB types, micronodular types, B1 , B2, B3, A or carcinoid TETs, wherein the frequency of Eg5 occurrence decreases with the order mentioned. Hence, it is more preferred to predict the likelihood that the patient will respond to the treatment with an Eg5 inhibitor if said patient suffers from TET C type having the highest risk of recurrence.
The patient can belong to any mammal species, for example a primate species, particularly humans, rodents, including mice, rats and hamsters, rabbits, horses, cattle, dogs, cats, etc. Animal models are of interest for experimental investigations, providing a model for the treatment of a human disease.
A "medicament", "drug", "pharmaceutical composition" or "pharmaceutical formulation" in the meaning of the invention is any agent in the field of medicine, which comprises one or more Eg5 inhibitors of the invention or preparations thereof and can be used in prophylaxis, therapy, follow-up or aftercare of patients who suffer from thoracic cancer diseases in such a way that a pathogenic modification of their overall condition or of the condition of particular regions of the organism could establish at least temporarily. The dose varies depending on the specific compound used, the specific disease, the patient status, etc. Typically, a therapeutic dose is sufficient considerably to reduce the undesired cell population in the target tissue, while the viability of the patient is maintained. The treatment is generally continued until a considerable reduction has occurred, for example at least about a 50 % reduction in the cell burden, and can be continued until essentially no undesired cells are detected in the body. Pharmaceutical formulations can be adapted for administration via any desired suitable method, for example by oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) methods. Such formulations can be prepared using all processes known in the pharmaceutical art by, for example, combining the active ingredient with the excipient(s) or adjuvant(s). The amount of excipient material that is combined with the active ingredient to produce a single dosage form varies depending upon the host treated and the particular mode of administration.
The treatment with said Eg5 inhibitory compound can be a first-line mono-therapy. The selected gene expression product is particularly the expressed protein Eg5. There are several compounds in the state of the art, which are suggested to possess Eg5 inhibiting activity and can be selected for testing in the scope of the present invention by the skilled artisan. As used herein, a compound with Eg5 inhibiting activity is an agent that blocks at least some of the biological effects of Eg5, which refers to any factor, agent, compound whether endogenous or exogenous in origin, which is capable of binding and interacting with Eg5 and thereby stopping certain biological effects of Eg5. Preferably, the treatment of a TET patient can be based on the prediction of a response to a compound of formula (I)
Figure imgf000006_0001
in which
W denotes CH or N,
R1, R2, R3, independently of one another, denote H1 R, A, aryl, heteroaryl, Hal, -(CY2)n-SA,
-(CY2VSCF3, -(CYa)n-SCN1 -(CY2Jn-CF3, -(CY2)n-OCF3, cycloalkyl, -SCH3, -SCN, -CF3, -OCF3, -OA, -(CY2)n-OH, -(CY2Jn-CO2R, -(CY2Jn-CN1 -(CY2Jn-HaI, - (CY2Jn-NR2, (CY2Jn-OA, (CY2Jn-OCOA, -SCF3, (CY2Jn-CONR2, -(CY2Jn-NHCOA, -(CY2Jn-NHSO2A, SF5, Si(CH3J3, CO-(CY2Jn-CH3, -(CY2)n-N-pyrrolidone,
CH(CH2JnNRCOOR, CHNRCOOR, NCO, CH(CH2JnCOOR, NCOOR, CH(CH2JnOH, N(CH2JnOH1 CHNH2, CH(CH2JnNR2, CH(CH2JnNR2, C(OH)R, CHNCOR, CH(CH2)n-aryl, CH(CH2)n-heteroaryl, CH(CH2JnR1, N(CH2JnCOOR, CH(CH2)nX(CH2)n-aryl, CH(CH2JnX(CH2)n-heteroaryl, N(CH2JnCONR2, XCONR(CH2JnNR2, N[(CH2)nXCOOR]CO(CH2)n-aryl, Nf(CH2JnXR]CO(CH2Jn- aryl, N[(CH2)nXR]CO(CH2)nX-aryl, N[(CH2)nXR]SO2(CH2)n-aryl,
N[(CH2)nNRCOOR]CO(CH2)n-aryl1 N[(CH2)nNR2]CO(CH2)n-aryl, N[(CH2)nNR2]CO(CH2)nNR-aryl, N[(CH2)nNR2]SO2(CH2)n-aryl, N[(CH2)nXR]CO(CH2)n-heteroaryl, N[(CH2)nXR]CO(CH2)nX-heteroaryl, N[(CH2)nXR]SO2(CH2)n-heteroaryl, N[(CH2)nNRCOOR]CO(CH2)n-heteroaryl, N[(CH2)nNR2]CO(CH2)n-heteroaryl, N[(CH2)nNR2]CO(CH2)nNR-heteroaryl, N[(CH2)πNR2]S02(CH2)n-heteroaryl, O(CH2)πNR2, X(CH2)nNR2, NCO(CH2)πNR2,
R1 and R2 together also denote -N-C(CF3)=N-, -N-CR=N-, -N-N=N-,
Y denotes H, A, Hal, A denotes alkyl or cycloalkyl having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 C atoms, in which one or more H atoms may be replaced by Hal,
Hal denotes F, Cl, Br or I, R denotes H or A, in the case of geminal radicals R together also denote -(CH2)5-,
-(CH2)4-, -(CH2)2-X-(CH2)2 or -(CH2)2-Z-(CH2)2,
R4, R5, independently of one another, denote H or an unsubstituted or mono- or poly- OR-, NO2-, HaI-, CF3-, OCF3-, CN-, NR2- or SR-, aryl- or heteroaryl-substituted N-pyrrolidone radical, -X-(CH2)2OR, -X-CO(CH2)nCH3, -X-(CH2)2NR2, S-aryl, O- aryl, CH2Si(CH3)3, or together denote -X(CR2J2-, -X-(CR2)3-, -X- (CHCH2OR)(CH2)2-, -X-(CHCH2NR2)(CH2),-, -X(CH2)2NR2, -(CR2)3-, -(CR2)4-, -CR=CR-CR=CR-, -XCHQ(CR2)2-, -XCHQCR2-, R-N-(C=X)-N-R,
-XC[(CH2)nOR]2CH2CH2-,
X denotes O, S or NR,
Q denotes CH2HaI, CHO, CORa, CH2R3 , CH2OCOR3, CH2NCOR1, CH2N(R1)2,
CH2OR1, CH2OCON(R1)2, CH2OCOOR1, CH2NHCON(R1)2, CH2NHCOOR1,
Ra denotes
Figure imgf000008_0001
Figure imgf000009_0001
OR, NHR2, NR2, NR(CH2)n-aryl, NR(CH2)nOR, COOR, N-pyrrolidone radical, OCOR, NR(CHa)nNR2, N[(CH2)nNR2]CO(CH2)n-aryl, N[(CH2)nNHCOOR]CO-aryl, R1, N[CH2(CH2)nOR]2, NR(CH2)πNCOOR, X(CH2)nX(CH2)nXR,
NR(CH2)nX(CH2)nOH, NR(CH2)nO(CH2)nOH, (CH2)nCOOR, O(CO)NR(CH2)nOR, O(CO)(CH2)nNR2, NR(CH2)nNR2, N[(CH2)nNR2]CO(CH2)n-aryl,
N[(CH2)nXR]CO(CH2)n-aryl, N[(CH2)nXR]CO(CH2)n-heteroaryl,
N[(CH2)nNR2]CO(CH2)n-heteroaryl, N[(CH2)nNR2]CO(CH2)nR1,
N(R)(CH2)nN(R)COOR, XCOO(CH2)nNR2, OSO2A, OSO2CF3, OSO2Ar,
OCONR2, OCH2(CH2)nNR2,
Z denotes CH2, X, CHCONH2, CH(CH2)nNRCOOR, CHNRCOOR, NCO,
CH(CH2)nCOOR, NCOOR, CH(CH2)nOH, N(CH2)nOH, CHNH2, CH(CH2JnNR2, CH(CH2)nNR2, C(OH)R1 CHNCOR, CH(CH2)n-aryl, CH(CH2)n-heteroaryl,
CH(CH2)nR1, N(CH2)nCOOR, CH(CHz)nX(CH2)n-aryl, CH(CH2)nX(CH2)n- heteroaryl, N(CH2)nCONR2, XCONR(CH2)nNR2, N[(CH2)nXCOOR]CO(CH2)n- aryl, N[(CH2)nXR]CO(CH2)n-aryl, N[(CH2)nXR]CO(CH2)nX-aryl,
N[(CH2)nXR]SO2(CH2)n-aryl, N[(CH2)nNRCOOR]CO(CH2)n-aryl,
N[(CH2)nNR2]CO(CH2)n-aryl, N[(CH2)nNR2]CO(CH2)nNR-aryl,
N[(CH2)nNR2]SO2(CH2)π-aryl, N[(CH2)nXR]CO(CH2)n-heteroaryl,
N[(CH2)nXR]CO(CH2)nX-heteroaryl, N[(CH2)nXR]SO2(CH2)n-heteroaryl, N[(CH2)nNRCOOR]CO(CH2)n-heteroaryl, N[(CH2)nNR2]CO(CH2)n-heteroaryl, N[(CH2)nNR2]CO(CH2)nNR-heteroaryl, N[(CH2)nNR2]S02(CH2)n-heteroaryl, O(CH2)nNR2, X(CH2)nNR2> NCO(CH2)nNR2,
R6 denotes aryl or heteroaryl, each of which is unsubstituted or mono- or
polysubstituted by aryl or heteroaryl, each of which may be substituted by Hal, NO2, CN, A, OR, OCOR, COR, NR2, CF3, OCF3, OCH(CF3)2, or by Hal, NO2, CN, OR, A, -(CY2)n-OR, -OCOR, -(CY2)n-CO2R, -(CY2)n-CN, -NCOR, -COR or
-(CY2)n-NR2,
R7 denotes (C=O)-R, (C=O)-NR2, (C=O)-OR, H or A, aryl denotes unsubstituted or substituted phenyl, naphthyl or biphenyl, heteroaryl denotes a mono- or bicyclic, unsubstituted or substituted aromatic heterocycle having one or more N, O and/or S atoms, m denotes 0, 1 or 2, and n denotes 0, 1 , 2, 3, 4, 5, 6 or 7, and solvates, tautomers, salts and stereoisomers thereof, including mixtures thereof in all ratios, wherein the compounds of the following formulae are excluded: 5-phenyl-3,4,4a,5,6, 10b-hexahydro-2H-pyrano[3,2-c]quinoline,
7-chloro-5-phenyl-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline,
9-chloro-5-phenyl-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline,
7-methyl-5-phenyl-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinorme,
5-phenyl-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinolin-7-ol,
