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Home , Anazocine, Butinazocine, Carbazocine, Cogazocine, Etazocine, Fluorophen

(19) TZZ _ __T

(11) EP 2 716 291 A1

(12) EUROPEAN PATENT APPLICATION

(43) Date of publication: (51) Int Cl.: 09.04.2014 Bulletin 2014/15 A61K 31/485 (2006.01) A61P 35/00 (2006.01)

(21) Application number: 12007179.0

(22) Date of filing: 17.10.2012

(84) Designated Contracting States: (72) Inventors: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB • Friesen, Claudia GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO 89075 Ulm (DE) PL PT RO RS SE SI SK SM TR • Miltner, Erich Designated Extension States: 89075 Ulm (DE) BA ME (74) Representative: Jostarndt, Hans-Dieter (30) Priority: 08.10.2012 EP 12006946 Jostarndt Patentanwalts-AG Brüsseler Ring 51 (71) Applicant: Universität Ulm 52074 Aachen (DE) 89081 Ulm (DE)

(54) Combination of and anticancer for cancer treatment

(57) The invention relates to novel strategies for the an receptor and an anticancer compound. treatment of cancer patients based on a combination of EP 2 716 291 A1

Printed by Jouve, 75001 PARIS (FR) 1 EP 2 716 291 A1 2

Description addition to surgery and or chemotherapy but for certain types of cancer such as early head and neck cancer may [0001] The invention relates to novel strategies for the be used alone. For painful bone metastasis it has been treatment of cancer patients based on a combination of found to be effective in about 70% of people. an agonist and an anticancer compound. 5 [0009] Despite the numerous therapeutic strategies there are still tumours which cannot be effectively treated Background of the invention with the current treatment options. In addition, the effec- tiveness of radiation- and chemotherapy is often limited [0002] Cancer can be defined as an abnormal growth by toxicity to other tissues in the body. Furthermore, an- of tissue characterized by a loss of cellular differentiation. 10 ticancer therapies are frequently ineffective due to resist- This term encompasses a large group of diseases in ance of the tumour cells to radio- and/or chemotherapy. which there is an invasive spread of undifferentiated cells [0010] Thus in oncology there is a great need for novel from a primary site to other parts of the body where further strategies, which render cancer treatments more effec- undifferentiated cellular replication occurs, which even- tive. In particular, it is the objective of the present inven- tually interferes the normal functioning of tissues and or- 15 tion to provide novel means for treating cancer patients. gans. [0003] Cancers are primarily an environmental dis- Summary of the invention ease with 90-95% of cases attributed to environmental factors and 5-10% due to genetics. Environmental, as [0011] This objective is solved by using a combination used by cancer researchers, means any cause that is 20 of opioid receptor and anticancer drugs for the not inherited genetically, not merely pollution. Common treatment of cancer wherein this combination is given in environmental factors that contribute to cancer death in- a specific administration scheme according to claim 1 of clude tobacco (25-30%), diet and obesity (30-35%), in- the invention. fections (15-20%), radiation (both ionizing and non-ion- [0012] In a first aspect the invention relates to a com- izing, up to 10%), stress, lack of physical activity, and 25 bination of an opioid receptor agonist and at least one environmental pollutants. anticancer agent for use in the treatment of cancer, [0004] With more than 3 million new cases and 1.7 wherein million deaths each year, cancer represents the second most important cause of death and morbidity in Europe. (a) said opioid receptor agonist is administered to a Ona global scale, canceraccounted for 7.4 milliondeaths 30 patient in one or more doses to establish a thera- (around 13% of the total) in 2004. peutically effective plasma level for a period of at [0005] Although more than 40% of cancer deaths can least one week,and be prevented, cancer is a leading cause of death, causing (b) at least one anticancer agent selected from the 20% of the total in the European Region. Noticeably, Eu- group consisting of chemotherapeutical agents, cy- rope comprising only one eighth of the total world popu- 35 totoxic agents, cytostatic agents, immunotoxic lation but has around one quarter of the global total of agents and/or radiotherapy is administered to estab- cancer cases: some 3.2 million new patients per year. lish a period with a therapeutically effective plasma [0006] The most common forms of cancer were pros- level, and tate, colorectal, breast, leukemia and lung cancer. The (c) said periods of a) and b) overlap. risk of getting cancer before the age of 75 years is 26.5%, 40 or around one in four. However, because the population [0013] This combination therapy is based on the un- of Europe is ageing, the rate of new cases of cancer is expected finding that opioid receptor agonists together also expected to increase. with anticancer agents kill cancer cells more effectively. [0007] Each cancer is characterized by the site, nature, Furthermore, the inventors could show that the interac- and clinical cause of undifferentiated cellular prolifera- 45 tion between opioid receptor agonists and anticancer tion, whereby the underlying mechanism for the initiation agents represents a self-reinforcing feedback loop as il- of cancer is not completely understood. lustrated by Figure 26. In the first path of this loop opioid [0008] Cancer is usually treated with chemotherapy, receptor agonists enhance the cellular uptake and inhibit radiation therapy and surgery. Chemotherapy in addition the efflux of anticancer drugs. In the second path of said to surgery has proven useful in a number of different can- 50 loop the accumulating anticancer drugs lead to an in- cer types including: breast cancer, colorectal cancer, creased expression of opioid receptors on the surface of pancreatic cancer, osteogenic sarcoma, testicular can- the cancer cell. Hence, both the opioid receptor agonist cer, ovarian cancer, and certain lung cancers. Radiation and the anticancer agent can exert their cytotoxic poten- therapy involves the use of ionizing radiation in an at- tial to a higher extent. tempt to either cure or improve the symptoms of cancer. 55 [0014] Furthermore the invention is based on the un- It is used in about half of all cases and the radiation can expected finding that the amount of opioid receptor ex- be from either internal sources in the form of brachyther- pressed on the cell surface of cancer cells is varying apy or external sources. Radiation is typically used in among the different cancer types and also exhibiting in-

2 3 EP 2 716 291 A1 4 ter-individual differences and that this surface-associat- included in this invention. ed opioid receptor expression can be increased by anti- [0018] The term "opioid receptor agonist" as used cancer agents. For example doxorubicin, idarubicin, epi- herein comprises full agonists as well as mixed ago- rubicin, daunorubicin, carbopaltin, oxaliplatin, cisplatin, nists/antagonists or partial agonists such as buprenor- etoposide, methotrexate, cytarabine, teniposide, rituxi- 5 phine. mab fludarabine, are able to induce an increasing of the [0019] The group of opioids includes natural number of opioid receptors which are expressed on the such as alkaloids like or dihydrocodein, as well cell surface of cancer cells. as semi-synthetic opiates, derived from the natural opi- [0015] By extensive in vitro and in vivo experiments it ates (e.g. or ), or fully syn- could be shown that different cancer types can be sub- 10 thetic opioids, such as or . It also jected to the combination therapy of the invention. Fur- includes endogenous opioid peptides, which may be pro- thermore also different anticancer drugs and different duced naturally in the body as endorphins, opioids proved to be active in the above described feed- or but which can also be synthesized. back loop. [0020] As used herein the term "anticancer " en- [0016] Therefore, the combination therapy of opioid re- 15 compasses all chemical or physical interventions that are ceptor agonist and anticancer drug according to the in- used for the treatment of cancer. It therefore includes vention can improve cancer therapy by one or more of chemotherapeutical agents such as cytotoxic agents or the following ways: immunotoxic agents but also radioactively labelled anti- bodies, peptides and chemical substances, which might • Due to the upregulation of opioid receptors, former 20 emit , beta and gamma rays as well as elec- opioid insensitive cancer types could be subjected trons..The radiotherapy further includes photons of suf- to an opioid receptor agonist therapy. ficiently high energy, charged particles such as electrons, • Due to the opioid-receptor-agonist-induced intracel- positrons, muons, protons, alpha particles, and heavy lular accumulation (by either an increased uptake of atomic nuclei from accelerators, but also neutrons and anticancer drugs or a reduced eflux or a combination 25 gamma rays. of both) of anticancer drugs the efficacy of the treat- [0021] The term "therapeutically effective plasma lev- ment is enhanced. el" is defined as a plasma level that is between the plasma • This could lead to therapy of cancer types which are level of the drug that causes a lethal effect and the min- non-treatable or not effectively treatable by conven- imum plasma level that causes a therapeutic effect. In tional therapeutic anticancer approaches. 30 the context of the invention the therapeutic effect of the • Furthermore, this might allow a dose reduction for opioid receptor agonist is given by the increase in cellular the anticancer drugs enhancing the safety and pa- uptake and/or the inhibition of the cellular efflux of the tient compliance of the chemotherapy. co-administered anticancer drug and/or the induction of • Finally, also resistant cancer cells could be re-sen- cell death by e.g. apoptosis, necrosis, mitotic catastrophe sitized for an anticancer treatment. 35 and autophagy. In the context of the invention the ther- • In addition, the numerous opioids and numerous an- apeutic effect of the anticancer drug is given by its ability ticancer drugs on the market open up the way for to kill cancer cells and/or to induce the opioid receptor new drug combinations which might represent im- expression on the cancer cells. proved treatment due to increased efficacy and/or [0022] There are two ways to look at the results of can- safety. 40 cer treatment. One common way is the measurement of cell death (increasing data means more cells are dead). [0017] In the context of the present invention the term The other way is to measure the viability of cells (de- "opioid receptor agonist" is defined as a chemical heter- creasing data means that less living cells are present or ogeneous group of natural, synthetic or semi-synthetic have lost their proliferation potential). substances, working agonistic at the same type of recep- 45 [0023] As used in the context of the present invention tor, the so called opioid receptor. According to the anal- the words "treat," "treating" or "treatment" refer to using gesia and side effect profile five types of opioid receptors, the combination of the present invention or any compo- the p-receptor (ligand = morphine), the κ[kappa]-receptor sition comprising them to either prophylactically prevent (ligand = ), the delta-receptor (ligand = deltor- a cancer, or to mitigate, ameliorate or stop cancer. They phine II), the σ[sigma]-receptor (ligand = SKF 10081), as 50 encompass either curing or healing as well as mitigation, well as the later-identified ORL1 -receptor (ligand = no- remission or prevention, unless otherwise explicitly men- ciceptin) are known. Corresponding to other receptor tioned. Also, as used herein, the word "patient" refers to systems, binding studies as well as functional investiga- a mammal, including a human. tions indicate that subtypes of opioid receptors exist. [0024] According to the invention the treatment specif- Within the m- and δi-receptor type 2 subtypes, them -1 55 ically refers to the inhibition of cancer cell proliferation and m-2 and δ -1 and δ -2 have been described. The κ- and/or growth. This activity can include e.g. cytostatic or receptor contains an additional κ-3 subtype. Especially cytotoxic activity as well arresting growth of cells and/or in regards to the m-opioid receptor its two subtypes are tumours. Cancer cell proliferation is the result of the in-

3 5 EP 2 716 291 A1 6 hibition of cell division. In particular opioid receptor ago- cancer agent and said opioid receptor agonist are ad- nists induce cell death in tumours. Cell death in the con- ministered simultaneously or successively. text of the invention includes all types of cells death. This [0030] In a preferred embodiment of the invention the can include necrotic as well as apoptotic cell death or periods of the therapeutically effective plasma levels of autophagy. In one embodiment of the invention the cell 5 the opioid receptor agonist and the anticancer agent, re- death is induced by the activation of the caspases-de- spectively overlap predominantly. pendent or caspases-independent pathway. However, [0031] In a further preferred embodiment of the inven- opioid receptor agonists can induce cell death via various tion the period of the therapeutically effective plasma lev- pathways. In a preferred embodiment of the invention, els of the anticancer agent is completely within the re- opioid receptor agonists induce apoptosis in cancer cells. 10 spective period of the opioid receptor agonist. [0025] As used herein, the term "cancer" which is syn- [0032] When administering two or more anticancer onymously used to the term "neoplasm" refers to diseas- agents the respective period for which a partial, predom- es in which abnormal cells divide without control and are inant or complete overlap is claimed, is given by the com- able to invade other tissues. Cancer cells can spread to bined periods of the two or more anticancer agents. other parts of the body through the blood and lymph sys- 15 [0033] In a further embodiment of the invention the opi- tems. oid receptor agonist is given in a way that the patient develops a habituation against said opioid receptor ag- • The terms "conventional therapy" and "conventional onist. It is thus preferable to wait with the anticancer treat- therapy regimen" in the context of the present inven- ment until the habituation period has begun or even tion are defined as the treatment programs (concern- 20 reaches a plateau. The habituation can be a result of a ing dose, iteration-time and duration) which are rec- decreased drug efficacy and/or a decrease in side effects ommended as therapeutic guidelines of associa- such as respiratory depression. Side effects of opioid re- tions, federations like Deutsche Krebshilfe, Deut- ceptor agonist are hypotension, respiratory depression, sche Krebsgesellschaft, Nationacl Cancer Institute vomiting, constipation, dizziness, sedation, and (NCI), National Comprehensive Cancer Network25 cardiac effects. This side effects have to be taken in ac- (NCCN) and respective health or cancer organiza- count for determine the therapy scheme with the opiod tions which could be private, non-governmental or receptor agonist and the cancer agent. This means that federal organizations. This also includes the treat- the opioid receptor agonist is given at a starting dose on ment programs for a (preferably single) anticancer a very low level i.e. 1% of the therapeutical dose and agent as prescribed by the manufacturer or distrib- 30 then increasing the dose depending to the guidelines of utor of the anticancer agent which are disclosed in the opioid receptor agonist known by a skilled person the respective instruction leaflets of the anticancer and published by the manufacture or distributor of the agents. opioid receptor agonist in an adequate time up to the therapeutic level which is required for the combination of [0026] The term "conventional therapy time" in the con- 35 anticancer agent and opioid receptor agonist. text of the present invention is defined as the time in a [0034] In a preferred embodiment of the invention the conventional therapy where an anticancer agent is ap- administration regimen und thus the period within a ther- plied to a patient without an opioid receptor agonist ac- apeutically effective plasma level of the anticancer agent cording the invention. The therapy time starts with the is defined by the conventional therapy regimen. first application of the anticancer agent, and may include 40 [0035] In a further aspect of the invention the patient iteration-periods which are specific for cancer and anti- treated with the combination according to the invention cancer agent (for example application of a dose two times has received a pre-treatment comprising an anticancer a day for a week than a pause of three weeks and then agent. again application of a dose two times a day for a week [0036] In a more preferred embodiment the pre-treat- followed by a pause of three weeks), up to time point, at 45 ment with the anticancer agent has been discontinued which the anticancer agent is below the therapeutic plas- or even terminated. ma level of the patient. [0037] In a further preferred embodiment the pre-treat- [0027] Cancer and its different types in the context of ment has been terminated due to resistance against the the present invention can be classified by the ICD-O anticancer treatment. Standard which is a specialised classification of the ICD- 50 [0038] In a preferred embodiment of the invention the 10 Standard Classes C00-C97 and DOO-D36. Alterna- period with a therapeutically effective plasma level of the tively, the classification of Boecker et al. 2008 in chapter anticancer agent lasts for at least 1 day, preferably 3 6 (Pathologie, Elsevier, Urban &Fischer,p 167-218) can days, and more preferably for at least 5 days. be used. [0039] In one embodiment of the invention the period [0028] In a further embodiment of the invention said 55 with a therapeutically effective plasma level for the opioid opioid receptor agonist is capable of inhibiting cell prolif- receptor agonist is at least two weeks, more preferably eration. four weeks and even more preferably represents a chron- [0029] In one embodiment of the invention said anti- ic treatment.