9-methoxy-5-phenyl-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline,
10-chloro-5-phenyl-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline,
9,10-dichloro-5-phenyl-3,4,4a,5,6, 10b-hexahydro-2H-pyrano[3,2-c]quinoline,
8-chloro-5-phenyl-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline,
8,9-dichloro-5-phenyl-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline,
4-phenyl-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline,
8-methoxy-4-phenyl-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline,
6-methyl-4-phenyl-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline,
4-phenyl-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinolin-6-ol,
8-chloro-4-phenyl-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline,
8-nitro-4-phenyl-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline,
1 -(2-phenyl-1 ,2,3,4-tetrahydroquinolin-4-yl)pyrrolidin-2-one,
3,3-dimethyl-2-phenyl-1 ,2,3,4-tetrahydroquinoline,
4-benzyloxy-3,3-dimethyl-2-phenyl-1 ,2,3,4-tetrahydroquinoline,
4-methoxy-2-phenyl-1 ,2,3,4-tetrahydroquinoline,
2-(4-fluorophenyl)-4-methoxy-1 ,2,3,4-tetrahydroquinoline,
2-furan-2-yl-4-methoxy-1 ,2,3,4-tetrahydroquinoline,
1-(3-pentyl-2-p-tolyM ,2,3,4-tetrahydroquinolin-4-yl)pyrrolidin-2-one, 1-(1-methyl-3-pentyl-2-phenyl-1 ,2,3,4-tetrahydroquinolin-4-yl)pyrrolidin-2-one, 3-methyl-2-phenyl-1 ,2,3,4-tetrahydro(1 ,5]naphthyridine,
4-ethoxy-2-phenyl-1 ,2,3,4-tetrahydroquinoline,
4-(4-chlorophenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline,
8-chloro-4-phenyl-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline,
5-(4-chlorophenyl)-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline,
5-(4-methoxyphenyl)-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline,
5-(3,4-dichlorophenyl)-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline,
5-(4-bromophenyl)-3,4,4a,5,6,10b-hexahydro-2l-l-pyrano[3,2-c]quinoline,
9-methyl-5-phenyl-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline,
9-chloro-5-phenyl-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline,
5-(4-chlorophenyl)-9-isopropyl-3,4,4a, 5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline.
Said compounds of formula (I) are described in EP 1761515 B1. Said publication, including the specification and all embodiments and figures thereof as well as the content of the claims are incorporated by reference as a whole in the disclosure of the invention. The prior teaching of this reference concerning compounds of formula (I) and sub-formulae thereof is valid without restrictions to the application of said compounds within the predictive method of the invention if expedient.
In a more preferred embodiment of the method according to the present invention, the intended treatment is with the Eg5 inhibitory compound 1-(2-dimethylamino-ethyI)-3- ((2R,4aS,5R,10bS)-5-phenyl-9-trifluoromethyl-3,4,4al5,6, 10b-hexahydro-2H-pyrano[3,2- c]quinoline-2-ylmethyl)-urea.
Furthermore, the Eg5 inhibitory compound may be administered in combination with other treatments. A synergistic effect may be achieved by using more than one compound in the pharmaceutical composition, i.e. the Eg5 inhibitor of the invention is combined with at least another agent as active ingredient. The active ingredients can be used either
simultaneously or sequentially. The treatment with said anti-Eg5 drug can be a combination therapy with a chemotherapeutic agent after the patient has developed a chemo-refractory tumor. The selected gene expression product is particularly the expressed protein Eg5.
The patient sample particularly derives from tumor tissue or plasma. It is preferred to withdraw the tissue sample of the patient by biopsy. Tissue samples are taken from the patient before treatment with said anti-Eg5 drug and optionally, additional samples can be withdrawn on treatment with said anti-Eg5 drug. In doing so, the expression levels of the gene expression product obtained on treatment are compared with the values obtained before starting treatment of said patient. Furthermore, the method can also comprise the step of exposing in-vivo the patient to the Eg5 inhibitory compound prior to step (a). That means the term "candidate" is broadly to construe comprising either designated patients or present patients.
In step (a) the expression level is determined on DNA/RNA or protein basis, preferably the level of the expressed Eg5 protein encoded by the eg5 gene is determined. In general, "a gene" is a region on the genome that is capable of being transcribed to RNA that either has a regulatory function, a catalytic function and/or encodes a protein. A gene typically has introns and exons, which may organize to produce different RNA splice variants that encode alternative versions of a mature protein. "Gene" contemplates fragments of genes that may or may not represent a functional domain. The term "gene product" denotes molecules that are formed from the substrate of said genes by biochemical, chemical or physical reactions, such as DNA synthesis, transcription, splicing, translation,
fragmentation or methylation. Preferred gene products of the invention are RNA, particularly mRNA and cRNA, cDNA and proteins. As obvious to the skilled artisan, the present invention shall not be construed to be limited to the full-length protein Eg5.
Physiological or artificial fragments of Eg5, secondary modifications of Eg5, species- dependent alterations as well as allelic variants of Eg5 are also encompassed by the present invention. In this regard an "allelic variant" is understood to represent the gene product of one of two or more different forms of a gene or DNA sequence that can exist at a genetic single locus. Preferably, full-length Eg5 or a physiological variant of this marker is detected in a method according to the present invention.
The determination may be performed by applying intact cells to a detection method of choice. It is preferred, however, to provide cellular extracts first. Cell lysis can be performed in suitable, well-known lysis buffers, which may cause an osmotic shock and perforate the cell membrane. The stability of the cell structure can also be destroyed by mechanical forces, such as ball mill, French press, ultrasonic, etc., by enzymatic degradation of cell wall and cell membrane, respectively, and/or by the action of tensides. The biomarker may be further purified to remove disturbing substances or the biomarker Eg5 can be concentrated in the sample. Downstream-processing and/or concentrating are preferably performed by the method of precipitation, dialysis, gel filtration, gel elution, or
chromatography, such as HPLC or ion exchange chromatography. It is recommended to combine several methods for better yields. Suitable tests for detecting Eg5 are known to those skilled in the art or can be easily designed as a matter of routine. Many different types of assays are known, examples of which are set forth below. Although the assay according to the invention may be any assay suitable to detect and/or quantify gene expression, Eg5 is preferably determined by means of substances specifically interacting with Eg5. The term "specific substances" as used herein comprises molecules with high affinity to at Eg5 in order to ensure a reliable binding. The substances are preferably specific to parts of the protein. Such parts represent a restriction to those regions which are sufficient for the expression of a specific function, i.e. the provision of a structural determinant for recognition. All truncations are inevitably limited by the requirement of preserving the unique recognition. However, the parts of the gene products can be very small. Preferably, the substances are mono-specific in order to guarantee an exclusive and directed interaction with the chosen single target.
The recognition of the biomarker protein or parts thereof can be realized by a specific interaction with substances on the primary, secondary and/or tertiary structure level of an amino acid sequence. In the context of the present invention, the term "recognition" - without being limited thereto - relates to any type of interaction between the specific substances and the target, particularly covalent or non-covalent binding or association, such as a covalent bond, hydrophobic/ hydrophilic interactions, van der Waals forces, ion pairs, hydrogen bonds, ligand-receptor interactions, interactions between epitope and antibody binding site, nucleotide base pairing, and the like. Such association may also encompass the presence of other molecules such as peptides, proteins or other nucleotide sequences. The specific substances are composed of biological and/or chemical structures capable to interact with the target molecule in such a manner that makes a recognition, binding and interaction possible. In particular, the substances are selected from the group of nucleic acids, peptides, carbohydrates, polymers, small molecules having a molecular weight between 50 and 1.000 Da and proteins, preferably nucleic acids and proteins. The specific substances express a sufficient sensitivity and specificity in order to ensure a reliable detection. A specific substance has at least an affinity of 10"7 M for its corresponding target molecule. The specific substance preferably has an affinity of 108 M or even more preferred of 10~9 M for its target molecule. As the skilled artisan will appreciate the term specific is used to indicate that other biomolecules present in the sample do not
significantly bind to the substance specific for Eg5. Preferably, the level of binding to a biomolecule other than the target molecule results in a binding affinity of only 10 % of the affinity of the target molecule, more preferably only 5 % or less. A most preferred specific substance will fulfill both the above minimum criteria for affinity as well as for specificity.
The proteins or peptides are preferably selected from the group consisting of antibodies, cytokines, lipocalins, receptors, lectins, avidins, lipoproteins, glycoproteins, oligopeptides, peptide ligands and peptide hormones. More preferably, antibodies are used as specific substance. "Antibody" denotes a polypeptide essentially encoded by an immunoglobulin gene or fragments thereof. According to the invention, antibodies are present as intact immunoglobulins or a number of well-characterized fragments. Fragments are preferably selected from the group consisting of Fab fragments, Fc fragments, single chain antibodies (scFv), variable regions, constant regions, H chain (H), and L chain (L), more preferably Fab fragments and scFv. Fragments, such as Fab fragments and Fc fragments, can be produced by cleavage using various peptidases. Furthermore, fragments can be engineered and recombinantly expressed, preferably scFv.
The term "nucleic acid" refers to a natural or synthetic polymer of single or double-stranded DNA or RNA alternatively including synthetic, non-natural or modified nucleotides, which can be incorporated in DNA or RNA polymers. Each nucleotide consists of a sugar moiety, a phosphate moiety, and either a purine or pyrimidine residue. The nucleic acids are preferably single or double stranded DNA or RNA, primers, antisense oligonucleotides, ribozymes, DNA enzymes, aptamers and/or siRNA, or parts thereof. The nucleic acids can be optionally modified as phosphorothioate DNA, locked nucleic acid (LNA), peptide nucleic acid (PNA) or spiegelmer. Particular preferred nucleic acid probes to be used as Eg5-specific substances are aptamers.
DNA aptamers and RNA aptamers have been found to express a high affinity for a wide variety of target molecules. Target structures may comprise proteins, peptides and small molecules, such as organic dyes, nucleotides, amino acids, vitamins, alkaloids, etc. More preferred are RNA aptamers since the 2'-hydroxyl group available in RNA promotes a couple of intra- and intermolecular contacts, the latter being between molecules of the same sequence, different sequences, or between RNA and any other molecule which is not composed of RNA. These nucleic acid ligands can be identified by an efficient in-vitro selection procedure - the so-called SELEX process (systematic evolution of ligands by exponential enrichment). Since RNA is very susceptible to nucleolytic degradation in biological solutions, RNA aptamers should be chemically modified using
phosphorothioates, locked nucleic acids, or Spiegelmers, for instance. L-RNA versions of aptamers called Spiegelmers are especially long-lived as they are essentially impervious to natural degradation processes. Because of their high affinity for a broad spectrum of structural targets, aptamers act very similar to antibodies. Aptamers can be synthesized using standard phosphoramidite chemistry. In addition, RNA aptamers having more than approximately 30 nucleotides can be favorably synthesized in large amounts by in-vitro transcription. Selection, synthesis, and purification of aptamers are well-known to those skilled in the art.
The specific substances can be labeled, in doing so the labeling depends on their inherent features and the detection method to be applied, i.e. the required sensitivity, ease of conjugation, stability requirements, and available instrumentation and disposal provisions. For the detection of specific incubation products, the applied methods depend on the specific incubation products to be monitored and are well-known to the skilled artisan.
Preferred examples of suitable detection methods according to the present invention are luminescence, particularly fluorescence, furthermore VIS coloring and/or radioactive emission. Luminescence concerns the emission of light as a result of chemiluminescence, bioluminescence or photoluminescence. Chemiluminescence involves the emission of visible light as a result of a chemical reaction, whereas bioluminescence requires the activity of luciferase. The presently preferred photoluminescence, which is also known as fluorescence stimulation, is caused by the absorption of photons, preferably provided by radiation, which is released again as photon with a shift in wavelength of 30 to 50 nm and within a period of approximately 10~8 seconds. The instruments for fluorescence detection include, but are not limited to typical benchtop fluorometers, fluorescence multi-well plate readers, fiber optic fluorometers, fluorescence microscopes and microchips/microfluidics systems coupled with fluorescence detection. VIS coloring denotes the visualization of any achromatic substance in order to be visible to the naked eye. Preferably, the intensity of coloring is measured by a photometer. Radioactive radiation of isotopes is measured by scintillation. The process of liquid scintillation involves the detection of beta decay within a sample via capture of beta emissions in a system of organic solvents and solutes referred to as the scintillation cocktail. The beta decay electron emitted by radioactive isotopes such as 3H, 14C, 32P, 33P and 35S in the sample excites the solvent molecule, which in turn transfers the energy to the solute. The energy emission of the solute (the light photon) is converted into an electrical signal by a photo-multiplier tube within a scintillation counter. The cocktail must also act as a solubilizing agent keeping a uniform suspension of the sample. Gamma ray photons often arise as a result of other decay processes (series decay) to rid the newly formed nucleus of excess energy. They have no mass and produce little if any direct ionization by collision along their path. Gamma photons are absorbed for detection and quantization by one or more of three mechanisms: The Compton effect, the photoelectric effect and pair production. A favorable gamma decay isotope of the present invention is 125I. A labeling method is not particularly limited as long as a label is easily detected. A "labeled specific substance" is one that is bound, either covalently through a linker or a chemical bond, or non-covalently through ionic, van der Waals, electrostatic, hydrophobic
interactions or hydrogen bonds, to a label such that the presence of the Eg5 protein may be detected by detecting the presence of the label bound to the biomarker.
The covalent linkage of an anti-Eg5 antibody to an enzyme may be performed by different methods, such as the coupling with glutaraldehyde. Both, the enzyme and the antibody are interlinked with glutaraldehyde via free amino groups, and the by-products of networked enzymes and antibodies are removed. In another method, the enzyme is coupled to the antibody via sugar residues if it is a glycoprotein, such as the peroxidase. The enzyme is oxidized by sodium periodate and directly interlinked with amino groups of the antibody. Other enzyme containing carbohydrates can also be coupled to the antibody in this manner, however sometimes a loss in activity is observed due to the oxidation, e.g. a diminished activity of alkaline phosphatase. Enzyme coupling may also be performed by interlinking the amino groups of the antibody with free thiol groups of an enzyme, such as β-galactosidase, or using a heterobifunctional linker, such as succinimidyl 6-(N-maleimido) hexanoate.
Specific immunological binding of an antibody to a protein can be detected directly or indirectly. Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody. An antibody labeled with iodine-125 (125I) can be used. A chemiluminescence assay using a chemiluminescent antibody specific for the protein marker is suitable for sensitive, non-radioactive detection of protein levels. An antibody labeled with fluorochrome is also suitable. Examples of fluorochromes include, without limitation, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R- phycoerythrin, rhodamine, Texas red, and lissamine. Indirect labels include various enzymes well known in the art, such as horseradish peroxidase (HRP), alkaline
phosphatase (AP), β-galactosidase, urease and the like. A horseradish-peroxidase detection system can be used, for example, with the chromogenic substrate
tetramethylbenzidine (TMB), which yields a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm. An alkaline phosphatase detection system can be used with the chromogenic substrate p-nitrophenyl phosphate, for example, which yields a soluble product readily detectable at 405 nm. Similarly, a β-galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl-β-D-galactopyranosxde
(ONPG), which yields a soluble product detectable at 410 nm. A urease detection system can be used with a substrate, such as urea-bromocresol purple.
In a preferred embodiment of the present invention, the antibodies are labeled with detectable moieties, which include, but are not limited to, radionuclides, fluorescent dyes, e.g. fluorescein, fluorescein isothiocyanate (FITC), Oregon Green™, rhodamine, Texas red, tetrarhodimine isothiocynate (TRITC), Cy3, Cy5, etc., fluorescent markers, e.g. green fluorescent protein (GFP), phycoerythrin, etc., auto-quenched fluorescent compounds that are activated by tumor-associated proteases, enzymes, e.g. luciferase, HRP, AP, etc., nanoparticles, biotin, digoxigenin and the like. In another preferred embodiment of the present invention, the nucleic acids are labeled with digoxigenin, biotin, chemi- luminescence substances, fluorescence dyes, magnetic beads, metallic beads, colloidal particles, electron-dense reagents, enzymes; all of them are well-known in the art, or radioactive isotopes. Preferred isotopes for labeling nucleic acids in the scope of the invention are 3H, 14C, 32P, 33P, 35S, or 125I, more preferred 32P, 33P, or 125I.