4 7 EP 2 716 291 A1 8

[0040] Within the context of the present invention the term "chronic treatment" is defined as a opioid receptor agonist treatment with an administration period above four weeks, which preferably lasts over several months. In a further embodiment this chronic treatment differs 5 from the conventional therapy regimen as prescribed or known to the person skilled in art. or is published in ther- apeutic guidelines of associations or federations like Deutsche Krebshilfe or Deutsche Krebsgesellschaft; NCCN, NCI or similar health or cancer organizations or 10 the guidelines of producer or distributer of drugs which are used for treatment of cancer [0041] Within the context of the present invention the wherein R 1 is an aliphatic ketone, a 3-acetoxypropyl res- use of at least one anticancer agent refers to the use of idue, a cyano group, or a 1-pyrrolidino-methylketone, 15 one or more anticancer agents to be given in combination -(C=O)C2H5, R2 and R3 are CH3 or together forming a with the opioid receptor agonist according the invention. heterocyle, preferably a morpholino group, and R4 is H Thus,the combinationincludes the useof one, two, three, or an alkyl residue, being preferably CH 3. four, five or even more anticancer agents. [0046] A non-limited list of examples for compounds [0042] Generally, it is known, that apoptosis can be of the group includes methadone, normeth- induced via two main biochemical pathways. The "death 20 adone, , , acetylmeth- receptor pathway" (or extrinsic pathway) includes the adol, , levoacetylmethadol, premeth- TNF-receptor-induced (tumour necrosis factor) model adone, racemoramid, , dextropropoxy- and the Fas-receptor-induced model (the Fas-receptor phene, , benzitramide, , lopera- is also known as Apo-1 or CD95). Bindings to these re- mide, themalon (which represents a 3,3-dithiophenylpro- ceptors result in the formation of death-inducing signal- 25 pylamine) and levomoramid. ling pathways in the cell, including the activation of cas- [0047] All these opioids can be used as salts. The ra- pase-8. The "mitochondrial pathway" (or intrinsic path- cemic form of D-/L-methadone is preferably provided in way) involves the release of cytochrom c from mitochon- form of a hydrochloride. In a preferred embodiment of dria, binding of Apaf-1 and activation of procaspase-9. the invention, the opioid methadone induces apoptosis Several regulators are known to activate or deactivate 30 in cancer cells via the mitochondrial pathway. the apoptosis pathways, such as the pro-apoptotic pro- [0048] In one embodiment of the invention the opioid teins Bax and Bak or the anti-apoptotic proteins Bcl-2, receptor agonist is selected from the list consisting of Bcl-XL or XIAP. compounds of the methadone group such as D/L-meth- [0043] In one embodiment of the invention the opioid adone, D-methadone, L-methadone, , receptor agonists induce apoptosis by cleavage of cas- 35 fentanyl derivatives such as fentanyl, sufentanyl, pase-3 and poly(ADP-ribose) polymerase (PARP) in the lofenantil, , , and car- tumour cell, and/or cleavage of caspase-9 and down reg- fentanyl; morphinane compounds such as morphine, co- ulation of X-linked inhibitor of apoptosis protein (XIAP), deine,heroine, dextrallorphane,, dex- and/or down regulation of the B-cell lymphoma-extra trophanol, , levalorphanol, , lev- 40 large protein (Bcl-XL). ofurethylnormorphanol, levomethorphane, levophena- [0044] According to a preferred embodiment of the in- cylmorphane, , methorphane, morphanol, vention, the opioid receptor agonist is a member of the , , and , benzomor- methadone group, comprising D-/L-methadone, lev- phane derivatives such as 5,9-DEHB, , anazo- omethadone, and piritramide. cine, , , , cogazo- [0045] In the context of the present invention the term 45 cine, , , , , "methadone group" relates to opioids which are deriva- ethylketocyclazocine, , , ibazoc- tives of 3,3-diphenylpropylamine. These compounds ine, ketazocine, ketocyclazozine, , moxazo- possess the 3,3-diphenylamine core structure as shown cine, , , , thiazoc- by the following formula (I): ine, , and 8-CAC; endogenous opi- 50 oids such as endorphins (which can be alpha-, beta-, gamma- or delta-endorphins), enkephalins such as Met- , Leu-enkephalin and methorphamid, dynor- phins such as A, or alpha-neoen- dorphin, , , , beta- 55 caomorphine-5, DALAMID, DADLE, DADL DSLT, DS- LET, DTLET, DAGO, DAMGO, DALCE, DAMME, DAL- DA, DPDPE, FK 33-824, [D-Met2,Pro5]enkephalin- amide, , and endomorphines such as endomo-

5 9 EP 2 716 291 A1 10 prhin-1 and endomoprhin-2; furthermore all fragments can be a radioactively labeled chemical compound, pep- derived from the protein (POMC) tide, protein or monoclonal antibody, wherein the radio- such as beta-, beta-LPH-[61-64], beta-active label could emit alpha, beta or gamma rays and LPH-[61-65]-NH2, (Met(O)65)-beta-LPH-[61-65], beta- also ionizing particles. LPH-[61-69], and beta-LPH-[61-69]; 4-phenylpiperidine 5 [0053] In a preferred embodiment of the invention the derivatives such as , , , anticancer agent is methotrexate, cytarabine, carbopla- , , , alpha-prodin, tri- tine, oxaliplatine, etoposide, vincristine, fludarabine es- meperidine, including 4-phenylpyrrolidine derivatives pecially cisplatin, doxorubicin, anthracyclin, idarubicin, such as and 4-phenylazepanderivates such as daunorubicin, epirubicin, or alpha-, beta-, or gamma ir- ; cyclohexane derivatives such as , U- 10 radiation. 50488, and . [0054] When treating a cancer entity for which an in- [0049] In a preferred embodiment of the invention the duction of the opioid receptor is desired the patient is opioid receptor agonists of the invention are capable of preferably treated with an anticancer agent selected from inhibiting cell proliferation. the group consisting doxorubicin, idarubicin, epirubicin, [0050] In a preferred embodiment the methadone,15 daunorubicin, carboplatin, oxaliplatin, cisplatin, etopo- preferably the D,L-methadone and most preferably the side, methotrexate, cytarabine, tenisposide, retuximab. hydrochloride form of D,L-methadone is given to the pa- [0055] In one embodiment of the invention the patient tient in particular to yield a plasma level which is between which is treated with the combination of the invention 0.05 mg/mL and 3 mg/mL. suffers from a neoplasm as classified according the In- [0051] In one embodiment of the invention the antican- 20 ternational statistical classification of Diseases and re- cer agent is selected from the list consisting of interca- lated health problems 10th Revision (ICD-10), wherein lating substances such as anthracycline, doxorubicin, the neoplasm is from the group consisting of malignant idarubizin, epirubizin,and daunorubizin; topoisomerase neoplasms of classes C00 to C97, in situ neoplasms of inhibitors such as irinotecan, topotecan, camptothecin, classes D00 to D09, benign neoplasms of classes D10 lamellarin D, etoposide, teniposide, mitoxantrone, amsa- 25 to D36, and neoplasms of uncertain or unknown behav- crine, ellipticines and aurintricarboxylic acid; nitrosourea iour of classes D37 to D48. compounds such as carmustine (BCNU), lomustine (CC- [0056] The classes are defined as follows: (C00) Ma- NU), streptozocin; nitrogen mustards such as cyclophos- lignant neoplasm of lip, (C01) Malignant neoplasm of phamide, mechlorethamine, uramustine, bendamustine, base of tongue, (C02) Malignant neoplasm of other and melphalan, chlorambucil, mafosfamide, trofosfamid and 30 unspecified parts of tongue, (C03) Malignant neoplasm ifosfamide; alkyl sulfonates such as busulfan and treo- of gum, (C04) Malignant neoplasm of floor of mouth, sulfan; alkylating agents such as procarbazin, dacar- (C05) Malignant neoplasm of palate, (C06) Malignant ne- bazin, temozolomid and thiotepa; platinum analogues oplasm of other and unspecified parts of mouth, (C07) such as cisplatin, carboplatin, nedaplatin, oxaliplatin, sat- Malignant neoplasm of parotid gland, (C08) Malignant raplatin, and triplatin tetranitrate; microtubule disruptive 35 neoplasm of other and unspecified major salivary glands, drugs such as vinblastine, colcemid and nocodazole; an- (C09) Malignant neoplasm of tonsil, (C10) Malignant ne- tifolates like methotrexate, aminopterin, dichlorometh- oplasm of oropharynx, (C11) Malignant neoplasm of na- otrexat, pemetrexed, raltitrexed and pralatrexate; purine sopharynx, (C12) Malignant neoplasm of piriform sinus, analogues like azathioprine, mercaptopurine, thiogua- (C13) Malignant neoplasm of hypopharynx, (C14) Malig- nine, fludarabine, fludarabine phosphate, pentostatin40 nant neoplasm of other and ill-defined sites in the lip, oral and cladribine; pyrimidine analogues like 5-fluorouracil, cavity and pharynx, (C15) Malignant neoplasm of Es- floxuridine, cytarabine, 6-azauracil, gemcitabine; steroid ophagus, (C16) Malignant neoplasm of Stomach, (C17) hormones like gestagene, androgene, glucocorticoids, Malignant neoplasms of small intestine, (C18) Malignant dexamethasone, prednisolone, and prednisone; anti- neoplasm of colon, (C19) Malignant neoplasm of rectos- cancer peptides including radioactively labeled paptides 45 igmoid junction, (C20) Malignant neoplasm of rectum, and peptide-drug conjugates; anti-cancer antibodies in- (C21) Malignant neoplasms of anus and anal canal, cluding radioactively-labelled antibodies and antibody- (C22) Malignant neoplasms of liver and intrahepatic bile drug conjugates such as bevacizumab, cetuximab, pan- ducts, (C23) Malignant neoplasm of gallbladder, (C24) itumumab, rituximab, ipilimumab, alemtuzumab, ofatu- Malignant neoplasm of other and unspecified parts of mumab, gemtuzumab-ozogamicin, brentuximab vedot- 50 biliary tract, (C25) Malignant neoplasm of pancreas, in, 90Y-ibritumomab tiuxetan, 131I-tositumomab, or tras- (C26) Malignant neoplasms of other and ill-defined Di- tuzumab, alpha, beta or gamma irradiation; including par- gestive Organs, (C30) Malignant neoplasm of nasal cav- ticle radiation. ity and middle ear, (C31) Malignant neoplasm of acces- The above listed anticancer agents comprise also mod- sory sinuses, (C32) Malignant neoplasm of larynx, (C33) ifications such as PEGylation and formulations such as 55 Malignant neoplasm of trachea, (C34) Malignant neo- the use of liposomes (i.e. PEGylated liposomal doxoru- plasm of bronchus and lung, (C37) Malignant neoplasm bicin). of thymus, (C38) Malignant neoplasm of heart, mediasti- [0052] In a further embodiment the anticancer agent num and pleura, (C39) Malignant neoplasms of other and

6 11 EP 2 716 291 A1 12 ill-defined sites in respiratory system and intrathoracic major salivary glands, (D12) Benign neoplasm of colon, organs, (C40-C41) Malignant neoplasms, bone and ar- rectum, anus and anal canal, (D13) Benign neoplasm of ticular cartilage, (C43) Malignant melanoma of Skin, other and ill-defined parts of digestive system, (D14) Be- (C44) Other malignant neoplasms of skin, (C45) Mes- nign neoplasm of middle ear and respiratory system, othelioma, (C46) Kaposi’s Sarcoma, (C47) Malignant ne- 5 (D15)Benign neoplasm of other and unspecified intratho- oplasm of peripheral nerves and autonomic nervous sys- racic organs, (D16) Benign neoplasm of bone and artic- tem, (C48) Malignant neoplasm of retroperitoneum and ular cartilage, (D17) Benign lipomatous neoplasm, (D18) peritoneum, (C49) Malignant neoplasm of other connec- Haemangioma and lymphangioma, any site, (D19) Be- tive and soft tissue, (C50) Malignant neoplasm of breast, nign neoplasm of mesothelial tissue, (D20) Benign neo- (C51) Malignant neoplasm of vulva, (C52) Malignant ne- 10 plasm of soft tissue of retroperitoneum and peritoneum, oplasm of vagina, (C53) Malignant neoplasm of cervix (D21) Other benign neoplasms of connective and other uteri, (C54) Malignant neoplasm of corpus uteri, (C55) soft tissue, (D22) Melanocytic naevi, (D23) Other benign Malignant neoplasm of uterus, part unspecified, (C56) neoplasms of skin, (D24) Benign neoplasm of breast, Malignant neoplasm of ovary, (C57) Malignant neo- (D25) Leiomyoma of uterus, (D26) Other benign neo- plasms of other and unspecified female and genital or- 15 plasms of uterus, (D27) Benign neoplasm of ovary, (D28) gans, (C58) Malignant neoplasm of placenta, (C60) Ma- Benign neoplasm of other and unspecified female genital lignant neoplasm of penis, (C61) Malignant neoplasm of organs, (D29) Benign neoplasm of male genital organs, prostate, (C62) Malignant neoplasm of testis, (C63) Ma- (D30) Benign neoplasm of urinary organs, (D31) Benign lignant neoplasm of other and unspecified male genital neoplasm of eye and adnexa, (D32) Benign neoplasm organs, (C64) Malignant neoplasm of kidney, except re- 20 of meninges, (D33) Benign neoplasm of brain and other nal pelvis, (C65) Malignant neoplasm of renal pelvis, parts of central nervous system, (D34) Benign neoplasm C66) Malignant neoplasm of ureter, (C67) Malignant ne- of thyroid gland, (D35) Benign neoplasm of other and oplasm of bladder, (C68) Malignant neoplasm of other unspecified endocrine glands, (D36) Benign neoplasm and unspecified urinary organs, (C69) Malignant neo- of other and unspecified sites, (D37) Neoplasm of uncer- plasms of eye and adnexa, (C70) Malignant neoplasm 25 tain or unknown behaviour of oral cavity and digestive of meninges, (C71) Malignant neoplasm of brain, (C72) organs, (D38) Neoplasm of uncertain or unknown behav- Malignant neoplasm of spinal cord, cranial nerves and iour of middle ear and respiratory and intrathoracic or- other parts of central nervous system, (C73) Malignant gans, (D39) Neoplasm of uncertain or unknown behav- neoplasm of thyroid gland, (C74) Malignant neoplasm of iour of female genital organs, (D40) Neoplasm of uncer- adrenal gland, (C75) Malignant neoplasm of other endo- 30 tain or unknown behaviour of male genital organs, (D41) crine glands and related structures, (C76) Malignant ne- Neoplasm of uncertain or unknown behaviour of urinary oplasm of other and ill-defined sites, (C77) Secondary organs, (D42) Neoplasm of uncertain or unknown behav- and unspecified malignant neoplasm of lymph nodes, iour of meninges, (D43) Neoplasm of uncertain or un- (C78) Secondary malignant neoplasm of respiratory and known behaviour of brain and central nervous system, digestive organs, (C79) Secondary malignant neoplasm 35 (D44) Neoplasm of uncertain or unknown behaviour of of other sites, (C80) Malignant neoplasm without speci- endocrine glands, (D45) Polycythaemia vera, (D46) My- fication of site, (C81) Hodgkin’s Disease, (C82) Follicular elodysplastic syndromes, (D47) Other neoplasms of un- non-Hodgkin’s lymphoma (nodular), (C83) Diffuse non- certain or unknown behaviour of lymphoid, haematopoi- Hodgkin’s lymphoma, (C84) Peripheral and cutaneous etic and related tissue, (D48) Neoplasm of uncertain or T-cell lymphomas, (C85) Other and unspecified types of 40 unknown behaviour of other and unspecified sites. non-Hodgkin’s lymphoma, (C88) Malignant immunopro- [0057] In a specific embodiment of the invention the liferative diseases, (C90) Multiple myeloma and malig- patient which is treated with the combination of the in- nant plasma cell neoplasms, (C91) Lymphoid leukemia, vention suffers from metastases. (C92) Myeloid leukemia, (C93) Monocytic leukemia, [0058] In a preferred embodiment of the invention the (C94) Other leukemias of specified cell type, (C95)45 patient which is treated with the combination of the in- Leukemia of unspecified cell type, (C96) Other and un- vention suffers from a neoplasm selected from list of specified malignant neoplasms of lymphoid, haemat- classes consisting of C25, C50, C56, C71, C91, and C92. opoietic and related tissue, (C97) Malignant neoplasms [0059] In a more preferred embodiment the patient suf- of independent (primary) multiple sites, (D00) Carcinoma fers from a neoplasm selected from the list comprising in situ of oral cavity, oesophagus and stomach, (D01) 50 of acute lymphoblastic leukemia (CD91.0), B-cell chronic Carcinoma in situ of other and unspecified digestive or- lymphatic leukemia (CD 91.2), acute promyelocytic gans, (D02) Carcinoma in situ of middle ear and respi- leukemia (CD92.4), all forms of glioblastoma (CD71), all ratory system, (D03) Melanoma in situ, (D04) Carcinoma forms of pancreatic cancer (CD25), all forms of ovarian in situ of skin, (D05) Carcinoma in situ of breast, (D06) cancer (CD56), classes of breast cancer (CD50) and tu- Carcinoma in situ of cervix uteri, (D07) Carcinoma in situ 55 mor stem cells such as glioblastoma intiating stem cells. of otherand unspecified genitalorgans, (D09) Carcinoma [0060] In a preferred embodiment the patient suffering in situ of other and unspecified sites, (D10) Benign neo- from acute lymphoblastic leukemia (CD91.0) is treated plasm of mouth and pharynx, (D11) Benign neoplasm of with the combination according the invention including