A variety of immunoassay techniques, including competitive and non-competitive immunoassays, can be used. The term "immunoassay" encompasses techniques including, without limitation, enzyme immunoassays (EIA), such as enzyme multiplied immunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA), IgM antibody capture ELISA (MAC ELISA) and microparticle enzyme immunoassay (MEIA), furthermore capillary electrophoresis immunoassays (CEIA), radioimmunoassays (RIA); immunoradiometric assays (IRMA), fluorescence polarization immunoassays (FPIA) and chemiluminescence assays (CL). If desired, such immunoassays can be automated. Immunoassays can also be used in conjunction with laser induced fluorescence. Liposome immunoassays, such as flow-injection liposome immunoassays and liposome immunosensors, are also suitable for use in the present invention. In addition, nephelometry assays, in which the formation of protein/antibody complexes results in increased light scatter that is converted to a peak rate signal as a function of the marker concentration, are suitable for use in the methods of the present invention.
In an embodiment of the present invention, antibodies are used as specific substances to Eg5 and the incubation products are detected by the labeling of the antibodies, preferably by ELISA1 RIA, fluoro immunoassay (FIA), soluble particle immune assay (SPIA) or western blotting. In general, all methods for detection include intensive washing steps to separate unbound and/or non-specifically bound antigens from the Eg5/antibody complex. Furthermore, the experimental procedure of any detection method is well-known to those skilled in the art. In a preferred embodiment of the invention, an ELISA is used for the detection of the incubation products. Component of ELISAs are enzymes which are bound to one partner of the immunological reaction. The tracer antigen (analyte derivative) of Eg5 is preferably labeled in the competitive ELISA using a single capture antibody (herein after referred to as primary), whereas the antibody is preferably labeled in the non-competitive ELISA preferably comprising the precipitation of the antigen-antibody complex by a second antibody (herein after referred to as secondary) which is directed to another epitope of Eg5 than the primary antibody. Complexes consisting of antigen and two antibodies are also called sandwich complexes. The detection comprises the subsequent enzymatic conversion of a substrate to a product, preferably a colored product, which is recognized by visual coloring, bioluminescence, fluorescence or the measurement of electrical signals (enzyme electrode). Favorable enzymes for labeling in the present invention are known to the skilled artisan, such as peroxidase (e.g. HRP), chloramphenicol acetyl transferase (CAT), green fluorescent protein (GFP), glutathione S-transferase (GST), luciferase, β- galactosidase and AP.
SPIA utilizes the color change of silver particle as result of agglutination. Neither a secondary antibody nor an indicator reaction are required making it particularly useful in the scope of the present invention. Similarly favorably is the latex agglutination test using antibodies which are bound to colored latex particles.
Another favorite detection method for specific incubation products of the invention is western blotting. Firstly, a gel is mixed and cast, samples previously prepared are loaded onto the gel and fractionated by electrophoresis. The proteins present in the
polyacrylamide gel are blotted onto a nitrocellulose membrane to which antibodies may be applied to detect the specific protein of interest, Eg5. Because the blotting process is not 100 % efficient, residual Eg5 in the gel may be non-selectively stained using Coomassie Blue. Western blotting is simply performed and advantageously when an exact
determination of the concentration is dispensable. There is a distinct number of specific antibodies against Eg5 existing. Antibodies are usually produced in mammal organisms when an immune response is caused by antigens being strange to the organism and having a molecular weight which exceeds 3.000 g/mol. Favorable host species for antibody production comprise goat, rabbit, and mouse. Further polyclonal and monoclonal antibodies can be selected against Eg5 originated form different species and fragments thereof. Popular techniques, such as the hybridoma technology, are well-known to the skilled artisan. The antibodies directed against Eg5 are applied as specific substances in the inventive method.
A signal from the direct or indirect label can be analyzed, for example, using a
spectrophotometer to detect color from a chromogenic substrate, using a radiation counter to detect radiation, such as a gamma counter for detection of 125I, or using a fluorometer to detect fluorescence in the presence of light of a certain wavelength. For detection of enzyme-linked antibodies, a quantitative analysis can be made using a spectrophotometer, such as an EMAX Microplate Reader (Molecular Devices; Menlo Park, CA) in accordance with the manufacturer's instructions. If desired, the assays of the present invention can be automated or performed robotically, and the signal from multiple samples can be detected simultaneously.
The antibodies can be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate (e.g. microtiter wells), pieces of a solid substrate material or membrane {e.g. plastic, nylon, paper) and the like. An assay strip can be prepared by coating the antibody or a plurality of antibodies in an array on a solid support. This strip can then be dipped into the test sample and processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot. The analysis can be carried out in a variety of physical formats. For example, the use of microtiter plates or automation could be used to facilitate the processing of large numbers of test samples. Alternatively, single sample formats could be developed to facilitate diagnosis or prognosis in a timely fashion. Useful physical formats comprise surfaces having a plurality of discrete, addressable locations for the detection of a plurality of different biomarkers. Such formats include protein microarrays or protein chips. In these embodiments, each discrete surface location may comprise antibodies to immobilize one or more protein markers for detection at each location. Surfaces may alternatively comprise one or more discrete particles (e.g. microparticles or nanoparticles) immobilized at discrete locations of a surface, where the microparticles comprise antibodies to immobilize one or more protein markers for detection. Optical images viewed and optionally recorded by a camera or other recording device (e.g. a photodiode and data storage device) are optionally further processed in any of the embodiments herein, e.g. by digitizing the image and storing and analyzing the image on a computer. A variety of commercially available peripheral equipment and software is available for digitizing, storing and analyzing a digitized video or digitized optical image. One conventional system carries light from the specimen field to a cooled charge-coupled device (CCD) camera, in common use in the art. A CCD camera includes an array of picture elements (pixels). The light from the specimen is imaged on the CCD. Particular pixels corresponding to regions of the specimen are sampled to obtain light intensity readings for each position. Multiple pixels are processed in parallel to increase speed. The apparatus and methods of the invention are easily used for viewing any sample, e.g. by fluorescent or dark field microscopic techniques.
In another embodiment of the present invention, analysis of the biomarker can be achieved, for example by high pressure liquid chromatography (HPLC) and/or mass spectrometry, e.g. matrix-assisted laser desorption/ionization mass spectrometry (MALDI- MS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI- TOF/MS), tandem MS, etc. Furthermore, electrochemical processes and probes are also well-established and described in WO 2003/060464 A2, for example, which is incorporated by reference herein.
An amount of signal or change in signal is correlated with an Eg5 expression level in the sample. The amount of emitted signal or change in signal observed in the presence of the inhibitor is indicative for the response of the patient. The change in signal is a change in the signal intensity and/or the signal lifetime. It does not matter whether the change in signal results in a decrease or increase of the signal. Even the loss of any signal is regarded as change in signal. The signal amount or change, respectively, can be then related to the expression level of the biomarker in the sample, i.e. the calibration curve enables the meter-reading of a matching concentration. Preferably, the calibration curve is based on the Lambert-Beer equation if using UV/VIS coloring or luminescence. The expression level of the biomarker is subsequently calculated by considering the molar part of Eg5 within the product complex if present. Preferably, the molar ratio of specific substance and Eg5 is 1 :1 , which is present in antibody/Eg5 complexes for instance, so that the molar concentration of the incubation products corresponds to the molar concentration of Eg5.
Efficacy of Eg5 inhibitory compounds is diagnosed by comparing the expression level of Eg5 in the sample with known Eg5 expression levels. It shall be understood that the known expression levels are statistically proven. This reference value is defined by one ore more of a specific functional or clinical property, and/or a specific, genetic or protein expression profile obtained from a reference patient or reference patient group. The reference value is an expression threshold value which is individually constituted or defined by specific clinical response parameters to be determined or by specific pre-treatment or treatment conditions. Suitable clinical response parameters are the progression free survival time (PFS), overall survival time (OS), partial response (PR), stable response (SR), progressive disease (PD) or combinations thereof. In case of option (i) under step (a) said reference patient or patient group does not express or express less prognostic gene product compared to the candidate patient. It goes without saying that the healthy reference person or patient, respectively, shows no pathological symptoms of thoracic cancer. In case of option (ii) under step (a) said reference patient or patient group does express the prognostic gene product having a similar expression level compared to the candidate patient. The term "similar" denotes any value that is to be considered as functionally equivalent within a certain statistical margin of deviation; the latter shall preferably not exceed 10 %. Herein, the term "functionally" is to be understood to cover the same stage of thoracic cancer. The stage can be easily determined by a skilled medical artisan by means of histological diagnostic findings.
In another preferred embodiment of the screening method of the present invention, the method comprises the further steps of:
(b) (i) exposing in-vivo the patient to the Eg5 inhibitory compound along with taking
another tissue sample from the patient on treatment, and/or
(ii) exposing ex-vivo the tissue samples specified in step (a) and/or another tissue sample, which is taken from the patient, to the Eg5 inhibitory compound,
(c) determining the expression level of the biomarker specified in step (a) in the tissue samples of step (b), and
(d) calculating the differences in expression levels determined in step (a) before starting the treatment of the patient and step (c) on treatment, wherein a decrease in the expression level of the biomarker obtained in step (c) indicates an increased likelihood that the patient responds therapeutically to the treatment compared to the expression level of the biomarker obtained in step (a). The invention also relates to a method for monitoring a thoracic tumor, which is associated with Eg5 expression, wherein an effective amount of at least one Eg5 inhibitory compound or a physiologically acceptable salt thereof is administered to a mammal in need of such treatment and an expression level of Eg5 is determined in a tissue sample taken from the mammal, and wherein a decrease in the expression level of Eg5 indicates an increased likelihood that the mammal responds to the treatment with the compound. The identification of Eg5 described above provides a powerful tool for assessing the progression of a state, condition or treatment. The present invention can be used as a clinical marker to monitor efficacy of an Eg5 inhibitor compound on each patient individually. Specifically, Eg5 can be identified in a patient prior to an event, such as a surgery, the onset of a therapeutic regime, or the completion of a therapeutic regime, to provide a base-line result. This base-line can then be compared with the result obtained using identical methods either during or after such event. This information can be used for both diagnostic and prognostic purposes. The information about the clinical marker can be additionally used to optimize the dosage and the regimen of an active compound by monitoring the induction and accumulation of Eg5 in the subject's biological sample.
Furthermore, the method of the present invention can be used to find a therapeutically effective compound and/or a therapeutically effective amount or regimen for the selected compound, thereby individually selecting and optimizing a therapy for a patient.
Accordingly, the inventive method of monitoring can be employed in human and veterinary medicine. Herein, the compounds can be administered before or following an onset of disease once or several times acting as therapy. The terms "effective amount" or "effective dose" or "dose" are interchangeably used herein and denote an amount of the
pharmaceutical compound having a prophylactically or therapeutically relevant effect on a disease or pathological conditions, i.e. which causes in a tissue, system, animal or human a biological or medical response which is sought or desired, for example, by a researcher or physician. The aforementioned medical products of the inventive use are particularly used for the therapeutic treatment. Monitoring is considered as a kind of treatment, wherein the compounds are preferably administered in distinct intervals, e.g. in order to booster the response and eradicate the pathogens and/or symptoms of thoracic tumors completely. Either the identical compound or different compounds can be applied. The medicament can also be used to reduce the likelihood of developing a thoracic cancerous disease or even prevent the initiation of such a diseases associated with Eg5 activity in advance or to treat the arising and continuing symptoms. In the meaning of the invention, prophylactic treatment is advisable if the subject possesses any preconditions for the aforementioned physiological or pathological conditions, such as a familial disposition, a genetic defect, or a previously passed disease. The prior teaching of the present specification concerning the predictive method is valid and applicable without restrictions to method of monitoring if expedient. Object of the invention is also the use of genetic biomarker eg5 or gene expression product Eg5 thereof for predicting in-vitro the pharmaceutical efficacy and/or clinical response of a patient suffering from an Eg5-expressing thoracic tumor to an Eg5 inhibitory compound intended to be used for the treatment of the tumor. Another object of the invention relates to the use of Eg5 or a nucleic acid encoding Eg5 as biomarker for assessing a thoracic tumor. It is still another object of the invention to use Eg5 or a nucleic acid encoding Eg5 as biomarker for assessing (i) a reduction of likelihood of developing the thoracic tumor and/or progressive growth of the thoracic tumor and/or (ii) a status of cell proliferation of the thoracic tumor and/or inhibition of the thoracic tumor.
The biomarker can be used for monitoring, determining and/or predicting. The different uses can be subsumed under the general term "assessing". It goes without saying that data are monitored over a specific period, while data are determined at a particular time. Both the period and the time can be easily designed by the skilled artisan depending on the experimental trials conducted. Moreover, Eg5 can be used as biomarker for predicting in- vitro the pharmaceutical efficacy and/or clinical response of a mammal suffering from thoracic cancer to an Eg5-inhibiting drug, which is intended to be administered and/or is administered in cancer treatment. The underlying treatment is particularly a first-line treatment, and the inhibitory drug with which the mammalian patient is to be treated, is administered in mono-therapy. In an alternative embodiment of the underlying treatment, said drug is combined with a chemotherapeutic agent, and said patient has developed chemo-refractory cancer. The prior teaching of the present specification concerning the predictive method is valid and applicable without restrictions to any of said uses if expedient.
The susceptibility of a certain cell to treatment with the compounds according to the invention can be determined by testing in vitro. Typically, a culture of the cell is combined with a compound according to the invention at various concentrations for a period which is sufficient to enable the active compound to induce cell death or inhibit migration, usually between approximately one hour and one week. For testing in-vitro, cultivated cells from a biopsy sample can be used. The viable cells remaining after the treatment are then counted. Accordingly, the present invention also relates to a method for screening compounds, which are therapeutic against a thoracic tumor, comprising the steps of.
(1) providing a cellular system or a sample thereof being capable of expressing Eg5, wherein the system is selected from the group of single cells, cell cultures, tissues, organs and mammals, whose expression level of Eg5 is determined, (2) incubating at least a portion of the system with compounds to be screened, and
(3) detecting the compounds with anti-tumor effect by determining the expression level of Eg5, wherein the effect and the expression level are inversely proportional. In the first step (1), a cellular system is provided. The cellular system is defined to be any subject provided that the subject comprises cells. Hence, the cellular system can be selected from the group of single cells, cell cultures, tissues, organs and mammals. The scope of the cellular system also comprises parts of such biological entities, i.e. samples of tissues, organs and mammals. It shall be understood that each cellular system in the aforementioned order represents a sample of the respective following system. Particularly, the cellular sample is taken in-vivo or in-situ from a mammal to be tested. The withdrawal of the cellular sample follows good medical practice. Biological samples may be taken from any kind of biological species, but the sample is especially taken from a laboratory animal or a human, more preferably a rat, mouse or human, most preferably a human. It is preferred to gather a tissue sample by biopsy, especially taken close to the location of ailment. The cellular system may also comprise a biological fluid, wherein the sample of body fluid preferably consists of blood, serum, plasma, saliva or urine. The sample may be purified to remove disturbing substances, such as inhibitors for the formation of hydrogen bonds.
The cell sample refers to any type of primary cells or genetically engineered cells, either in the isolated status, in culture or as cell line, provided that they are capable of expressing Eg5. The engineered cells are capable of expressing the Eg5 protein by transfection with appropriate vectors harboring the corresponding gene or parts thereof. Preferably, the recombinant cells are of eukaryotic origin.
The cell sample is stored, such as frozen, cultivated for a certain period or immediately subjected to step (2). Before incubating it with compounds to be screened, the cell sample is divided into multiple portions. At least two portions are provided; one is used for screening while the other one serves as control. Preferably, the number of portions for screening exceeds the number of control portions. Usually, numerous portions are subjected to a high-throughput screening.
The compounds to be screened in the inventive method are not restricted anyway. The compounds are composed of biological and/or chemical structures capable to interact with a target molecule. Herein, any component of Eg5 signaling shall be considered as "target molecule", which is not limited to the Eg5 protein target, but may also comprise the coding gene or a gene product thereof, or a regulator protein, or a component of a signal transduction pathway comprising said gene or gene products thereof. Consequently, the specific interaction of compounds may involve either the mere targeting or the induction of alterations in cell function, or it may even include both effects simultaneously. In particular, the compounds are selected from the group of nucleic acids, peptides, carbohydrates, polymers, small molecules having a molecular weight between 50 and 1.000 Da and proteins. These compounds are often available in libraries. It is preferred to incubate a single compound within a distinct portion of the cell sample. However, it is also possible to investigate the cooperative effect of compounds by incubating at least two compounds within one portion. A further portion of cells is simultaneously incubated in the absence of the compounds.
The term "incubation" denotes the contacting of the compounds with the cells for a distinct period, which depends on the kind of compounds and/or target. The incubation process also depends on various other parameters, e.g. the cell type and the sensitivity of detection, which optimization follows routine procedures known to those skilled in the art. The incubation procedure can be realized without a chemical conversion or may involve a biochemical reaction. Adding chemical solutions and/or applying physical procedures, e.g. impact of heat, can improve the accessibility of the target structures in the sample. Specific incubation products are formed as result of the incubation.