7 13 EP 2 716 291 A1 14 an the anticancer agent selected from the list consisting from glioblastoma (CD71) is treated with the combination of methotrexate, cytarabine, carboplatin, oxaliplatin, vin- according the invention including an the anticancer agent cristine, fludarabine, being preferably cisplatin, anthra- selected from the list consisting of methotrexate, cytara- cycline doxorubicin, idarubicin, daunorubicin, epirubicin, bine, carboplatin, oxaliplatin, vincristine, fludarabine, be- etoposide or alpha, beta or gamma irradiation. 5 ing preferably cisplatin, anthracycline doxorubicin, ida- [0061] In a further preferred embodiment the patient rubicin, daunorubicin, epirubicin, etoposide or alpha, be- suffering from acute lymphoblastic leukemia (CD91.0) is ta or gamma irradiation. treated with the combination according the invention in- [0070] In a further preferred embodiment the patient cluding an opioid receptor agonist selected from the list suffering from glioblastoma (CD71) is treated with the consisting of D,L-methadone, buprenorphine, fentanyl, 10 combination according the invention including an opioid and morphine, being preferably D,L-morphine. receptor agonist selected from the list consisting of D,L- [0062] In a even more preferred embodiment of the methadone, buprenorphine, fentanyl, and morphine, be- invention the embodiment the patient suffering from ing preferably D,L-methadone. acute lymphoblastic leukemia (CD91.0) is treated with [0071] In a even more preferred embodiment of the the combination comprising D,L-methadone and etopo- 15 invention the embodiment the patient suffering from side or D,L-methadone and doxorubicine. glioblastoma(CD71) is treated with the combination com- [0063] In a preferred embodiment the patient suffering prising D,L-methadone and doxorubicin. from B-cell chronic lymphatic leukemia (CD 91.2) is treat- [0072] In a preferred embodiment the patient suffering ed with the combination according the invention including from glioblastoma inititating stem cells are treated with an the anticancer agent selected from the list consisting 20 the combination according the invention including an the of methotrexate, cytarabine, carboplatin, oxaliplatin, vin- anticancer agent selected from the list consisting of meth- cristine, fludarabine, being preferably cisplatin, anthra- otrexate, cytarabine, carboplatin, oxaliplatin, vincristine, cycline doxorubicin, idarubicin, daunorubicin, epirubicin, fludarabine, being preferably cisplatin, anthracycline etoposide or alpha, beta or gamma irradiation. doxorubicin, idarubicin, daunorubicin, epirubicin, etopo- [0064] In a further preferred embodiment the patient 25 side or alpha, beta or gamma irradiation. suffering from B-cell chronic lymphatic leukemia (CD [0073] In a further preferred embodiment the patient 91.2) is treated with the combination according the in- suffering from glioblastoma inititating stem cells are treat- vention including an opioid receptor agonist selected ed with the combination according the invention including from the list consisting of D,L-methadone, buprenor- an opioid receptor agonist selected from the list consist- phine, fentanyl, and morphine, being preferably D,L-30 ing of D,L-methadone, buprenorphine, fentanyl, and mor- methadone. phine, being preferably D,L-methadone. [0065] In a even more preferred embodiment of the [0074] In a even more preferred embodiment of the invention the embodiment the patient suffering from B- invention the embodiment the patient suffering from cell chronic lymphatic leukemia (CD 91.2) is treated with glioblastoma inititating stem cells are treated with the the combination comprising D,L-methadone and fludara- 35 combination comprising D,L-methadone and doxoru- bine, or buprenorphine and doxorubicin or fentanyl and bicin. doxorubicin or morphine and doxorubicin. [0075] In a preferred embodiment the patient suffering [0066] In a preferred embodiment the patient suffering from pancreatic cancer (CD25) is treated with the com- from acute promyelocytic leukemia (CD92.4) is treated bination according the invention including an the antican- with the combination according the invention including 40 cer agent selected from the list consisting of methotrex- an the anticancer agent selected from the list consisting ate, cytarabine, carboplatin, oxaliplatin, vincristine, of methotrexate, cytarabine, carboplatin, oxaliplatin, vin- fludarabine, being preferably cisplatin, anthracycline cristine, fludarabine, being preferably cisplatin, anthra- doxorubicin, idarubicin, daunorubicin, epirubicin, etopo- cycline doxorubicin, idarubicin, daunorubicin, epirubicin, side or alpha, beta or gamma irradiation. etoposide or alpha, beta or gamma irradiation. 45 [0076] In a further preferred embodiment the patient [0067] In a further preferred embodiment the patient suffering from pancreatic cancer (CD25) is treated with suffering from acute promyelocytic leukemia (CD92.4) is the combination according the invention including an opi- treated with the combination according the invention in- oid receptor agonist selected from the list consisting of cluding an opioid receptor agonist selected from the list D,L-methadone, buprenorphine, fentanyl, and morphine, consisting of D,L-methadone, buprenorphine, fentanyl, 50 being preferably D,L-methadone. and morphine, being preferably D,L-methadone. [0077] In a even more preferred embodiment of the [0068] In a even more preferred embodiment of the invention the embodiment the patient suffering from pan- invention the embodiment the patient suffering from creatic cancer (CD25) is treated with the combination acute promyelocytic leukemia (CD92.4) is treated with comprising D,L-methadone and cisplatin. the combination comprising D,L-methadone and doxo- 55 [0078] In a preferred embodiment the patient suffering rubicin or buprenorphine and doxorubicin or fentanyl and from ovarian cancer (CD56) is treated with the combina- doxorubicin or morphine and doxorubicine. tion according the invention including an the anticancer [0069] In a preferred embodiment the patient suffering agent selected from the list consisting of methotrexate,

8 15 EP 2 716 291 A1 16 cytarabine, carboplatin, oxaliplatin, vincristine, fludarab- neuromas, intestinal ganglioneuromas, hyperplastic cor- ine, being preferably cisplatin, anthracycline doxorubicin, neal nerve tumour, marfanoid habitus tumour, Wilms’ tu- idarubicin, daunorubicin, epirubicin, etoposide or alpha, mour, seminoma, ovarian tumour, leiomyomata, cervical beta or gamma irradiation. dysplasia and in situ carcinoma, neuroblastoma, retino- [0079] In a further preferred embodiment the patient 5 blastoma, soft tissue sarcoma, malignant carcinoid, top- suffering from ovarian cancer (CD56) is treated with the ical skin lesion, mycosis fungoide, rhabdomyosarcoma, combination according the invention including an opioid Kaposi’s sarcoma, osteogenic and other sarcoma, ma- receptor agonist selected from the list consisting of D,L- lignant hypercalcemia, renal cell tumour, polycythermia methadone, buprenorphine, fentanyl, and morphine, be- vera, adenocarcinoma, glioblastoma multiforme, leuke- ing preferably D,L-methadone. 10 mia, lymphomas, malignant melanomas, and epidermoid [0080] In a even more preferred embodiment of the carcinomas. inventionthe embodiment thepatient suffering fromovar- [0086] In a further preferred embodiment of the inven- ian cancer (CD56) is treated with the combination com- tion the patient to be treated suffers from a neoplasm prising D,L-methadone and cisplatin. selected from the group consisting of pancreatic carci- [0081] In a preferred embodiment the patient suffering 15 noma,hepatoblastoma, coloncarcinoma, (small cell lung from breast cancer (CD50) is treated with the combina- cancer, melanoma, mamma carcinoma, ovarian carcino- tion according the invention including an the anticancer ma, prostate carcinoma, glioblastoma, acute lymphob- agent selected from the list consisting of methotrexate, lastic leukaemia, acute myeloid leukaemia, chronic my- cytarabine, carboplatin, oxaliplatin, vincristine, fludarab- eloid leukaemia, chronic lymphocytic leukaemia, pro- ine, being preferably cisplatin, anthracycline doxorubicin, 20 forms of leukaemia, hairy cell leukaemia, Hodgkin’s dis- idarubicin, daunorubicin, epirubicin, etoposide or alpha, ease, Non-Hodgkin lymphoma, lymphoma, tumor stem beta or gamma irradiation. cells, glioblastoma-initiating stem cells and multiple my- [0082] In a further preferred embodiment the patient eloma. suffering from breast cancer (CD50) is treated with the [0087] In another embodiment of the invention the pa- combination according the invention including an opioid 25 tient exhibits either an intrinsic or an acquired resistance. receptor agonist selected from the list consisting of D,L- [0088] Accordingly, in the context of the present inven- methadone, buprenorphine, fentanyl, and morphine, be- tion a "resistance" can either be total or partly; in other ing preferably D,L-methadone. words, the patients considered treatable according to the [0083] In a even more preferred embodiment of the invention can exhibit a reduced sensitivity or even a full invention the embodiment the patient suffering from30 lack of sensitivity to conventional anticancer treatments. breast cancer (CD50) is treated with the combination These patients can also be determined as "non-respond- comprising D,L-methadone and cisplatin. ers" or "poor-responders". [0084] In another embodiment the cancer to be treated [0089] A further synonym for a "resistant" cancer or is a neoplasm according the International classification tumour is a "refractory" type of cancer, which can also of Diseases for Oncology ICD-O in the actual version 35 be either completely or partly refractory. Intrinsic resist- ICD-O-3 from 2000. Alternatively, the cancer to be treat- ancecan thus also be determined as a "primary refractory ed is a cancer as included in the TNM Classification of cancer". A particular form of refractory or resistant cancer Malignant Tumours (TNM), which represents a cancer cells are the so called "kinetically refractory cells"; a phe- staging system that describes the extent of cancer in a nomenon known e.g. from leukaemia cells, when the patient’s body. In a further alternative, the cancer to be 40 cells are at first killed, but reproduce fast that an effective treated is disclosed by Boecker et al., 2008 in chapter 6 treatment is hardly possible. (Pathologie, Elsevier, Urban & Fischer, p. 167-218), [0090] As used in the context of the present invention which is incorporated by reference in its entirety. the term "conventional" treatment or therapy refers to the [0085] In a preferred embodiment of the invention the currently accepted and widely used therapeutic treat- patient that is treated with said combination suffers from 45 ment of a certain type of cancer, based on the results of non-solid tumours from the group consisting of leukae- past researches and/or regulatory approval. mia, breast cancer, skin cancer, bone cancer, prostate [0091] Conventional anticancer drugs include cytotox- cancer, liver cancer, lung cancer, brain cancer, cancer ic and cytostatic agents, which kill the cancer cells or of the larynx, gallbladder, pancreas, rectum, parathyroid, reduce and/or stop their growth or proliferation. The thyroid, adrenal, neural tissue, head and neck, colon, 50 modes of action of these anticancer drugs can vary; ex- stomach, bronchi, kidneys, basal cell carcinoma, squa- amples are antimetabolites (e.g. cytarabine, methotrex- mous cell carcinoma of both ulcerating and papillary type, ate, mercaptopurine or clofarabine), DNA cross-linking metastatic skin carcinoma, osteosarcoma, Ewing’s sar- agents (e.g. cisplatine and its derivates), DNA intercalat- coma, veticulum cell sarcoma, myeloma, giant cell tu- ing substances (e.g. doxorubicin), Topoisomerase poi- mour, small-cell lung tumour, islet cell tumour, primary 55 sons (e.g. etoposide), kinase inhibitors (e.g. cetuximab), brain tumour, acute and chronic lymphocytic and granu- steroids (e.g. dexamethasone) or mitotic inhibitors (e.g. locytic tumours, hairy-cell tumour, adenoma, hyperpla- vincristine). One example for a conventional anticancer sia, medullary carcinoma, pheochromocytoma, mucosal treatment of leukaemia is the administration of doxoru-

9 17 EP 2 716 291 A1 18 bicin or rituximab. or lower than the recommended dose for the respective [0092] The conventional radiotherapy can also include cancer. The recommended dose is given by a conven- radiation therapy, which means the use of high-energy tional cancer therapy without the administration of an opi- radiation from x-rays, alpha, beta and gamma rays, Au- oid receptor agonist. Preferably, the respective dose of ger electrons, Ultraviolet rays, neutrons, protons, and 5 the anticancer agent from the perspective of the skilled other sources to kill cancer cells and shrink tumours. Ra- person represents a suboptimal or sub therapeutic dose, diation may originate from an outside the body device which have the advantage for the patient to have less (external-beam radiation therapy), or it may originate side effects. The main effect is that the uptake of the from radioactive sources placed in the body in the vicinity dosis of the anticancer drug is increased in the cancer of the cancer cells (internal radiation therapy). Systemic 10 cells, while the plasma concentration is on the level of radiation therapy uses a radioactive substance, such as the conventional therapy. This has the effect that non a radiolabeled monoclonal antibody, that travels in the responder to conventional therapy could be treated. blood stream to the target tissue. Radio resistant cancer [0098] In a further preferred embodiment of the inven- cells do not or only partly respond to these treatments. tion the anticancer agent that is administered together [0093] As outlined in detail above, according to one 15 with the opioid receptor agonist is given at a dose, which embodiment of the invention the opioid receptor agonists is equal than or lower than the recommended dose for are applied for overcoming or "breaking" the intrinsic or the respective cancer, wherein the period of effective acquired resistance of cancer cells to conventional anti- plasma levels of the anticancer agent is completely within cancer treatments and/or radiation treatment or apopto- the period of effective plasma levels of the opioid receptor sisresistance. Inone embodiment ofthe inventioncancer 20 agonist. The recommended dose is given by a conven- cells considered treatable according to the invention ex- tional cancer therapy without the administration of an opi- press an opioid receptor, in particular the m opioid recep- oid receptor agonist. tor. [0099] In a further preferred embodiment of the inven- [0094] According to the invention, the terms "resist- tion the opioid receptor agonist is D/L-methadone and ance", "radioresistance" or "chemoresistance" are de- 25 the anticancer agents are methotrexate and dexameth- fined as a reduced sensitivity of a cancer cell to at least asone. oneconventional cancer therapy, i.e.either an anticancer [0100] In a further embodiment of the invention, the drug or radiotherapy. A patient suffering from such a can- opioids or opioid receptor agonist can be used as a com- cer is determined as a "resistant" cancer patient. Since posite with at least one anticancer drug. the resistance can be intrinsic or acquired the observed 30 [0101] A "composite" within the context of the present reduction in sensitivity is either compared to fully sensi- invention relates to a pharmaceutical preparation com- tive "normal" cancer cells, which are responsive to the prising a therapeutically effective amount of any of the therapeutically effective dosage of the applied anticancer opioid receptor agonist (component A) as defined ac- drug and/or radiation compared to the original sensitivity cording to the invention and at least one further antican- upon therapy onset. In the later case the resistance man- 35 cer substance (component B). This "composite" can con- ifests either in a diminished amount of tumour regression stitute a single composition or at least two compositions, for the same dose (either of the radiation or the anticancer which can be administered to the patients either concom- drug) or an increased dose which is necessary for an itantly or subsequently. The above mentioned substanc- equal amount of tumour regression. es are preferably combined with methadone, more pref- [0095] In another embodiment of the invention the pa- 40 erably with the hydrochloride form of D/L-methadone. tient exhibits one or more of the subsequent resistances: [0102] The composite of the invention can be advan- apoptosis resistance, multi-drug resistance, anticancer tageous for the effective treatment of cancer cells, since drug resistance, cytotoxic drug resistance, resistance to it can exhibit synergistic effects compared to the single reactive oxygen species, resistance to DNA damaging compositions. In particular composite with methadone agents, resistance to toxic antibodies, doxorubicin resist- 45 as component A and one of the agents as component B ance, single or cross resistance, irradiation resistance as follows is preferred: methotrexate, cytarabine, cispl- (e.g. alpha, beta, gamma or Auger electrons). atine, carboplatin, oxaliplatin, etoposide, vincristine, - [0096] In a particular embodiment the patient is resist- orubicin, idarubicin, epirubicin, daunorubicin, fludarbine.. ant to one or more of the following drug substances: Moreover, combinatorial treatment also comprising irra- methotrexate, cytarabine, cisplatine, oxaliplatin, etopo- 50 diation treatments is possible. side, vincristine, paclitaxel, carboplatin, teniposide, dex- [0103] In a preferred embodiment of the invention opi- amethasone, prednisolone, cyclophosphamide, diphos- oids are used to treat either resistant or sensitive non- phamide, doxorubicin, epirubicin, daunorubicin, idaru- solid cancers, i.e. all haematological malignancies affect- bicin, mercaptopurine, fludarabine, HER2, and anti- ing blood, bone marrow and lymph nodes, including CD20. 55 acute lymphoblastic leukaemia, acute myeloid leukae- [0097] In one embodiment of the invention the antican- mia, chronic myeloid leukaemia, chronic lymphocytic leu- cer agent that is administered together with the opioid kaemia and all pro-forms of leukaemia, hairy cell leukae- receptor agonist is given at a dose, which is equal than mia, Hodgkin’s disease, Non-Hodgkin lymphoma, lym-