In step (3), the identification of effective compounds in the meaning of the invention is indirectly performed by determining the presence of Eg5. The determination is performed at a specified moment and correlated to the signal strength at the beginning of the experiment and the positive/negative control. Either the control system is not incubated with the compounds (negative control) or the control system is incubated with a standard compound having no Eg5 inhibiting activity (negative control) or a standard compound having Eg5 inhibiting activity (positive control). Pair-wise comparisons are made between each of the treatments. A pair-wise comparison involves the protein presence data for the given biomarker Eg5 under a given treatment condition compared to the protein presence data for this protein under a second treatment condition. The comparison is performed using suitable statistical technique with the assistance of known and commercially available programs. Efficacy of compounds is diagnosed by comparing the expression level of Eg5 in the sample with known Eg5 expression levels of either non-treated cells and/or treated cells. It shall be understood that the non-treated cells may be the cells of step (1) and the Eg5 expression level thereof. The expression levels represent a statistically proven level or range. Any measured expression level, which differs from the Eg5 level of untreated cells, indicates an abnormality of the tested cell sample, whereas a compound cannot be classified as inhibitor at an Eg5 expression level that is comparable to the concentration level of untreated cells. Using this method, the inventors demonstrated sensitivity to submicromolar or even nanomolar concentrations. The calibration plot reveals that the method can be applied in a dynamic range that spans over a couple of magnitude.
Eg5 inhibition refers to any observable or measurable decrease in the levels of Eg5 expression in comparison to a control system. The measurement of levels of expression may be carried out using any techniques that are capable of protein biomarkers in a biological sample. Examples of these techniques are discussed above. It is another embodiment of the present invention that in the case of modulating the Eg5 expression, Eg5 concentration under-runs at least twice the Eg5 concentration in the control system, preferably at least 10 times, more preferably at least 25 times, most preferably at least 40 times. In a highly preferred embodiment of the invention, Eg5 inhibition results in the disappearance of Eg5. By means of the inventive method, any partial inhibition or even the aforementioned thresholds can be achieved. The inverse proportionality can follow either a linear or a non-linear function.
Among those compounds being revealed to reduce Eg5 surface expression, each or some representatives are selected for further analysis. In a preferred embodiment of the present invention, the screening method involves another step (4), which comprises the detection of the specific interaction of compounds with a eg5 gene or a gene product thereof, or a regulator protein, or a component of a signal transduction pathway comprising said gene or a gene product thereof, provided that the aforementioned interaction results in the decrease or even inhibition of Eg5 activity. It is preferred to detect the specific binding of compounds to the Eg5 protein target. Preferably, the compounds showing the greatest discrepancy to the control are chosen. They are analyzed for specificity to exclude another signal transduction, which is not initiated by the binding to the Eg5 protein target of the invention or associated molecules thereof, and additionally tested for such cross-reactivity in order to prevent adverse reactions or other effects by linked pathways if simultaneous docking to further target structures occurs. Several methods are known in the field of the art for detecting specific and/or mono-specific binding, such as gel shift experiments, Biacore measurements, X-ray structure analysis, competitive binding studies, and the like. In a preferred embodiment of the screening method, the mono-specific binding of substances to the target structures of the invention is detected. The prior teaching of the present specification concerning the predictive and monitoring method in general and the determination of Eg5 expression levels in particular is considered as valid and applicable for the screening method if expedient.
The invention also teaches an embodiment of the method for screening therapeutic compounds against a thoracic tumor, wherein in step (1) mammals are provided, whose expression level of Eg5 is determined in a biological sample taken from the mammals, in step (2) the compounds are administered to the mammals, and in step (3) the compounds with anti-tumor effect are detected by determining the expression level of Eg5 in biological samples taken from the mammals, wherein a decrease in the expression level of Eg5 indicates an increased likelihood that the compounds are therapeutic against the thoracic tumor. The mammal of step (1) is preferably a non-human organism, more preferably a laboratory animal, most preferably species such as mice or rats that may be genetically modified. The mammal suffers from a rare thoracic tumor that is associated with an increased Eg5 level. This expression level of the biomarker Eg5 on protein basis is measured in a biopsy sample, such as a tissue sample from tumor tissue or plasma of said mammalian patient, and set as base-line.
In step (2), it is possible to contact mice or rats, for example, with the compound candidates by injection, infusion, oral or rectal intake. It is preferred to incubate a single compound within a distinct portion of the non-human organisms. Step (2) can also be performed in-vitro by exposing ex-vivo a sample, such as a tissue sample from tumor or plasma of the mammalian patient to said anti-Eg5 drug. A human patient is preferred if performing step (2) in-vitro.
In step (3), the Eg5 expression level is measured again in an identical manner to step (1), and differences in the expression levels measured in step (1) and (3) are calculated. Any difference is inherently based on a change to the initial base-line, thereby confirming an interaction of a defined screening compound with Eg5. The desired increase in levels of Eg5 expression indicates a successful Eg5 inhibition, which is to be correlated to the therapeutic effect. Such a relationship can be established by monitoring the chronological sequence of the effect against a single or multiple dose of the defined screening compound by using the treated mammal only. The effect is determined either by means of qualitative parameters, e.g. decreasing severity of symptoms, or quantitative parameters, e.g. reduced rate of cell growth rate, diminished tumor size, etc. The reference value is set as described above. A decrease in the expression level of the biomarkers Eg5 obtained in step (3) compared to step (1) indicates an increased likelihood that said mammal responds therapeutically to the treatment with said screening compound. This decrease may approach a threshold under the proviso that the comparison is performed with a healthy control mammal (i.e. lacking any proliferative abnormality), which inherently exhibits a down-regulated Eg5 activity. Accordingly, the determination of step (3) is favorably performed in comparison with another mammal showing non-proliferative and/or proliferative effects. The comparative mammal is not exposed to compounds to be screened, but treated in an identical manner to measure Eg5 levels. Step (3) preferably comprises the further sub-steps of:
(31) correlating an amount of signal or change in signal with a Eg5 expression level in the system, and
(3") detecting a level of anti-tumor activity by comparing the Eg5 expression level with another Eg5 expression level in a system of non-TET and/or TET cells.
It is additionally preferred within this embodiment that at least two subjects of a non-human organism suffering of a TET are provided as sample, a subset of them the compounds are administered, and the protein processing pattern is correlated to the symptoms of the disorder in subjects to which compounds have been administered and subjects to which no compounds have been administered. It goes without saying that the basic principles of the general screening method are valid and applicable without restrictions to any special embodiment hereunder, if expedient. With the therapeutic effect, the qualitative level is incorporated into step (3). A
"therapeutically relevant effect" relieves to some extent one or more symptoms of a disease or returns to normality, either partially or completely, one or more physiological or biochemical parameters associated with or causative of the disease or pathological conditions. In addition, the expression "therapeutically effective amount" denotes an amount which, compared with a corresponding subject who has not received this amount, has the following consequence: improved treatment, healing, prevention or elimination of a disease, syndrome, condition, complaint, disorder or side-effects or also the reduction in the advance of a disease, complaint or disorder. The expression "therapeutically effective amount" also encompasses the amounts which are effective for increasing normal physiological function. Testing of several compounds makes the selection of that compound possible that is best suited for the treatment of the mammal subject. The in-vivo dose rate of the chosen compound is advantageously pre-adjusted to the proliferation of the specific cells with regard to their in-vitro data. Therefore, the therapeutic efficacy is remarkably enhanced.
In the scope of the present invention, a method for predicting the likelihood that a patient suffering from TET will respond to the treatment with therapeutic agents by applying the unique biomarker Eg5 is provided for the first time. The present invention teaches the induction and accumulation of Eg5 in TET cells and hence, Eg5 can be explored as biomarker for the evaluation in patients with advanced TET treated with therapeutic agents. Eg5 is not only of predictive value in these rare thoracic cancers, but in addition to the robust biomarker function it also acts as therapeutic target. Without being fixed to the following theory, an Eg5 inhibitor may hypothetically down-regulate the expression of Eg5 on the transcriptional or translational level with the consequence that the protein amount decreases or the protein even disappears from the cell surface and/or the tumor cells are killed. Accordingly, Eg5 has several advantages as target and biomarker for therapeutic TET agents. The presence at tumor cell surface of Eg5 reflects the agent activity over a period, thus truly representing the consequence of Eg5 inhibition and better correlating with efficacy of the testing agent. Suppression of Eg5 is of benefit in the correlation with the in- vivo anti-angiogenic and anti-tumor activity. The analysis of the Eg5 expression levels is also very suitable for large-scale screening tests. The novel marker allows the identification of novel Eg5-inhibitors. Compounds can be identified and evaluated with a specific cellular mechanism of action and additionally, their potential to exert anti-TET effects can be favorably proved. Any method of the invention can be performed in a simple and fast manner. In addition, an appropriate kit is cost-efficiently produced. Eg5 can be easily detected in tumor samples. All detecting substances are characterized by a high affinity, specificity and stability; low manufacturing costs and convenient handling. These features form the basis for a reproducible action, wherein the lack of cross-reactivity is included, and for a reliable and safe interaction with their matching target structures.
All the references cited herein are incorporated by reference in the disclosure of the invention hereby.