10 19 EP 2 716 291 A1 20 phoma and multiple myeloma. and haemoglobin content; (d) genomic and proteomic [0104] In a further aspect the invention provides a assays which include the analysis of the activation of method for the selection of a combination of an opioid stress pathways using DNA microarrays and protein receptor agonist and one or more anticancer drugs. This chips. method comprises the following steps: 5 [0108] In a further preferred embodiment the cell via- bility is measured by the propidium iodide assay and the (a) providing an vitro culture of cancer cells, cell lines apoptosis is measured by determination of hypodiploid or primary cells, preferably isolated from a cancer DNA (subG1) and FSC/SSC analyses by flow cytometry. biopsy or from a liquid sample (such as e.g. blood, [0109] In step (d) the cultured cells are preferably treat- amniotic fluid, pleural fluid, peritoneal fluid, or cere- 10 ed in parallel experiments comprising the use of the opi- brospinal fluid); oid alone, the anticancer agent alone and a combination (b) optionally testing the cells from step (a) for ex- of the two substances. In a further embodiment the po- pression of opioid receptors; tency of the effect is analysed by studying the dose de- (c) treating the cells from step (a) with an opioid ag- pendency of the respective effect. In alternative experi- onist, or at least one anticancer drug or a combina- 15 ments several anticancer agents can be combined to in- tion thereof; crease the anti-apoptotic effect or opioid receptor expres- (d) analysing the cells for cell death and/or expres- sion or to reduce the side effect profile. In a further em- sion of opioid receptors bodiment the initial selection of the test compounds will (e) selecting the opioid receptor/anticancer drug depend on the characteristics of the tumour. Furthermore combination and preferably a dose for said combi- 20 also the patient characteristics can be taken in consid- nation based on the desired extent of cell death/vi- eration including the age, the sex, the body weight, co- ability or inhibiting proliferation; and/or morbidities, individual metabolic capabilities, allergies (f) selecting the anticancer agent and preferably a and incompatibilities, genetic predisposition, the course dose for said anticancer agent which shows the de- of the disease and the family history. sired extent of induction of opioid receptors. 25 [0110] For the in vitro analysis the opioid receptor ag- onists as described above can be used for testing. Pref- [0105] The in vitro cultured cancer cells can be an im- erably, D,L-methadon, L-methadon, fentanyl, buprenor- mortalized cell line, xenografted cells, a secondary or a phin, morphine, , oxycodone, tramadol and tap- primary cancer cell line or primary cells. In a preferred entadol are used. embodiment the cell line and/or cells is derived from a 30 [0111] Ina preferred embodiment, an anti canceragent cancer biopsy, in more preferred embodiment the biopsy is chosen which is well known to have an effect on the or blood sampling or cerebrospinal fluid sampling or pleu- respective cancer cell type, cell line or cells. ral fluid sampling or amniotic fluid sampling or peritoneal [0112] When testing an anticancer agent alone, the fluid sampling is taken from the patient to be treated with cultured cells are analysed for opioid receptor expression the combination according the invention. The cancer cell 35 prior anticancer treatment and after the anticancer treat- line can represent a homogenous cell line based only on ment under conditions which allow a comparison of the one cancer cell type or a heterogeneous cancer cell line opioid receptor expression levels. Said comparison al- comprising of different cell types. lows to identify anticancer agents which increase the opi- [0106] The analysis of the opioid receptor expression oid receptor expression on the respective cancer cell. in step (b) can be performed by techniques which are 40 [0113] The selection in step (e) prioritizes the drug known to the person skilled in art. A non-limiting list of combination and/or the respective doses in order to max- examples include immunofluorescence using an anti- imise the efficacy while retaining a side effect profile body or antibody fragment directed against said opioid which is acceptable for the patient. receptor, the immunoprecipitation of the opioid recep- [0114] The selection in step (f) prioritizes an anticancer tors, or the use of labelled opioid receptor ligands such 45 agent with regard to its ability to increase the opioid re- as -fluorescein. ceptor expression on the cancer cell. As a consequence [0107] For the analysis of the cell viability and apopto- the anti-apoptotic effect of the opioid agonist, as well as sis in step (c) there are several techniques which are the anti-apoptotic affect of the anticancer agent is max- known to the person skilled in art. A non-limiting list of imised. examples include (a) cytolysis or membrane leakage as- 50 [0115] In the case that in step (c) the cell culture was says such as the lactate dehydrogenase assay, the pro- treated with a combination of opioid receptor agonist and pidium iodide assay, the Trypan blue assay, the 7-Ami- anticancer agent the prioritization of the combination noactinomycin D assay, (b) mitochondrial activity or cas- which is used, is done under the aspect which combina- pase assays such as the Resazurin and Formazan tion of doses has the better lethal effect on the cells in (MTT/XTT) can assay for various stages in the apoptosis 55 culture. The combination with the highest lethal effect or process that foreshadow cell death, (c) functional assays if observable, the dose with a up to 10% less effect in which in the case of red blood cells measure the cell lethality of cells compared to the combination with the deformability, osmotic fragility, haemolysis, ATP level, highest lethal effect on cells in culture but with the lower

11 21 EP 2 716 291 A1 22 dose of opioid-receptor agonist should be used. Figure Cell lines for example shows that with doxorubicin in conven- tional therapeutical dose as described in the instruction [0121] The human B-cell leukemia (BCP-ALL) cell leaflet a D,L-methadone dose of 0,1 mg/mL would be pref- lines Tanoue, Reh and Nalm6 were obtained from the erable. 5 DSMZ (Braunschweig, Germany) and cultured in RPMI [0116] In a further aspect the invention provides a 1640 (Invitrogen) containing 10% heat inactivated FCS method for selection of an opioid receptor agonist for the (Lonza, Verviers, Belgium), 1 mmol/L glutamine (Invitro- treatment of cancer comprising the following steps: gen), 1% penicillin/streptomycin (Invitrogen), 25 mmol/L HEPES (Biochrom) at 37°C, 95% air/ 5% CO 2. In exper- (a) providing an vitro culture of cancer cells, cell lines 10 imental settings, the leukemia cells were seeded in a or primary cells, preferably isolated from a cancer density of 10,000 cells/mL. biopsy or from a liquid sample (such as e.g. blood, amniotic fluid, pleural fluid, or peritoneal fluid or cer- Testing opioid receptor signaling ebrospinal fluid); (b) optionally testing the cells from step (a) for ex- 15 [0122] Stimulationof opioidreceptors (OR)by agonists pression of opioid receptors; like D,L-methadone leads to an activation of the inhibitory (c) treating the cells from step (a); Gi-protein. The αi-subunit inactivates adenylyl cyclase (d) analysing the cells for cell death/viability or inhi- (AC) resulting in a reduction of cAMP levels within the bition of proliferation; cell which in turn leads to apoptosis which might be me- (e) selecting the opioid receptor/anticancer drug20 diated by several different modulators. Also the βγ-sub- combination and preferably a dose for said combi- units of the Gi-protein modulate the activity of different nation based on the desired extent of cell death; effectors like the inhibition of Ca2+- and the activation of and/or K+-channels. Naloxone as opioid in- (f) selecting the opioid receptor agonist and prefer- hibits competitively opioid receptors. PTx (pertussis tox- 25 ably a dose for said opioid receptor agonist which in) inactivates Gi-proteins and blocks downregulation of shows the desired extent of induction of cell death. cAMP. IBMX (lsobutyl-1-methylxanthine) inhibits phos- phodiesterase and increases cAMP levels [0117] For this method the steps (a) to (d) can be per- formed by methods and strategies as described above. Serum concentrations of methadone [0118] The analysis of the opioid receptor expression 30 allows a selection of cancer type which might be treated [0123] Determination of methadone in serum samples with an opioid receptor agonist. Due to the in vitro treat- was carried out after liquid/liquid extraction using a mass ment with an opioid receptor agonist, the individual dose spectrometer equipped with a gas chromatograph for the cancer in vivo treatment can be determined. (GC/MS). As internal standard d9-methadone was add- 35 ed. The mass selective detector was operated in electron 1. EXPERIMENTAL PROCEDURES impact mode. Data were acquired in the selected-ion monitoring mode. The analytes were identified with the Drugs and reagents following masses m/z 294, 223, 72 (target ion) for meth- adone and m/z 303, 226, and 78 for d 9-methadone with [0119] For the in vitro experiments, D,L-methadone hy- 40 a limit of detection of 0,8ng/ml and a limit of quantification drochloride (D,L-methadone) and doxorubicin were pur- of 1,2ng/ml. chased from Sigma (Taufkirchen, Germany), naloxone from Fagron GmbH&Co. KG (Barsbüttel, Germany), and Serum concentrations of doxorubicin pertussis toxin (PTX) from Calbiochem (Nottingham, UK). Prior to each experiment these substances were 45 [0124] Determination of doxorubicin and its main me- freshly dissolved in sterile distilled water to ensure the tabolites in serum were performed as described previ- constant quality of the preparations. 3-Isobutyl-1-meth- ously (Hilger et al., 2005; Richly et al., 2006) Using this ylxanthine (IBMX, Sigma) was freshly dissolved in 0.01 validated method, the quantification of doxorubicin, dox- N NaOH. orubicinol, and 7-deoxy-doxorubicinolon was possible [0120] For in vivo application, we used D,L-methadone 50 with a LLQ of 0.2ng/ml. (Methaddict, Hexal, Germany) as 5 mg tablets purchased from the local pharmacy. The tablets were pulverized and Patient-derived-ALL xenografts solubilized freshly before use in 10% Tween 80 in saline. Doxorubicin (Hexal) was purchased as injection solution [0125] For in vivo use ALL-SCID6 model was chosen. (5mg/ml) and diluted freshly with saline to the appropriate 55 Fragments from in vivo passaged tumours were trans- concentrations. planted at day zero subcutaneously to 32 male NOD/SCID/IL2ry null (NSG) mice. After randomization oral treatment (by gavage) with D,L-methadone was in-

12 23 EP 2 716 291 A1 24 itiated one day later and performed daily until the end of lysed with Nicoletti-buffer containing sodium citrate the experiment with increasing doses:st week 1 20 (0.1%), Triton X-100 (0.1%) and propidium iodide (50 mg/kg/d, 2ndweek 30 mg/kg/d, rd 3week 40 mg/kg/d, mg/mL) as described by Nicoletti (Nicoletti et al., 1991). 4thweek 60 mg/kg/d, 5 th -10thweek 2 x 60 mg/kg/inj.. The Apoptotic cells were determined by hypodiploid DNA dose adaptation was necessary to avoid toxic deaths be- 5 (subG1) or forward scatter/side scatter analysis (Carbon- cause of an overdosage of D,L-methadone. The maxi- ari et al., 1994). The percentage of specific apoptosis mum tolerated dose of D,L-methadone in the employed was calculated as follows: 100 x [experimental dead cells mouse strain is 2x60mg/kg/inj.. At day 46, 53, 60 and 76 (%) - spontaneous dead cells in medium (%)] / [100% doxorubicin 3mg/kg was administered i.v.. Tumour size -spontaneous dead cells in medium (%)]. The spontane- was measured twice weekly at two dimensions and tu- 10 ous dead cells were in the rage of 5 to 10% using cell mour volumes were calculated according to the formula lines. The viability of the untreated patient cells (sponta- (length x width2)/2. Mean tumour volumes and standard neous dead cells) was less than 35% at 24h and 48h. deviations were calculated per group. Treated to control values (T/C) in percent were calculated by relating mean General caspase inhibition by zVAD.fmk tumour volumes of each group at each measurement day 15 to the controls. Individual body weight was determined [0130] For inhibition of apoptosis, leukemia cells were twice per week as parameter for tolerability and body treated with the pancaspase inhibitor of caspases, weight changes in percent were calculated by relating zVAD.fmk (benzoylcarbonyl-Val-Ala-Asp-fluoromethyl- the mean values of each group to the first measurement ketone; Enzyme-Systems-Products, Dubli, USA) as de- day. 20 scribed (Friesen et al., 2007). 50 mM zVAD.fmk was add- [0126] Serum from D,L-methadone treated mice was ed to the cells 1 h before stimulation with D,L-methadone taken 0.5, 1, 4 and 24 hours after last D,L-methadone and doxorubicin. After different time points, the percent- treatment at day 76, respectively, and stored at -20°C age of apoptotic cells was determined by FSC/SSC anal- until the determination of methadone concentration. Mice ysis via flowcytometry(Carbonari et al., 1994). were sacrificed at day 77 for ethical reasons. 25 [0127] For the in vitro investigations, cell suspensions Western blot analysis of human xenograft-derived-ALL-cells from patients with T-cell(ALL-SCID6, ALL-SCID3), B-cell (ALL-SCID7) and [0131] Western blot analyses were performed as de- B-cell precursor (BCP, pre-B-ALL-SCID) acute leukemia scribed (Classen et al., 2003; Friesen et al., 2004). Whole were gained and cultivated in vitro and were phenotypic 30 cell lysates were immunodetected for PARP, caspase- and genotypic characterized as described (Borgmann et 3, caspase-9, caspase-2, XIAP, Bcl-x L and β-actin using al., 2000). All animal experiments were approved by the rabbit-anti-PARP-polyclonal-antibody (1:5000, Roche), local responsible authorities (LaGeSo Berlin) and per- mouse-anti-caspase-2-monoclonal-antibody (1:1000, formed according to the guidelines for animal welfare in BD-Transduction-Laboratories, Heidelberg, Germany), oncological experiments (Workman et al. 2000). 35 anti-XIAP-monoclonal-antibody (1:1000, BD-Transduc- tion-Laboratories), mouse-anti-caspase-3-monoclonal- Flow cytometric assay for determination of cell sur- antibody (1:1000, Cell-Signaling, Boston, MA/USA), rab- face opioid-receptors bit-anti-caspase-9-polyclonal-antibody (1:1000, Cell- Signaling) rabbit-anti-Bcl-XL-polyclonal-antibody [0128] Cells were washed in PBS supplemented with 40 (1:1000, Santa-Cruz, Heidelberg, Germany) and mouse- 1% FCS, centrifuged and resuspended in PBS/1% FCS anti-β-actin-monoclonal-antibody (1:5000, Sigma). As containing naloxone-fluoresceine (0.05mM, Invitrogen) secondary antibodies peroxidase-conjugated-goat-anti- (Hedin et al., 1997). After 30min of incubation at RT, the mouse IgG or peroxidase-conjugated-goat-anti-rabbit cells were washed twice with PBS/1% FCS, centrifuged IgG (1:5000, Santa-Cruz) were used for the enhanced and resuspended in icecold PBS/1% FCS. Flowcytome- 45 chemoluminescence system (ECL, Amersham-Pharma- try analysis was performed using FACSCalibur (BD, Hei- cia, Freiburg, Germany). Equal protein loading was con- delberg, Germany). trolled by β-actin detection.