It is to be understood that this invention is not limited to the particular methods,
compounds, specific substances and uses described herein, as such matter may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is only defined by the appended claims. As used herein, including the appended claims, singular forms of words such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise. Thus, e.g., reference to "a sample" includes a single or several different samples, whereas reference to "samples" shall be applicable mutatis mutandis, and reference to "a method" includes reference to equivalent steps and methods known to a person of ordinary skill in the art, and so forth. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which this invention belongs. The term "Eg5" shall comprise the eg5 gene and the Eg5 protein, unless the context clearly dictates otherwise. The techniques that are essential according to the invention are described in detail in the specification. Other techniques which are not described in detail correspond to known standard methods that are well known to a person skilled in the art, or the techniques are described in more detail in cited references, patent applications or standard literature.
Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable examples are described below. The following examples are provided by way of illustration and not by way of limitation. Within the examples, standard reagents and buffers that are free from
contaminating activities (whenever practical) are used. The examples are particularly to be construed such that they are not limited to the explicitly demonstrated combinations of features, but the exemplified features may be unrestrictedly combined again if the technical problem of the invention is solved.
Figure 1 shows the percentage of positive and negative Eg5 cases according to histological subtypes.
Figure 2 shows the nuclear staining in TET B3 subtype (a) and micronodular type (b).
EXAMPLE: Eg5 immunostaining in thymic epithelial tumors staged according to the WHO. TETs have shown modest responses to standard chemotherapy. The aim of this study was to identify the presence of biomarkers/ therapeutic targets with potential predictive value in these rare thoracic cancers. Candidate markers were selected for evaluation in patients with advanced TET. 92 TETs (4 of type A, 15 of type AB, 6 of type B1 , 26 of type B2, 18 of type B3, 12 of type C, 5 micronodular TETs, 1 sarcomatoϊd thymoma and 5 carcinoid TETs) including 3 paired primary/secondary tumors (of type B2, B3, C respectively) were centrally reviewed according to the WHO classification. Each tumor was assessed by immunohistochemistry (IHC) with a binary score (0 if negative, 1 if positive). IHC was performed on paraffin embedded tumor sections with the primary antibody 20/Eg5 BD Transduction Lab (1/50). Each tumor was analyzed without intensity score. The staining was considered positive when more than 10% of tumor cells were stained. EG5 analysis revealed nuclear and cytoplasmic staining. The staining was present in all the TET types except of the sarcomatoϊd thymoma, which was negative. It was found 75 % of Eg5 positively in C types, 64 % in AB types, 60 % in micronodular types, 50 % in B1 types, 46 % in B2 types, 41 % in B3 types, 25 % in A types and 25 % in carcinoid TETs. EG5 positively was most frequently observed in the C types (TET patients with the highest risk of recurrence). This exploratory study demonstrates the presence of the molecular Eg5 target in a rare thoracic cancer that might be useful in future treatment options.

Claims

1. An in-vitro method for predicting the likelihood that a patient suffering from a thoracic tumor, who is a candidate for treatment with an Eg5 inhibitory compound, will respond to the treatment with the compound, comprising the step of:
(a) determining an expression level of a prognostic gene or a gene expression product thereof (biomarker), which is eg5 gene or Eg5 protein, in one or more tissue samples taken from the patient, wherein (i) a higher expression level of the eg5 gene or Eg5 protein indicates that the patient is likely to respond to the treatment compared to a reference value of at least one healthy reference person and/or (ii) a similar expression level of the eg5 gene or Eg5 protein indicates that the patient is likely to respond to the treatment compared to a reference value of at least one reference patient suffering from the thoracic tumor in the same stage and susceptible to the treatment.
2. The method according to claim 1 , wherein the patient suffers from a thymic epithelial tumor (TET). 3. The method according to claim 1 or 2, wherein the treatment is with the Eg5 inhibitory compound of formula (I)
Figure imgf000032_0001
in which W denotes CH or N,
R1, R2, R3, independently of one another, denote H, R, A, aryl, heteroaryl, Hal,
-(CY2)n-SA, -(CY2)n-SCF3, -(CY2)n-SCN, -(CY2)π-CF3, -(CY2)n-OCF3, cycloalkyl, -SCH3, -SCN, -CF3, -OCF3, -OA, -(CY2)n-OH, -(CY2)n-CO2R, -(CY2)n-CN, -(CY2Jn-HaI, -(CY2Jn-NR2, (CY2)n-OA, (CY2)n-OCOA, -SCF3,
(CY2)n-CONR2, -(CY2)n-NHCOA, -(CY2Jn-NHSO2A, SF5, Si(CH3)3, CO- (CYz)n-CH3, -(CY2)n-N-pyrrolidone, CH(CH2)nNRCOOR, CHNRCOOR, NCO, CH(CH2)nCOOR, NCOOR, CH(CH2)nOH, N(CH2)nOH, CHNH2, CH(CHz)nNR2, CH(CH2)nNR2, C(OH)R, CHNCOR, CH(CH2)n-aryl, CH(CH2)n-heteroaryl, CH(CH2)nR1, N(CH2)nC00R, CH(CH2)nX(CH2)n- aryl, CH(CH2)nX(CH2)n-heteroaryl, N(CHz)nCONR2, XCONR(CH2)nNR2, N[(CH2)nXCOOR]CO(CH2)n-aryl, N[(CH2)nXR]CO(CH2)n-aryl,
N[(CH2)nXR]CO(CH2)nX-aryl, N[(CH2)nXR]SO2(CH2)n-aryl, N[(CH2)nNRCOOR]CO(CH2)n-aryl, N[(CH2)nNR2]CO(CH2)n-aryl,
N[(CH2)nNR2]CO(CH2)nNR-aryl, N[(CH2)nNR2]SO2(CH2)n-aryl,
N[(CH2)nXR]CO(CH2)n-heteroaryl, N[(CH2)nXR]CO(CH2)nX-heteroaryl, N[(CH2)nXR]S02(CH2)n-heteroaryl, N[(CH2)nNRCOOR]CO(CH2)n- heteroaryl, N[(CH2)nNR2]CO(CH2)n-heteroaryl,
N[(CH2)nNR2]CO(CH2)nNR-heteroaryl, N[(CH2)nNR2]SO2(CH2)n- heteroaryl, O(CH2)nNR2, X(CH2)nNR2, NCO(CH2)nNR2, R1 and R2 together also denote -N-C(CF3)=N-, -N-CR=N-, -N-N=N-,
Y denotes H, A, Hal,
A denotes alkyl or cycloalkyl having 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 C atoms, in which one or more H atoms may be replaced by Hal,
Hal denotes F, Cl, Br or I,
R denotes H or A, in the case of geminal radicals R together also denote -
(CH2)5-, -(CH2)4-, -(CH2)2-X-(CH2)2 or -(CH2)2-Z-(CH2)2,
R4, R5, independently of one another, denote H or an unsubstituted or mono- or poly-OR-, NO2-, HaI-, CF3-, OCF3-, CN-, NR2- or SR-, aryl- or heteroaryl- substituted N-pyrrolidone radical, -X-(CH2J2OR, -X-CO(CH2)nCH3, -X-(CH2)2NR2, S-aryl, O-aryl, CH2Si(CH3)3, or together denote -X(CRz)2-, -X-(CR2),-, -X-(CHCH2OR)(CH2)2-, -X-(CHCH2NR2)(CH2)2-,
-X(CH2)2NR2, -(CR2)3-, -(CR2)4-, -CR=CR-CR=CR-, -XCHQ(CR2)2-, -XCHQCR2-, R-N-(C=X)-N-R, -XC[(CH2)nOR]2CH2CH2-,
X denotes O, S or NR, Q denotes CH2HaI, CHO, CORa, CH2R3, CH2OCOR9, CH2NCOR1, CH2N(R1)2, CH2OR1, CH2OCON(R1)2, CH2OCOOR1, CH2NHCON(R1)2, CH2NHCOOR1,
Ra denotes
Figure imgf000034_0001
Figure imgf000035_0001
OR, NHR2, NR2, NR(CH2)n-aryl, NR(CH2)nOR, COOR, N-pyrrolidone radical, OCOR, NR(CH2)nNR2) N[(CH2)nNR2]CO(CH2)n-aryl, N[(CH2)nNHCOOR]CO-aryl, R1, N[CH2(CH2)nOR]2, NR(CH2)nNCOOR, X(CH2)nX(CH2)nXR, NR(CH2)nX(CH2)nOH, NR(CH2)nO(CH2)nOH, (CH2JnCOOR, O(CO)NR(CH2)nOR, O(CO)(CH2)nNR2, NR(CH2)nNR2, N[(CH2)nNR2]CO(CH2)n-aryl, N[(CH2)nXR]CO(CH2)n-aryl,
N[(CH2)nXR]CO(CH2)n-heteroaryl, N[(CH2)nNR2]CO(CH2)n-heteroaryl, N[(CH2)nNR2]CO(CH2)nR1, N(R)(CH2)nN(R)COOR, XCOO(CH2)nNR2, OSO2A, OSO2CF3, OSO2Ar, OCONR2, OCH2(CH2)nNR2, denotes CH2, X, CHCONH2, CH(CH2)nNRCOOR, CHNRCOOR1 NCO,
CH(CH2)nCOOR, NCOOR1 CH(CH2)nOH, N(CH2)nOH, CHNH2, CH(CH2)nNR2, CH(CH2)nNR2, C(OH)R, CHNCOR, CH(CH2)n-aryl, CH(CH2)n-heteroaryl, CH(CH2)nR1, N(CH2)nCOOR, CH(CH2)nX(CH2)n- aryl, CH(CH2)nX(CH2)n-heteroaryl, N(CH2)nCONR2, XCONR(CH2)nNR2, N[(CH2)nXCOOR]CO(CH2)n-aryl, N[(CH2)nXR]CO(CH2)n-aryl,