Induction of apoptosis Analysis of doxorubicin uptake and efflux 50 [0129] ALL cells were treated with D,L-methadone ( ≤3 [0132] For analysis of doxorubicin uptake, the BCP- mg/mL therapeutic plasma concentration) alone or in ad- leukemia cell line Tanoue was seeded in a density of dition to doxorubicin in 175 cm 2 flasks or 96-well plates. 100,000 cells/mL in 175cm2 flasks and was either left Further experiments were performed simultaneously af- untreated or incubated with 0.3mg/mL doxorubicin or a ter addition of 60 mg/mL naloxone, 200 mM IBMX or 200 55 combination of 0.3mg/mL doxorubicin and 3 mg/mL D,L- ng/mL PTX. After different points in time, apoptosis rates methadone at 37°C/5%CO2. After 24h, cells were were measured by flowcytometry (Carbonari et al., 1994; washed twice with ice-cold PBS/1% FCS. Relative dox- Nicoletti et al., 1991). To determine apoptosis, cells were orubicin uptake in cells was analyzed using flowcytom-

13 25 EP 2 716 291 A1 26 etry. D,L-methadone on BCP-ALL-cell lines (Tanoue, Reh, [0133] For analysis of doxorubicin efflux, cells were Nalm6) expressing opioid-receptors in a moderate level washed to remove doxorubicin from medium after incu- on their cell surface (Fig.2A) was tested. bation for 24h. Next, cells were incubated with fresh me- [0138] These BCP-ALL-cell lines could only be killed dium without doxorubicin or fresh medium containing 5 slightly by D,L-methadone (Fig.2b) as observed for the

3mg/mL D,L-methadone at 37°C/5%CO2 without doxo- pre-B-ALL-SCID (Fig.1B). In order to show if different rubicin to measure doxorubicin efflux. After different time substances will act synergistically, the cell lines Tanoue, points cells were harvested, washed and relative doxo- Reh, Nalm6 and pre-B-ALL-SCID were treated with dif- rubicin contentin leukemia cellswas analyzed using flow- ferent concentrations of D,L-methadone and doxorubicin cytometry. 10 alone or in combination with each other (Fig.2B, 2C). It was observed that the combination treatment strongly II. Examples induced cell kill in BCP-ALL-cell lines as well as in xe- nograft-derived-BCP-ALL-patient-cells (pre-B-ALL- Example 1: D,L-Methadone induces cell death in xe- SCID) (Fig.2B,C). nograft-derived ALL-cells depending on opioid re- 15 [0139] In order to analyze the molecular pathways of ceptor expression cell killing in more detail and to find out how the combi- nation treatment with D,L-methadone and doxorubicin [0134] To show the clinical relevance of D,L-metha- induced apoptosis, it was analyzed at first which effector done in treatment of leukemia and the role of opioid re- molecules of apoptosis signaling are activated in BCP- ceptor triggering in cell death induction, the anti-cancer 20 ALL-cells upon this combination treatment compared to effect of D,L-methadone was analyzed in different xe- cells treated with D,L-methadone or doxorubicin alone. nograft-derived ALL-cells. The xenografts were originally 120h after treating the BCP-ALL-cell line Tanoue with established from patients with T-cell (ALL-SCID6, ALL- D,L-methadone in addition to doxorubicin, the activation SCID3), B-cell (ALL-SCID7) (Borgmann et al., 2000) and of the caspase cascade in BCP-ALL-cells was observed. B-cellprecursor (BCP, pre-B-ALL-SCID)acute leukemia. 25 The analysis revealed a strong activation of caspase-3, At first, the opioid-receptor expression on xenograft-de- caspase-9, and caspase-2 and cleavage of the prototype rived-ALL-cells was measured. It was observed that the substrate of caspase-3, poly-(ADP-ribose)-polymerase ALL-SCID6, ALL-SCID3 and the ALL-SCID7 leukemia (PARP) (Fig.3A). cells displayed opioid-receptors in high amounts[0140] The role of the caspase cascade in apoptosis (Fig.1A), whereas the pre-B-ALL-SCID expressed only 30 induction was further investigated with the broad-spec- moderate levels of opioid-receptors (Fig.1A). trum inhibitor of caspases zVAD.fmk. BCP-ALL-cells [0135] To analyze if cell death induction using D,L- were pre-incubated with or without 50mM of zVAD.fmk methadone depends on the levels of opioid receptor ex- and treated with D,L-methadone in addition to doxoru- pression, ALL-SCID6, ALL-SCID3, ALL-SCID7 and pre- bicin. zVAD.fmk strongly decreased cell death after com- B-ALL-SCID were treated with different concentrations 35 bination treatment with D,L-methadone and doxorubicin of D,L-methadone (Fig.1B). in BCP-ALL-cells (Fig.3B) underlining the dependence [0136] Therapeutic plasma concentrations of D,L- on caspases activation. methadone (≤3mg/mL) were used but also a higher con- [0141] The apoptotic machinery is tightly controlled by centration of 10 mg/mL D,L-methadone was used, be- anti-apoptotic factors like XIAP and Bcl-x L(Fulda, 2009a; cause levels of D,L-methadone in lymphatic tissue and 40 Fulda, 2009b) which we found to be strongly downregu- marrow may be higher, but have not been measured lated in BCP-ALL-cells treated with D,L-methadone in (Singh et al., 2011). It was found that therapeutic plasma addition to doxorubicin (Fig.3C). These results indicate concentrations of D,L-methadone (≤3mg/mL) induced a that the combination of D,L-methadone and doxorubicin strong cell death in xenograft-derived ALL-cells express- sensitizes BCP-ALL-cells for apoptosis via the activation 45 ing high amounts of opioid-receptors on their cell surface of caspases and downregulation of XIAP and Bcl-x L. (Fig.1A,B). In comparison to these observations, the pre- B-ALL-SCID having a moderate opioid-receptor level EXAMPLE 3: Doxorubicin strongly induces opioid- (Fig.1A) could only be slightly killed with therapeutic con- receptor expression in leukemia cells. centrations of D,L-methadone (Fig.1B). This clearly re- veals that apoptosis induction by D,L-methadone is de- 50 [0142] The efficiency of cell death induction and acti- pend on the level of opioid-receptor expression. vation of effector molecules in apoptosis pathways after treating leukemia cells with D,L-methadone seems to de- EXAMPLE 2: Combination treatment with D,L-Meth- pend on the amount of opioid-receptors displayed on the adone and doxorubicin kills and activates caspases cell’s surface. Combination treatment with D,L-metha- in ALL-cells with moderate opioid receptor expres- 55 done and doxorubicin profoundly kills leukemia cells with sion moderate opioid receptor expression, which could only be killed slightly by D,L-methadone or doxorubicin alone. [0137] In analogous studies, the cytotoxic potential of Chemotherapeutics enhance theexpression of receptors

14 27 EP 2 716 291 A1 28 like CD95 in leukemia cells (Posovszky et al., 1999). To leukemia cells (Naderi et al., 2009; Safa et al., 2010a). analyze whether doxorubicin might influence the opioid- Pertussis toxin (PTX) inactivates G i-proteins and blocks receptor expression, the BCP-ALL-cell line Tanoue was downregulation of cAMP (Law et al., 1985) (Fig. 7). IBMX treated with doxorubicin for 96h. Afterwards, the relative however increases cAMP levels as a result of phosphodi- amount of opioid-receptors compared to untreated cells 5 esterase inhibition (Fig. 7). To analyze the critical role of was measured by flowcytometry. It was found that dox- cAMP in opioid receptor activation-induced apoptosis, orubicin strongly increased opioid-receptor expression theBCP-ALL-cell line Tanouewas treated withD,L-meth- (Fig. 4A) suggesting that D,L-methadone can bind in adone, doxorubicin, and IBMX or PTX either alone or in higher amounts to cells co-treated with doxorubicin. This different combinations with each other (Fig.5C,D). After effect could presumably result in the higher cytotoxic po- 10 96h it was found that upregulation of cAMP by IBMX tential of the combination treatment with D,L-methadone (Fig.5C) and blocking downregulation of cAMP by PTX and doxorubicin. (Fig. 5D) strongly reduced the apoptosis rates of combi- nation treatment with D,L-methadone and doxorubicin. EXAMPLE 4: Opioids like D,L-methadone enhances In addition, the upregulation of cAMP by IBMX also de- the uptake of doxorubicin and inhibits its efflux 15 creased doxorubicin-induced apoptosis (Fig.5C). These results indicate that the activation of opioid receptor cou-

[0143] Opioids are substrates of the in multi-drug re- pled Gi-proteins is essential for the induction of apoptosis sistances-involved efflux pump P-glycoprotein (P-gp). To which might be regulated via the intracellular cAMP lev- analyze whether D,L-methadone might influence the up- els. take and/or efflux of doxorubicin in leukemia cells, the 20 BCP-ALL-cell line Tanoue was incubated for different in- EXAMPLE 6: D,L-methadone alone or in addition to tervals with doxorubicin alone or with a combination of doxorubicininhibits tumourgrowth in vivo in an ALL- doxorubicin and D,L-methadone. After 24h (Oh), an en- xenograft-model hanced doxorubicin concentration in the cells co-incu- bated with doxorubicin and D,L-methadone (Fig. 4B) was 25 [0147] In vitro results demonstrated that D,L-metha- observed. After removing doxorubicin from the superna- done could induce apoptosis in several leukemia cell tant, fresh medium was added without doxorubicin and lines and increased the cytotoxicity of doxorubicin. To D,L-methadonewas applied. After8h and 24h, D,L-meth- confirm the clinical relevance of the anti-cancer potential adone reduced the doxorubicin efflux strongly (Fig. 4B) of D,L-methadone alone or in combination with doxoru- indicating that D,L-methadone increases doxorubicin up- 30 bicin and to verify the results obtained so far an ALL- take and inhibits doxorubicin efflux out of leukemia cells. xenograft study was undertaken. This explains how D,L-methadone as well as doxorubicin [0148] For the in vivo study, a patient-derived-ALL-xe- mutually increase their cytotoxic potential. nograft-model (ALL-SCID6) was used. Its phenotypic and genotypic identity with the original patient sample EXAMPLE 5:Apoptosis induction by D,L-methadone 35 was proven (Borgmann et al., 2000). The experiment and doxorubicin depends critically on opioid recep- started at day 0 with subcutaneous inoculation of ALL- tor activation and cAMP concentration SCID6 fragments from anin vivo passage into male NOD/SCID/IL2ry null (NSG) mice. After randomization, [0144] To further analyze the role of opioid-receptor D,L-methadone was orally administered starting at day triggering in apoptosis induction and consequently acti- 40 one after ALL-inoculation with increasing doses. When vation of apoptotic pathways, the BCP-ALL-cell line tumours were palpable, doxorubicin treatment was initi- Tanoue was treated with D,L-methadone, doxorubicin or ated. D,L-methadone and doxorubicin treatment led to a with the opioid-receptor antagonist naloxone alone or in significant inhibition of tumour growth at comparable lev- different combinations with each other (Fig. 5A,B). els (Fig.6). [0145] After 96h it was found that blocking opioid-re- 45 [0149] Combination treatment with D,L-methadone ceptorsby naloxonestrongly reduced theapoptosis rates anddoxorubicin had a similar anti-tumour efficacyas D,L- of the combination treatment with D,L-methadone and methadone or doxorubicin alone until day 70. At later doxorubicin (Fig.5A). In addition, opioid-receptor block- time points, the tumour inhibition was longer lasting dur- ing by naloxone drastically reduced the activation of cas- ing the combined treatment of D,L-methadone and dox- pase-9,caspase-2 and caspase-3 and cleavageof PARP 50 orubicin. The therapy was well-tolerated with body weight after treating BCP-ALL-cells with D,L-methadone in ad- changes of -10% for the combination and -8% or -4% for dition to doxorubicin (Fig.5B). This indicates that opioid- the D,L-methadone or doxorubicin treatment, respective- receptor triggering is critically involved in apoptosis in- ly. To analyze D,L-methadone serum concentrations in duction and in caspase activation (Fig.7). mice, 0.5, 1, 4 and 24 hours after the last D,L-methadone [0146] Opioid receptor stimulation activates inhibitory 55 application, serum was taken and D,L-methadone quan- Gi-proteins which in turn block adenylyl cyclase activity tified by mass spectrometry. The serum concentrations reducing cAMP (Fig. 7). cAMP is an inhibitor of DNA- of methadone were found between 28 ng/mL and 138 damage- as well as doxorubicin-induced apoptosis in ng/mL in the time course of 0.5 until 4 hours after D,L-

15 29 EP 2 716 291 A1 30 methadone application indicating that levels comparable EXAMPLE 11: Sensitization of mamma carcinoma with the in vitro concentrations could be reached. The cells for treatment with different anticancer agents. serum concentrations of doxorubicin were found be- tween 156 ng/mL and 198 ng/mL. These results demon- [0154] As shown by flow cytometry, the Her2/Neu-re- strate that D,L-methadone and the co-treatment using 5 sistent mamma carcinoma cell line JIMT-1 expresses the doxorubicinand D,L-methadonesignificantly inhibited tu- m-opioid receptor (s. Fig. 20). As shown by FACS anal- mour growth in vivo. ysis the combination treatment of D,L-methadone and doxorubicin dose-dependently induces apoptosis in EXAMPLE 7: D,L-methadone sensitizes glioblasto- JIMT-1 cells (see Figure 21). It could be shown that cell ma cells for doxorubicin treatment. 10 death inductionof JIMT-1 cells using D,L-methadoneand doxorubicin cotreatment depends on caspase activation [0150] As shown by flow cytometry, the glioblastoma (s. Figures 22 and 23). cell lines A172 and U118MG (s. Fig. 8) as well as primary glioblastoma cells (s. Fig. 11A) and glioblastoma-initiat- EXAMPLE 12: Sensitization of cancer cells for dox- ing stem cells (s. Fig.12A) express opioid receptors. In 15 orubicin treatment by the opioid fentanyl. all these cells and cell lines the combination treatment of D,L-methadone and doxorubicin dose-dependently in- [0155] As exemplified for the T-cell derived leukemia duces apoptosis (see Figures 9, 10, 11B and 12B). As cell line CEM it could be shown that also the opioid fen- exemplified for the glioblastoma cell line A172 it could be tanyl was able to sensitize the CEM cells for treatment shown that cell death induction of glioma cells using D,L- 20 using doxorubicin (s. Fig. 24). In a further in vitro exper- methadone and doxorubicin cotreatment depends on iment, the opioid buprenorphine sensitized leukemia caspase activation (s. Fig. 13). Furthermore, it could be cells (HL-60) for apoptosis due to doxorubicin (s. Fig. 25). shown that D,L-methadone reversed deficient activation of apoptosis pathways by doxorubicin in glioblastoma- EXAMPLE 13: Combination treatment with D,L-Meth- initiating stem cells (s. Fig. 14). 25 adone and cisplatin (CDDP) kills and activates cas- pases in different leukemia cells. EXAMPLE 8: Effect of D,L-methadone on doxoru- bicin uptake and efflux. [0156] The cell death potential of D,L-methadone on different leukemia-cell lines was shown on human T cell [0151] In vitro results using the glioblastoma cell line 30 leukemia, human acute myeloid leukemia, human B cell A172 demonstrated that D,L-methadone could enhance precursor leukemia, human B cell leukemia. All tested the uptake and also inhibit the efflux of doxorubicin (s. cell lines expressing opioid-receptors in a moderate level Fig. 15). This gives an explanation for the sensitization on their cell surface (Fig.27). of cancer cell towards treatment with anticancer drugs. [0157] These leukemiacells could only be killed slightly 35 by D,L-methadone (Fig. 28, white bars ). In order to show EXAMPLE 9: Effect of doxorubicin or cisplatin on opi- that different anticancer agents (substances) will act syn- oid receptor expression on cancer cells. ergistically, human T cell leukemia, human acute myeloid leukemia, human B cell precursor leukemia and human [0152] As shown for the glioblastoma cell line A172, B cell leukemia were treated with different concentrations doxorubicin leads to a 6-fold increase in opioid receptor 40 of D,L-methadone (- CDDP) or cisplatin alone or with expression (s. Fig. 16). It could be shown that this mech- D,L-methadonein additionto cisplatin (+CDDP) (Fig.28). anism holds also true for other cancer types and antican- The combination treatment strongly induced cell death cer drugs since in the promyelocytic leukemia cell line in different leukemias depending on different concentra- HL60, the cisplatin-treatment leads to a 2.1-fold increase tions of cisplatin or/and D,L-methadone. in opioid receptor expression (s. Fig. 19). 45 [0158] The molecular pathways of cell killing was shown in more detail and it was shown how the combi- EXAMPLE 10: Sensitization of leukemia, pancreatic nation treatment with an opioid receptor agonist i.e. D,L- and ovarian cancer cells for treatment with different methadone and an anticancer agent i.e. cisplatin induce anticancer agents. apoptosis., First the effector molecules of apoptosis sig- 50 naling was shown, that are activated in different leukemia [0153] In further in vitro analyses it could be demon- cells (human T cell leukemia, human acute myeloid strated that D,L-methadone sensitizes leukaemia cancer leukemia, human B cell precursor leukemia). Combina- cells (Nalm-6), pancreatic cancer cells (Nalm6) and ovar- tion treatment of D,L methadone in combination with Cis- ian cancer cells (A2780) for etoposide or cisplatin treat- platin (+ CDDP) was compared to cells treated with D,L- ment (s. Fig. 17). Furthermore, also chronic lymphocytic 55 methadone (- CDDP) or cisplatin alone. It was shown leukaemia cells (CLL) could be sensitized by D,L-meth- that treating the different leukemia cells with D,L-meth- adone for apoptotic treatment using Fludarabine (s. Fig. adone in addition to cisplatin (+ CDDP), the activation of 18). the caspase cascade in leukemia cells was induced. A

16 31 EP 2 716 291 A1 32 strong activation of caspase-3 (active caspase-3 p19, al., 1999). To show that cisplatin has an influence to the p17), caspase-9 (active caspase-9 p37), and caspase-2 opioid-receptor expression, the different leukemia cells and cleavage of the prototype substrate of caspase-3, (human T cell leukemia, human acute myeloid leukemia, poly-(ADP-ribose)-polymerase (PARP) (cleavage p85 human B cell precursor leukemia) were treated with cis- and or a downregulation of PARP p116) was induced 5 platin. Afterwards, the relative amount of opioid-recep- depending on the combination treatment (Fig.29). tors compared to untreated cells was measured by flow- [0159] The role of the caspase cascade in apoptosis cytometry. It was shown that cisplatin strongly increased induction was further investigated with the broad-spec- opioid-receptor expression (Fig. 31). Therefore opioid re- trum inhibitor of caspases zVAD.fmk. Different leukemia ceptor-agonists like D,L-methadone can bind in higher cells (human T cell leukemia, human acute myeloid10 amounts to cells, co-treated with cisplatin or other anti- leukemia, human B cell precursor leukemia) were pre- cancer agents which are able to induce a higher level of incubated with 50mM of zVAD.fmk (+ zVAD.fmk, white expressed opioid receptors. This effect results in the bars) or without zVAD.fmk (-zVAD.fmk, black bars) and higher cell death potential of the combination treatment treated with D,L-methadone in addition to cisplatin. of an opioid receptor agonist i.e D,L-methadone and an zVAD.fmk strongly decreased cell death after combina- 15 anticancer agent i.e. cisplatin. tion treatment with D,L-methadone and cisplatin (Fig.30) [0162] It is shown in the invention that the opioid re- underlining the dependence on caspases activation. ceptor agonist which has a longer minimal duration of [0160] The apoptotic machinery is tightly controlled by effectiveness like D,L methadone has a better result than anti-apoptotic factors like XIAP and Bcl-xL and pro-ap- one, that has a shorter minimal duration of effectiveness optotic factors like Bax (Fulda, 2009a; Fulda, 2009b). 20 like morphine compared to D,L methadone (Figure 33 XIAP was strongly downregulated (p57) and or cleaved and Figure 34. (p30) in different leukemia cells treated with D,L-metha- done in addition to cisplatin (+CDDP) (Fig.29) depending EXAMPLE 15: Apoptosis induction by D,L-metha- on different concentrations of cisplatin or/and different done and doxorubicin depends critically on opioid concentration of D,L-methadone. A strong upregulation 25 receptor activation in glioblastomas of Bax (p21) is induced in human T cell leukemia induced after treatment with D,L-methadone in addition to cispl- [0163] To show the role of opioid-receptor triggering in atin (+ CDDP). The combination of D,L-methadone and apoptosis induction in glioblastomas which is a solid tu- cisplatin sensitizes different leukemia cells for apoptosis mor, glioblastoma cells were treated with D,L-metha- via the activation of caspases and by downregulation and 30 done, doxorubicin or with the opioid-receptor antagonist inhibition of anti-apoptotic factors such as XIAP and up- naloxone alone or in different combinations with each regulation of pro-apoptotic factors such as Bax. So it other (Fig. 32). shown that the opioid receptors are receptors which in- [0164] After 120h and 144h it was shown that blocking duce cell death and acitivate apoptosis pathways involv- opioid-receptors by naloxone strongly reduced the ap- ing caspase activation, downregulation and or clevage 35 optosisrates of thecombination treatment withD,L-meth- of PARP, and or downregulation of anti-apoptotic factors, adone and doxorubicin (Fig.32). and or upregulation of pro-apoptotic factors, and or down- regulation and inhibition of inhibitory apoptotic proteins EXAMPLE 16: Different duration of effectiveness of (IAP). Therefore the opioid receptors are a new unknown different opioids induces different rates of apoptosis way with an new mechanism of inducing cell death, be- 40 using D,L-methadone or morphine in combination side the common known cell death receptors/death in- with anticancer drugs such as doxorubicin in gliob- ducing ligands systems and mechanisms like the lastomas or in leukemias CD95/CD95L-System. [0165] The efficiency of cell death induction after treat- EXAMPLE 14: Cisplatin strongly induces opioid-re- 45 ing glioblastoma cells or leukemia cells with opioids de- ceptor expression in leukemia cells. pends on the duration of effectiveness of the opioids. The minimal duration of effectiveness of methadone is 5-7 [0161] The efficiency of cell death induction and acti- hours and the minimal duration of effectiveness of mor- vation of effector molecules in apoptosis pathways after phineis 2-4hours. Combinationtreatment withD,L-meth- treating leukemia cells with opioid-receptor-agonists i.e. 50 adone and doxorubicin strongly induced high cell death D,L-methadone depend on the amount of opioid-recep- rates in glioblastoma cells (Figure 33 A) and leukemia tors displayed on the cell’s surface. Combination treat- cells (Figure 34 A). In contrast, combination treatment ment with D,L-methadone and cisplatin profoundly kills with morphine and doxorubicin induced lower cell death leukemia cells with moderate opioid receptor expression, rates in glioblastoma cells (Figure 33 B) and leukemia which could only be killed slightly by D,L-methadone or 55 cells (Figure 34 B). This indicates that the rates of induc- cisplatin alone. Chemotherapeutics enhance the expres- tion of cell death after combination treatment of opioids sion of the receptor CD95 (FAS,APO-1) in leukemia cells with anticancer drugs depend also on the duration of ef- which is a special known death receptor (Posovszky et fectiveness of opioids. The effect is found also at other

17 33 EP 2 716 291 A1 34 anticancer agents. totic proteins XIAP and Bcl-x L involved in the occurrence of resistances in many malignancies like ALL or NHL Example 17 Combination treatment using D,L-meth- (Addeo et al., 2005) is markedly enhanced. This suggests adone in addition to doxorubicin mediated cell pro- that combination treatment of D,L-methadone and dox- liferation inhibition and G2/M cell cycle arrest in 5 orubicin strongly increases apoptosis induction and glioblastoma cells. could improve their anti-tumour efficacy synergistically. [0170] Resistance to conventional chemotherapeutic [0166] Cell proliferation is governed by the eukaryotic drugs is a limiting factor in the effectiveness of therapies cell cycle (Sherr CJ. Cancer cell cycles. Sciencewhereby multidrug resistances as a result of the overex- 1996;274:1672-7), which is regulated not only by growth 10 pression of drug transporters such as P-gp are also well- factors but also by a variety of signals that act to inhibit characterized. While in healthy cells the P-gp expression cell cycle progression. Most of cancer cells have 4 cell belongs to the normal cellular defense system, in human division cycle stages: gap 1 (G1), synthesis (S), G2, and cancer cells the overexpression of P-gp correlates with mitosis (M). Chromosomal DNA replicates during the S decreased survival and poor outcome (Diestra et al., phase. As glioblastoma cells divide, the cell cycle should 15 2003). D,L-methadone could be shown to be a substrate move from the S stage to the G2/M stage. This tightly of P-gp inhibiting its action (Crettol et al., 2007). As shown controlled temporal order is imposed by the sequential herewith, co-treatment of doxorubicin with D,L-metha- activation of a number of protein kinases known as cyclin- done enhances doxorubicin cell-uptake and furthermore dependent kinases (CDKs), by the formation of complex- inhibits doxorubicin-efflux out of leukemia cells, suggest- es with various cyclins. Opioid receptor agonist for ex- 20 ing that D,L-methadone sensitizes leukemia cells for dox- ample Methadone in combination with doxorubicin inhib- orubicin-induced apoptosis by increasing concentrations its proliferation of cancer cells such as glioblastoma cells of doxorubicin within the cells. and induces S/G2-M cell cycle arrest in glioblastoma [0171] Combination treatment using D,L-methadone cells. and doxorubicin induced apoptosis and caspase activa- 25 tion in BCP-ALL-cells expressing moderate amounts of DISCUSSION opioid-receptors on their surface. The enhanced toxicity ofthis combination treatment was foundto be additionally [0167] The examples provide evidence that D,L-meth- associatedwith an increasedexpression of opioid-recep- adone induces apoptosis, activates caspases and in- tors after doxorubicin treatment. Therefore, D,L-metha- creases doxorubicin-induced cell death in leukemia cells 30 done can bind in higher amounts to cells co-treated with depending on opioid-receptor activation inducing the doxorubicin. These results indicate that the enhanced downregulation of cAMP. In addition, it could be demon- toxicity in the combination treatment with D,L-methadone strated for the first time, that D,L-methadone can strongly and doxorubicin is associated with the upregulation of reduce tumour growth of ALL in a xenograft-modelin opioid-receptor expression mediated by doxorubicin and vivo. Noticeably, this tumour-killing effect could be en- 35 furthermore with an increased uptake and decreased ef- hanced by the combination of D,L-methadone with the flux of doxorubicin mediated by D,L-methadone. Both anticancer drug doxorubicin. agents can hence exert their cytotoxic potential to a high- [0168] Methadone is a m-opioid receptor agonist bind- er extent. ing to m-opioid receptors if presented on cells. It was [0172] Opioid receptors signal by catalysing ligand-de- 40 found that D,L-methadone kills strongly xenograft-de- pendent nucleotide exchange on Gi, thereby inhibiting rived ALL-cells expressing high levels of opioid recep- adenylyl cyclase and modulating N-type calcium chan- tors. In contrast, D,L-methadone induces cell death only nels as well as G protein-gated inwardly rectifying potas- slightly in xenograft-derived ALL-cells and -cell lines ex- sium (GIRK)-type potassium channels leading to chang- pressing moderate opioid receptor amounts indicating es in cell signalling (Fig. 7). Dependence of apoptosis that D,L-methadone-induced apoptosis seems to de-45 induction on opioid-receptor triggering is underlined by pend on critical levels of opioid receptor expression in their inhibition. Blocking opioid-receptor signaling with leukemia cells. the opioid receptor antagonist naloxone inhibited combi- [0169] Combination treatment may prove to be advan- nation treatment with D,L-methadone and doxorubicin- tageous in malignancies that still partially respond to ei- induced apoptosis and caspase activation in a high rate, ther treatment alone as different therapeutics are known 50 suggesting that opioid-receptor triggering by D,L-meth- to interact with each other amplifying weaker death sig- adone is involved in apoptosis induction and caspase nals. Combination treatment with D,L-methadone and activation (Fig. 7). Based on this mechanism of action doxorubicin enhances the anti-tumour efficacy of both every opioid receptor agonist independent of the individ- agents synergistically in BCP-ALL-cells expressing mod- ual opioid receptor should kill tumour cells by apoptosis erate levels of opioid-receptors and increases strongly 55 since all opioid receptors are linked to the adenylyl cy- caspase activation playing a critical role in apoptosis in- clase via the Gi pathway. duction in sensitive and resistant cancer cells (Fulda, [0173] Further experiments prove the general applica- 2009c). In addition, the downregulation of the anti-apop- bility of the above described combination therapy:

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Broad spectrum of cancers. Several diverse can- new drug combinations which might represent im- cer types can be treated with the combination of opi- proved treatment due to increased efficacy and/or oid receptor agonists such as safety.