N[(CH2)nXR]CO(CH2)nX-aryl, N[(CH2)πXR]SO2(CH2)π-aryl, N[(CH2)nNRCOOR]CO(CH2)n-aryl, N[(CH2)nNR2]CO(CH2)n-aryl, N[(CH2)nNR2]CO(CH2)nNR-aryl, N[(CH2)nNR2]SO2(CH2)n-aryl, N[(CH2)nXR]CO(CH2)n-heteroaryl, N[(CH2)nXR]CO(CH2)nX-heteroaryl, N[(CH2)nXR]S02(CH2)n-heteroaryl, N[(CH2)nNRCOOR]CO(CH2)n- heteroaryl, N[(CH2)nNR2]CO(CH2)n-heteroaryl,
N[(CH2)nNR2]CO(CH2)nNR-heteroaryl, N[(CH2)nNR2]SO2(CH2)n- heteroaryl, O(CH2)nNR2, X(CH2)nNR2, NCO(CH2)nNR2, R6 denotes aryl or heteroaryl, each of which is unsubstituted or mono- or polysubstituted by aryl or heteroaryl, each of which may be substituted by HaI1 NO2, CN, A, OR, OCOR, COR, NR2, CF3, OCF3, OCH(CF3)2, or by Hal, NO2, CN, OR, A, -(CY2)n-0R, -OCOR, -(CY2)n-CO2R, -(CY2)n- CN, -NCOR, -COR or -(CY2)n-NR2,
R7 denotes (C=O)-R, (C=O)-NR2, (C=O)-OR, H or A, aryl denotes unsubstituted or substituted phenyl, naphthyl or biphenyl, heteroaryl denotes a mono- or bicyclic, unsubstituted or substituted aromatic
heterocycle having one or more N, O and/or S atoms, m denotes O, 1 or 2, and n denotes O, 1 , 2, 3, 4, 5, 6 or 7, and solvates, tautomers, salts and stereoisomers thereof, including mixtures thereof in all ratios, where the compounds of the following formulae are excepted:
S-phenyl-S.^a.S.Θ.IOb-hexahydro^H-pyranoβ^-cjquinoline,
7-chloro-5-phenyl-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline,
9-chloro-5-phenyl-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline,
7-methyl-5-phenyl-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline,
5-phenyl-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinolin-7-ol,
9-methoxy-5-phenyl-3,4,4a,5,6, 10b-hexahydro-2H-pyrano[3,2-c]quinoline,
10-chloro-5-phenyl-3,4,4a,5,6, 10b-hexahydro-2H-pyrano[3,2-c]quinoline,
9,10-dichloro-5-phenyl-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline,
8-chloro-5-phenyl-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline,
8,9-dichloro-5-phenyl-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline,
4-phenyl-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline,
8-methoxy-4-phenyl-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline,
6-methyl-4-phenyl-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline,
4-phenyl-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinolin-6-ol,
8-chloro-4-phenyl-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline, 8-nitro-4-phenyl-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline,
1 -(2-phenyl-1 ,2,3,4-tetrahydroquinolin-4-yl)pyrrolidin-2-one,
3,3-dimethyl-2-phenyl-1 ,2,3,4-tetrahydroquinoline,
4-benzyloxy-3,3-dimethyl-2-phenyl-1 ,2,3,4-tetrahydroquinoline,
4-methoxy-2-phenyl-1,2,3,4-tetrahydroquinoline,
2-(4-fluorophenyl)-4-methoxy-1 ,2,3,4-tetrahydroquinoline,
2-furan-2-yl-4-methoxy-1 ,2,3,4-tetrahydroquinoline,
1-(3-pentyl-2-p-tolyl-1 ,2,3,4-tetrahydroquinolin-4-yl)pyrrolidin-2-one,
1-(1-methyl-3-pentyl-2-phenyl-1 ,2,3,4-tetrahydroquinolin-4-yl)pyrrolidin-2-one, 3-methyl-2-phenyl-1 ,2,3,4-tetrahydro(1 ,5]naphthyridine,
4-ethoxy-2-phenyl-1,2,3,4-tetrahydroquinoline,
4-(4-chlorophenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline,
8-chloro-4-phenyl-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline,
5-(4-chlorophenyl)-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline,
5-(4-methoxyphenyl)-3,4,4a,5,6, 10b-hexahydro-2H-pyrano[3,2-c]quinoline,
δ-CS^-dichlorophenyO-S^^a.S.β.iOb-hexahydro^H-pyranotS^-clquinoline,
5-(4-bromophenyl)-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline,
9-methyl-5-phenyl-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline,
9-chloro-5-phenyl-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline,
5-(4-chlorophenyl)-9-isopropyl-3,4,4al5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline.
4. The method according to any of claims 1 to 3, wherein the treatment is with the Eg5 inhibitory compound 1-(2-dimethyIamino-ethyl)-3-((2R,4aS,5R,10bS)-5-phenyl-9- trifluoromethyl-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline-2-ylmethyl)-urea.
5. The method according to any of claims 1 to 4, wherein in step (a) the expression level is determined on DNA/RNA or protein basis, preferably the level of the expressed Eg5 protein encoded by the eg5 gene is determined.
6. The method according to any of claims 1 to 5, wherein in step (a) the tissue samples are taken from the patient before the treatment with the Eg5 inhibitory compound.
7. The method according to any of claims 1 to 5, which comprises the step of exposing in- vivo the patient to the Eg5 inhibitory compound prior to step (a).
8. The method according to any of claims 1 to 6, which comprises the further steps of:
(b) (i) exposing in-vivo the patient to the Eg5 inhibitory compound along with taking another tissue sample from the patient on treatment, and/or
(ii) exposing ex-vivo the tissue samples specified in step (a) and/or another tissue sample, which is taken from the patient, to the Eg5 inhibitory compound,
(c) determining the expression level of the biomarker specified in step (a) in the tissue samples of step (b), and
(d) calculating the differences in expression levels determined in step (a) before
starting the treatment of the patient and step (c) on treatment, wherein a decrease in the expression level of the biomarker obtained in step (c) indicates an increased likelihood that the patient responds therapeutically to the treatment compared to the expression level of the biomarker obtained in step (a).
9. The method according to any of claims 1 to 8, wherein the tissue samples are taken from tumor tissue or plasma of the patient by biopsy.
10. A method for monitoring a thoracic tumor, which is associated with Eg5 expression, wherein an effective amount of at least one Eg5 inhibitory compound or a
physiologically acceptable salt thereof is administered to a mammal in need of such treatment and an expression level of Eg5 is determined in a tissue sample taken from the mammal, and wherein a decrease in the expression level of Eg5 indicates an increased likelihood that the mammal responds to the treatment with the compound.
1 1. Use of genetic biomarker eg5 or gene expression product Eg5 thereof for predicting in- vitro the pharmaceutical efficacy and/or clinical response of a patient suffering from an
Eg5-expressing thoracic tumor to an Eg5 inhibitory compound intended to be used for the treatment of the tumor.
12. Use of Eg5 or a nucleic acid encoding Eg5 as biomarker for assessing a thoracic tumor.
13. Use according to claim 12 for assessing (i) a reduction of likelihood of developing the thoracic tumor and/or progressive growth of the thoracic tumor and/or (ii) cell proliferation of the thoracic tumor and/or inhibition of the thoracic tumor.
14. A method for screening compounds, which are therapeutic against a thoracic tumor, comprising the steps of:
(1) providing a cellular system or a sample thereof being capable of expressing Eg5, wherein the system is selected from the group of single cells, cell cultures, tissues, organs and mammals, whose expression level of Eg5 is determined,
(2) incubating at least a portion of the system with compounds to be screened, and
(3) detecting the compounds with anti-tumor effect by determining the expression level of Eg5, wherein the effect and the expression level are inversely proportional.
15. The method according to claim 14, wherein in step (1) mammals, preferably laboratory mammals, are provided, whose expression level of Eg5 is determined in a biological sample taken from the mammals, in step (2) the compounds are administered to the mammals, and in step (3) the compounds with anti-tumor effect are detected by determining the expression level of Eg5 in biological samples taken from the mammals, wherein a decrease in the expression level of Eg5 indicates an increased likelihood that the compounds are therapeutic against the thoracic tumor.
PCT/EP2010/003904 2009-07-24 2010-06-25 Eg5 as biomarker in rare thoracic cancer WO2011009523A1 (en)

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