Broad spectrum of opioids. In accordance with the 5 FIGURES Gi-associated mechanism of action, several struc- turally and pharmacologically distinct opioids like [0177] D,L-methadone, buprenorphine and fentanyl could sensitize the cancer cells for anticancer drugs. Fig. 1: D,L-methadone kills ALL cells ex vivo de- 10 pending on critical levels of opioid receptor ex- Broad spectrum of anticancer drugs. For several pression structurally and pharmacologically distinct antican- cer drugs it could be shown that they increase opioid (A) Human ALL-SCID6 and ALL-SCID3, ALL- receptor expression and show increased influx/de- SCID7 and pre-B-ALL-SCID derived from xe- creased efflux due to the co-applied opioid agonist. 15 nografted mice display different levels of opioid- receptors on their cell surface. ALL-SCID6, ALL- SUMMARY SCID3 and ALL-SCID7 were stained with naloxone-fluoresceine measuring opioid-recep- [0174] It has to be emphasized that the interaction be- tor expression (OR, thick black curve) and ana- tween opioids and anticancer agents represents a self- 20 lyzed by flowcytometry. Controls (Co) are exhib- reinforcing feedback loop as illustrated by Figure 26. In ited as thin black curves. the first path of this loop opioids enhance the cellular (B) ALL-SCID6, ALL-SCID3, ALL-SCID7 and uptake and inhibit the efflux of anticancer drugs. On the pre-B-ALL-SCID were treated with different con- second path of said loop the accumulating anticancer centrations of D,L-methadone (as indicated). Af- drugs lead to an increased expression of opioid recep- 25 ter 24h and 48h, the percentages of apoptotic tors. Hence, both agents can exert their cytotoxic poten- cellswere measured by FSC/SSC-analysis.The tial to a higher extent. percentage of specific apoptosis was calculated [0175] The present examples could verify the clinical as follows: 100 x [experimental dead cells(%) - relevance with patient-derived ALL-cellsex vivo and spontaneous dead cells in medium (%)] / [100% could show for the first time that D,L-methadone as mon- 30 - spontaneous dead cells in medium(%)]. Col- otherapy or in combination with doxorubicin leads to a umns, mean of triplicates; bars, SD<10%. strong tumour growth inhibition in a patient-derived leukemia model. Both the anti-leukemic efficacy and the Fig. 2: Combination treatment with D,L-metha- side effects of D,L-methadone alone or in combination done and doxorubicin induces apoptosis in ALL- with doxorubicin were comparable with those of doxoru- 35 cells expressing moderate amounts of opioid re- bicin alone. However, only the combination treatment ceptors was able to achieve a longer lasting growth inhibition. The serum concentrations of methadone in mice corre- (A) Different BCP-ALL-cell lines (Tanoue, lated with the concentrations showing in vitro cytotoxicity. Nalm6 and Reh) express a moderate number of [0176] In sum, a combination therapy of opioids and 40 opioid-receptors on their cell surface. Tanoue, anticancer drugs could improve the cancer therapies in Nalm6 and Reh were stained with naloxone-flu- several ways: oresceine measuring opioid-receptor expres- sion (OR, thick black curve) and analyzed by • Due to the upregulation of opioid receptors, former flowcytometry. Controls (Co) are exhibited as opioid insensitive cancer types could be subjected 45 thin black curves. to an opioid therapy. (B) BCP-ALL-cell lines (Tanoue, Nalm6 and • Due to the opioid-induced intracellular accumulation Reh) were treated with different concentrations of anticancer drugs the efficacy of the treatment is of D,L-methadone (as indicated) alone (-Doxo, enhanced. white columns), with doxorubicin alone or with • This could lead to therapy of cancer types which are 50 D,L-methadone (as indicated) in addition to dox- difficult to treat. orubicin (+Doxo,black columns). For thecell line • Furthermore, this might allow a dose reduction for Tanoue, we used doxorubicin in a concentration the anticancer drugs enhancing the safety and pa- of 0.06mg/mL, for Nalm6 and Reh a concentra- tient compliance of the chemotherapy. tion of 0.01mg/mL. 120h after stimulation, per- • Finally, also resistant cancer cells could be re-sen- 55 centages of apoptotic cells were measured by sitized for an anticancer treatment. hypodiploid DNA analysis. • In addition, the numerous opioids and numerous an- (C) D,L-methadone strongly enhances doxoru- ticancer drugs on the market open up the way for bicin sensitivity of xenograft-derived-BCP-ALL-

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patient-cells ex vivo. Xenograft-derived-BCP- pression whereas D,L-methadone enhances ALL-cells (pre-B-ALL-SCID) were treated with doxorubicin uptake and inhibits its efflux different concentrations of D,L-methadone (as indicated) alone (-Doxo, white columns) with (A) Doxorubicin enhances opioid receptor ex- 0.01mg/mL doxorubicin alone or with D,L-meth- 5 pression on the cells’ surface. The BCP-ALL- adone in addition to doxorubicin (+Doxo, black cell line Tanoue was treated for 96h with columns).48h after stimulation, the percentages 0.06mg/mL doxorubicin. After staining of doxo- of apoptotic cells were measured by FSC/SSC- rubicin-treated (+Doxo) and untreated cells analysis. The percentage of specific apoptosis (-Doxo) with naloxone-fluoresceine relative flu- was calculated as described in Fig.1B. Col-10 orescence intensities were determined flowcy- umns, mean of triplicates; bars, SD<10%. tometrically. X-fold increase in opioid receptor expression is shown after subtracting the cells’ Fig. 3: D,L-methadone in combination with dox- autofluorescence (-Doxo) and doxorubicin fluo- orubicin restores deficient activation of apoptot- rescence (+Doxo). ic pathways in BCP-ALL-cells expressing mod- 15 (B) D,L-methadone enhances doxorubicin up- erate amounts of opioid receptors in vitro take and inhibits its efflux. The BCP-ALL-cell line Tanoue was either pre-treated with 0.3mg/mL (A) D,L-methadone and doxorubicin co-treat- doxorubicin (Doxo) alone or with a combination ment provokes caspases activation. The BCP- of doxorubicin and 10mg/mL D,L-methadone ALL-cell line Tanoue was treated with D,L-meth- 20 (Doxo + methadone) for 24h. Maximal doxoru- adone (as indicated) alone (-Doxo), with bicin cell uptake was analyzed via doxorubicin 0.06mg/mL doxorubicin (+Doxo) alone or with fluorescence in cells using flowcytometry after D,L-methadone (as indicated) in addition to dox- 24h (Oh, max. uptake). After washing doxoru- orubicin (+Doxo). After 120h Western blot anal- bicin-treated cells, cells were either left untreat- yses for caspase-2, caspase-9, caspase-3 and 25 ed (Doxo) or treated with 10mg/mL D,L-metha- PARP were performed. Downregulation of pro- done (Doxo + Methadone) and incubated for dif- caspase-2 was detected at ∼48 kDa. The active ferent points in time (8h, 24h). Doxorubicin efflux fragment of caspase-9 was detected at ∼37kDa, was analyzed via doxorubicin fluorescence in the active fragment of caspase-3 at ∼19 kDa and cells using flowcytometry after 8h and 24h. Val- ∼17 kDa and PARP cleavage at ∼85 kDa. Equal 30 ues are mean fluorescence intensities +/-SE. protein loading was controlled by anti-β-actin antibody. Fig. 5: Combination treatment with D,L-metha- (B) D,L-methadone and doxorubicin-induced done and doxorubicin induced apoptosis de- apoptosis depends on caspase activation. Pre- pends on opioid-receptor triggering via down- incubation of the cell line Tanoue with 50 mM of 35 regulation of cAMP the caspase inhibitor zVAD.fmk for 1 h (white columns) or without pre-treatment (black col- (A) Inhibition of opioid-receptor triggering inhib- umns) was followed by addition of D,L-metha- its apoptosis induction mediated by combination done (as indicated) in combination with treatment with D,L-methadone and doxorubicin. 0.06mg/mL doxorubicin. Apoptosis induction40 The BCP-ALL-cell line Tanoue was incubated was detected 120h after stimulation by with 60mg/mL naloxone (Naloxone), m 3g/mL FSC/SSC-analysis. The percentage of specific D,L-methadone (D,L-Methadone) and apoptosis was calculated as described in 0.06mg/mL doxorubicin (Doxo) alone or in dif- Fig.1B. Columns, mean of triplicates; bars, ferent combinations as indicated. After 96h, the SD<10%. 45 percentages of apoptotic cells were measured (C) Downregulation of XIAP and Bcl-xL by D,L- by FSC/SSC-analysis. methadone and doxorubicin co-treatment. The (B) Inhibition of opioid-receptor triggering inhib- cell line Tanoue was treated with D,L-metha- its caspase activation mediated by combination done (as indicated) alone (-Doxo), with treatment with D,L-methadone and doxorubicin. 0.06mg/mL doxorubicin (+Doxo) alone or with 50 The BCP-ALL-cell line Tanoue was incubated D,L-methadone (as indicated) in addition to dox- with 60mg/mL naloxone (Naloxone), m 3g/mL orubicin (+Doxo). After 120h Western blot anal- D,L-methadone (D,L-Methadone) and

yses for XIAP and Bcl-x L were performed. XIAP 0.06mg/mL doxorubicin (Doxo) alone or in dif- was detected at 58 kDa and Bcl-xL at ∼30 kDa. ferent combinations as indicated. Western blot Equal protein loading was controlled by anti-β- 55 analyses for caspase-2, caspase-9, caspase-3 actin antibody. and PARP were performed after 96h of incuba- tion. Downregulation of procaspase-2 was de- Fig. 4: Doxorubicin enhances opioid receptor ex- tected at ∼48 kDa. The active fragment of cas-

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pase-9 was detected at ∼37 kDa, of caspase-3 A172 were stained with naloxone fluorescein meas- at -19 kDa and ∼17 kDa and PARP cleavage at uring opioid receptor expression (OR, thick black ∼85 kDa. Equal protein loading was controlled curve) and analysed by flow cytometry. Controls (Co) by anti-β-actin antibody. are exhibited as thin black curves. (C) Increasing cAMP levels via repression of 5 phosphodiesterase activity inhibits apoptosis. Fig. 9: D,L-methadone sensitizes glioblastoma The BCP-ALL-cell line Tanoue was incubated cells for doxorubicin treatment. Glioblastoma for 96hwith 200 mM3-Isobutyl-1-methylxanthine cells A172 were incubated with 3 mg/mL D,L-metha- (IBMX), 3mg/mL D,L-methadone (D,L-Metha- done alone, with 0.1mg/mL doxorubicin (0.1mg/mL done) and 0.06 mg/mL doxorubicin (Doxo) alone 10 Doxo) alone or with 3 mg/mL D,L-methadone in com- or in different combinations as indicated. bination with 0.1mg/mL doxorubicin (Doxo + D,L- (D) Uncoupling inhibitory G-proteins from opioid Methadone). Control represents untreated glioblas- receptors inhibits apoptosis by preventing inhi- toma cells. After 144h light microscopy pictures were bition ofadenylyl cyclase. The BCP-ALL-cell line taken. Cotreatment of A172 with 3 mg/mL D,L-meth- Tanoue was incubated with 20ng/mL pertussis 15 adone and 0.1 mg/mL doxorubicin led to detachment toxin (PTX), m 3g/mL D,L-methadone (D,L- ofthe cells from theground, membrane-blebbing and Methadone) and 0.06 mg/mL doxorubicin (Doxo) cell-shrinkage. alone or in different combinations as indicated. After 96h, the percentages of apoptotic cells Fig. 10: Combination treatment with D,L-metha- weremeasured by FSC/SSC-analysis. The frac- 20 done and doxorubicin induces apoptosis in tion of apoptotic cells were determined by glioblastoma cells. A172 and U118MG glioblasto- FSC/SSC-analysis. The percentage of specific ma cells were treated with different concentrations apoptosis was calculated as described in of D,L-methadone (10, 3, 1mg/mL) alone (Medium, Fig.1B. Columns, mean of triplicates; bars, white columns), with doxorubicin (0.1mg/mL Doxo, SD<10%. 25 black columns) alone or with different concentrations of D,L-methadone (10, 3, 1 mg/mL) in addition to dox- Fig. 6: D,L-methadone inhibits growth of leuke- orubicin (0.1mg/mL Doxo, black columns). After mia xenografts and increases doxorubicin sen- 120h and 144h the percentages of apoptotic cells sitivity Fragments of an in vivo passage of a patient- were measured by hypodiploid DNA analysis. The derived TALL (ALL-SCID6, see also Fig. 1) were 30 percentage of specific apoptosis was calculated as transplanted into male NSG mice. Mice were treated follows: 100 x [experimental dead cells (%) - spon- with D,L-methadone alone (n=8, orally day 1-76, taneous dead cells in medium (%)] / [100% - spon- D,L-Methadone), with doxorubicin alone (n=8, i.v. taneous dead cells in medium (%)]. Columns, mean day 46,53,60,76, Doxorubicin) or with a combination of triplicates; bars, SD <10%. Similar results were treatment with D,L-methadone and doxorubicin35 obtained in three independent experiments. (n=8, D,L-Methadone + Doxo). D,L-methadone was used in weekly increasing doses from 20 up to Fig. 11: D,L-methadone sensitizes primary hu- 120mg/kg/day and doxorubicin in a dose of 3mg/kg. man glioblastoma cells for doxorubicin treat- As control group xenografted mice were treated i.v. ment. (A) Primary human glioblastoma cells were with 10% Tween 80 in saline (n=8, Vehicle). For 76 40 stained with naloxone fluorescein measuring opioid days after transplantation all mice were monitored receptor expression (OR, thick black curve) and an- for tumour growth, body weight and health condition. alyzed by flow cytometry. Control (Co) is exhibited *significant to vehicle (p<0.05, Mann-Whitney U as thin black curve. (B) Primary human glioblastoma test). cells were treated with different concentrations of 45 D,L-methadone (3, 1mg/mL) alone (Medium, white Fig. 7: Opioid receptor signaling. Stimulation of columns) with 0.1mg/mL doxorubicin (0.1mg/mL opioid receptors (OR) by agonists like D,L-metha- Doxo, black columns) alone or with D,L-methadone done leads to an activation of the inhibitory Gi-pro- (3, 1mg/mL) in addition to 0.1mg/mL doxorubicin tein. The αi-subunit inactivates adenylyl cyclase (0.1mg/mL Doxo, black columns). After 120h the per- (AC) resulting in a reduction of cAMP levels within 50 centages of apoptotic cells were measured by hy- the cell which in turn leads to apoptosis which might podiploid DNA analysis. The percentage of specific be mediated by several different modulators. Also apoptosiswas calculated as describedin Fig.1c.Col- the βγ-subunits ofthe G i-proteinmodulate the activity umns, mean of triplicates, bars, SD <10%. Similar of different effectors like the inhibition of Ca2+- and results were obtained in three independent experi- the activation of K+-channels. 55 ments

Fig. 8: Opioid receptor expression on glioblast- Fig. 12: D,L-methadone sensitizes glioblastoma- oma cells. The glioblastoma cell lines U118MG and initiating stem cells for doxorubicin treatment.

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(A) Glioblastoma-initiating stem cells were stained for XIAP and Bcl-xL were performed. XIAP was de- with naloxone fluorescein measuring opioid receptor tected at 58kDa and Bcl-xL was detected at 30kDa. expression (OR, thick black curve) and analyzed by Equal protein loading was controlled by anti-β-actin flow cytometry. Control (Co) is exhibited as thin black antibody. curve. (B) Glioblastoma-initiating-stem cells were 5 treated with different concentrations of D,L-metha- Fig. 14: D,L-methadone reversed deficient acti- done (10, 3, 1mg/mL) alone (Medium, white col- vation of apoptosis pathways by doxorubicin in umns), with 0.1mg/mL doxorubicin (0.1 mg/mL Doxo, glioblastoma-initiating-stem cells. (A) Glioblast- black columns) alone or with D,L-methadone (3, oma-initiating-stem cells were treated with D,L- 1mg/mL) in addition to mg/mL 0.1 doxorubicin 10 methadone (3mg/mL) alone, with 0.1 mg/mL doxoru- (0.1mg/mL Doxo, black columns). After 144h the per- bicin (0.1mg/mL Doxo) alone or with D,L-methadone centages of apoptotic cells were measured by hy- (3mg/mL) in addition to doxorubicin m (0.1g/mL podiploid DNA analysis. The percentage of specific Doxo). After 144h Western blot analyses for cas- apoptosiswas calculated as described inFig.1c. Col- pase-10, -2, -9, -3 and PARP were performed. Down- umns, mean of triplicates; bars, SD <10%. Similar 15 regulation of procaspase-10 was detected at results were obtained in three independent experi- ∼58kDa and of procaspase-2 at ∼48kDa. The active ments. fragment of caspase-9 was detected at ∼37kDa, the active fragment of caspase-3 at ∼19kDa and ∼17kDa Fig. 13:Cell death induction of glioblastoma cells and PARP cleavage at ∼85kDa. Equal protein load- using D,L-methadone and doxorubicin cotreat- 20 ing was controlled by anti- β-actinantibody. (B)Gliob- ment depends on caspases activation. (A) D,L- lastoma-initiating-stem cells were treated with D,L- methadone restored deficient caspases activation methadone (3mg/mL) alone, with 0.1 mg/mL doxoru- by doxorubicin in glioblastoma cells. A172 were bicin (0.1mg/mL Doxo) alone or with D,L-methadone treated with different concentrations of D,L-metha- (3mg/mL) in addition to doxorubicin (0.1 mg/mL Doxo) 25 done (3, 1mg/mL) alone, with 0.1mg/mL doxorubicin for 144h. Western blot analyses for XIAP, Bcl-x L and (0.1mg/mL Doxo) alone or with different concentra- Bcl-xS were performed. XIAP was detected at tions of D,L-methadone (3, 1mg/mL) in addition to 58kDa, Bcl-x L was detected at 30kDa and Bcl-x Swas doxorubicin (0.1mg/mL Doxo). After 144h Western detected at 27kDa . Equal protein loading was con- blot analyses for caspase-10, -2, -9, -3 and PARP trolled by anti-β-actin antibody. were performed. Downregulation of procaspase-10 30 was detected at ∼58kDa and of procaspase-2 at Fig. 15: D,L-methadone enhances doxorubicin ∼48kDa. The active fragment of caspase-9 was de- uptake and inhibits ist efflux. (A) D,L-methadone tected at ∼37kDa, the active fragment of caspase-3 enhances doxorubicin-accumulation in the glioblas- at ∼17kDa and PARP cleavage at∼ 85kDa. Equal toma cell line A172. A172 were incubated with protein loading was controlled by anti-β-actin anti- 35 0.3mg/mL doxorubicin alone or in combination with body. (B) Inhibition of caspases activation with the 10mg/mL D,L-methadone. After 4, 8 and 24h incu- broad spectrum inhibitor of caspases zVAD.fmk bation the fluorescence intensity of doxorubicin blocks apoptosis induced by cotreatment of D,L- (Doxo) using flow cytometry analysis were deter- methadone and doxorubicin in A172 cells. Glioblas- mined. In the graphic the relative doxorubicin-uptake toma cells A172 were treated with different concen- 40 is shown. Columns, mean of triplicates; bars, SD trations of D,L-methadone (10, 3, 1mg/mL) in com- <10%. Similar results were obtained in three inde- bination with 0.1mg/mL doxorubicin (+ 0.1mg/mL pendent experiments. (B) A172 were incubated with Doxo) in the absence (Medium, black columns) or 0.3mg/mL doxorubicin for 4h. At distinct points in time presence of 50mmol/L of zVAD.fmk (white columns, (4, 8 and 24h) after washing the doxorubicin-con- 50mmol/L zVAD.fmk). After 120h and 144h, the per- 45 taining medium away (Oh) the fluorescence intensity centages of apoptotic cells were measured by hy- of doxorubicin using flow cytometry analysis was de- podiploid DNA analysis. The percentage of specific termined. In thegraphic the relative doxorubicin-con- apoptosiswas calculated as described inFig.1c. Col- tent is shown. Columns, mean of triplicates; bars, umns, mean of triplicates; bars, SD <10%. Similar SD <10%. Similar results were obtained in three in- results were obtained in three independent experi- 50 dependent experiments. ments. (C) Downregulation of XIAP and Bcl-xL in glioblastoma cells by using D,L-methadone in com- Fig. 16: 1 Doxorubicin enhances opioid receptor bination with doxorubicin. Glioblastoma cells A172 expression on the cell surface. were treated with different concentrations of D,L- methadone (3, 1mg/mL) alone, with 0.1mg/mL dox- 55 (A) The glioblastoma cell line A172 was treated orubicin (0.1mg/mL Doxo) alone or with D,L-metha- for 106 h with 0.1 mg/mL doxorubicin. After stain- done (3, m 1g/mL) in addition to doxorubicin ing of doxorubicin-treated (doxorubicin) and un- (0.1mg/mL Doxo) for 144h. Western blot analyses treated cells with naloxone-fluorescein

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(naloxone) relative fluorescence intensities metastasis of a 62-year old patient with breast can- were determined flowcytometrically. (B) Tabel- cer who was clinically resistant to Herceptin displays lary summary of untreated and doxorubicin con- opioid-receptors on its cell surface. JIMT-1 cells trol cells, naloxone treated cells, whereas were stained with naloxone-fluorescein measuring 5 D(naloxone-control) represents the median fluores- opioid-receptor expression (OR, thick black curve) cence intensities after subtracting the cells’ and analyzed by flow cytometry. Controls (Co) are autofluorescence (control). exhibited as thin black curves.

Fig. 17: D,L-methadone sensitizes leukemia can- Fig. 21: Cell cycle analysis and apoptosis of the cer cells (Nalm-6), pancreatic cancer cells10 Her2/neu-resistent mamma carcinoma cell line (Colo357) and ovarian cancer cells (A2780) for JIMT-1 treated with a combination of D,L-meth- etoposide or cisplatin treatment. The Nalm6, adone and doxorubicine. The human JIMT-1 cell Colo357 and A2780 cells were treated with different line was treated with 1, 3 or 10 mg/mL of methadone concentrations of D,L-methadone (10, 3, m1g/mL) alone (1,3,10 Met -Doxo) , with 0.015 mg/mL of dox- alone (Medium, white columns), with 0.03m g/mL 15 orubicin (Doxo) or a combination of 0.015 mg/mL of Etoposide or 0.3 mg/mL cisplatin alone or with D,L- doxorubicin with 1, 3 or 10m g/mL of methadone methadone (10, 3, 1 mg/mL) in addition to 0.03 mg/mL (1,3,10 Met +Doxo). (A) A FACS analysis of the cells Etoposide (0.03 mg/mL Etoposide, black columns) revealed that the combination of both substances or 0.3 mg/mL cisplatin (0.3 mg/mL cisplatin, black col- dose-dependently increased apoptosis of the JIMT- umns). After 120 to 144h the percentages of apop- 20 1 cells at 96 hours after treatment. (B) A FACS anal- totic cells were measured by hypodiploid DNA anal- ysis performed at 96 hours (left side of the figure) ysis. The percentage of specific apoptosis was cal- and120 hours (right side of the figure)after treatment culated as described in Fig.1c. Columns, mean of showed further increased levels of apoptosis due to triplicates; bars, SD <10%. Similar results were ob- combination treatment. 0 or 0,015 mg/ml doxorubicin tained in three independent experiments. 25 (black bars), 1 mg/mL methadone plus 0 or 0,015 mg/ml doxorubicin (white bars), 3 mg/mL methadone Fig. 18: D,L-methadone sensitizes chronic lym- plus 0 or 0,015 mg/ml doxorubicin (hatched bars), 10 phocytic leukemia (CLL) cells for Fludarabine mg/mL methadone plus 0 or 0,015 mg/ml doxorubicin treatment. The CLL cells were treated with different (doted bars). concentrations of D,L-methadone (10, 3, m1g/mL) 30 alone (Medium, white columns), with m 0.1M Fig. 22: Cell death induction of JIMT-1 cells using Fludarabine (0.1 mM Fludarabine, black columns) D,L-methadone and doxorubicin cotreatment alone or with D,L-methadone (30, 10, 5, 3, 1, 0.5, depends on caspases activation. Inhibition of cas- 0.3, 0.1 mg/mL) in addition to 0.1 mM FLudarabine pase activation with the broad spectrum caspase in- (0.1 mM Fludarabine, black columns). After 72h the 35 hibitor zVAD.fmk blocks apoptosis induced by percentages of apoptotic cells were measured by cotreatment of D,L-methadone and doxorubicin in hypodiploid DNA analysis. The percentage of spe- JIMT-1 cells. The human cell line JIMT-1 was treated cific apoptosis was calculated as described in with different concentrations of D,L-methadone (10, Fig.1B. Columns, mean of triplicates; bars, SD 3, 1mg/mL) in combination with 0.015 mg/mL doxo- <10%. Similar results were obtained in three inde- 40 rubicin (+ 0.015 mg/mL Doxo) in the absence (left pendent experiments. sided diagrams) or presence ofm mol/L 50 of zVAD.fmk (diagrams on the right side). At 96 hours Fig. 19: Cisplatin enhances opioid receptor ex- (A) or 120 hours (B) after addition of the drug com- pression in HL60 cells bination, the cells were analysed using flow cytom- Cisplatin enhances opioid receptor expression on 45 etry. the surface of the promyelocytic leukemia cell line HL60. The HL60 celll line was treated for 24h with Fig. 23: Cell death induction of JIMT-1 cells using 0.3 mg/mL cisplatin. After staining of cisplatin-treated D,L-methadone and doxorubicin cotreatment (+Cisplatin) and untreated cells (-Cisplatin) with depends on caspases activation. (A) D,L-metha- naloxone-fluoresceine relative fluorescence intensi- 50 done restored deficient caspases activation by dox- ties were determined flowcytometrically. 2.1-fold in- orubicin in JIMT-1. A172 were treated with different crease in opioid receptor expression is shown after concentrations of D,L-methadone (10, 3, m1g/mL) subtracting the cells autofluorescence. alone, with 0.015 mg/mL doxorubicin alone or with different concentrations of D,L-methadone (10, 3, Fig. 20: The Her2/neu-resisten mamma carcino- 55 1mg/mL) in addition to 0.015 mg/ml doxorubicin. After ma cell line JIMT-1 expresses high levels of the 96h Western blot analyses for caspase--8, -9, -3 and m-opioid receptor PARP were performed. The active fragment of cas- The human JIMT-1 cell line, derived from a pleural pase-8 was detected at ∼43kDa, the active fragment

23 45 EP 2 716 291 A1 46 of caspase-9 was detected at∼ 37kDa, the active D,L-methadone strongly enhances cisplatin sensi- fragment of caspase-3 at ∼17kDa and PARP cleav- tivity of different leukemia cells. Different leukemia age at ∼85kDa. Equal protein loading was controlled cells (human T cell leukemia, human acute myeloid by anti-β-actin antibody. (B) Downregulation of XIAP leukemia, human B cell precursor leukemia and hu- 5 and Bcl-xL in JIMT-1 cells by using D,L-methadone man B cell leukemia) were treated with different con- in combination with doxorubicin. Mamma carcinoma centrations of D,L-methadone (as indicated) alone cells JIMT-1 were treated with different concentra- (- CDDP, white columns) with cisplatin alone or with tions of D,L-methadone (10, 3, 1mg/mL) alone, with D,L-methadone in addition to cisplatin (+ CDDP, 0.015 mg/mL doxorubicin alone or with D,L-metha- black columns). After time of incubation, the percent- done (10, 3, 1g/mL)m in addition to doxorubicin 10 ages of apoptotic cells were measured by FSC/SSC- (0.015 mg/mL Doxo) for 96h. Western blot analyses analysis. The percentage of specific apoptosis was for XIAP and Bcl-xL were performed. XIAP was de- calculated as described in Fig.1B. Columns, mean tected at 57kDa and Bcl-xL was detected at 21kDa. of triplicates; bars, SD<10%. Equal protein loading was controlled by anti-β-actin antibody. 15 Fig. 29: D,L-methadone in combination with cis- platin restores deficient activation of apoptotic Fig. 24: Induction of apoptosis in T-Cell leukemia pathways in leukemia cells CEM cells by a combination of doxorubicin and D,L-methadone and cisplatin co-treatment provokes fentanyl. Human T-Cell leukemia CEM cell line caspases activation and induces downregulation (10000 cells/100 ml) were treated with 30, 10, 5, 3, 20 and cleavage of XIAP and upregulation of Bax. Dif- 1, 0.5, 0.3, 0.1 mg/mL fentanyl alone (white bars) or ferent leukemia cells (human T cell leukemia, human in addition to 0.02mg/mL doxorubicin (black bars). acute myeloid leukemia, human B cell precursor After 48h and 72h quantification of apoptosis was leukemia) were treated with D,L-methadone (as in- measured by flow cytometry. dicated) alone (- CDDP), with cisplatin (CDDP) alone 25 or with D,L-methadone (as indicated) in addition to Fig. 25: Induction of apoptosis in human acute cisplatin (+ CDDP). After time of incubation Western myeloid leukemia HL-60 cells by a combination blot analyses for caspase-2, caspase-9, caspase-3, of doxorubicin and buprenorphine. Human acute PARP, XIAP and Bax were performed. Downregu- myeloid leukemia HL-60 cell line (5000 cells/100 ml) lation of procaspase-2 was detected at ∼48 kDa. The were treated with 20,10, 5, 3, 1, 0.5, 0.3, 0.1 mg/mL 30 active fragment of caspase-9 was detected at∼ 37 buprenorphine alone (white bars) or in addition of kDa, the active fragment of caspase-3 at∼ 19 kDa 0.003mg/mL doxorubicin (black bars). After 144h or and or ∼17 kDa, PARP at ∼116 kDa, PARP cleavage 168h quantification of apoptosis was measured by at ∼85 kDa, XIAP was detected at ∼58 kDa and XIAP flow cytometry. cleavage at _∼30 kDa and Bax at∼ 21 kDa. Equal 35 protein loading was controlled by anti-β-actin anti- Fig. 26: Schematic diagram showing the mutual body. positive interaction between opioids and anti- cancer drugs. On one side, opioids enhance the Fig. 30 D,L-methadone and cisplatin-induced ap- cellular uptake and inhibit the efflux of anticancer optosis depends on caspase activation. Pre-in- drugs. On the other sides anticancer drugs lead to 40 cubation of different leukemia cells (human T cell an increased expression of opioid receptors. Hence, leukemia, human acute myeloid leukemia, human B both agents can exert their cytotoxic potential to a cell precursor leukemia) with 50mM of the caspase higher extent. inhibitor zVAD.fmk for 1h (+ zVAD.fmk, white col- umns) or without pre-treatment (- zVAD.fmk, black Fig. 27: Opioid receptor expression on different 45 columns) was followed by addition of D,L-metha- leukemia done (as indicated) in combination with cisplatin (as Different leukemia cells (human T cell leukemia, hu- indicated). Apoptosis induction was detected after manacute myeloid leukemia, human Bcell precursor time of incubation by FSC/SSC-analysis. The per- leukemia and human B cell leukemia) express dif- centage of specific apoptosis was calculated as de- ferent moderate number of opioid-receptors on their 50 scribed in Fig.1B. Columns, mean of triplicates; bars, cell surface. Leukemia cells were stained with SD<10%. naloxone-fluoresceine measuring opioid-receptor expression (OR, thick black curve) and analyzed by Fig. 31 Cisplatin enhances opioid receptor ex- flowcytometry. Controls (Co) without naloxone are pression exhibited as thin black curves. 55 (A) Cisplatin enhances opioid receptor expres- Fig. 28: Effect of combination therapy of opioid re- sion on the cells’ surface of different leukemia ceptor agonist and anticancer agent cells (human T cell leukemia, human acute my-

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eloid leukemia and human B cell precursor as described in Fig.1B. Columns, mean of triplicates; leukemia) were treated with cisplatin (as indicat- bars, SD<10%. ed). After staining of cisplatin-treated cells (CD- DP) and untreated cells (Co) with naloxone-flu- Fig. 34: Different duration of effectiveness of dif- oresceine relative fluorescence intensities were 5 ferent opioids induces different rates of cell determined flowcytometrically. X-fold increase death in leukemia cells in opioid receptor expression compared to the untreated control group is shown after subtract- A) Leukemia cells (human B cell leukemia) were ing the cells’ autofluorescence and cisplatin flu- treated with different concentrations of D,L- orescence. 10 methadone (as indicated) alone (-Doxo, white columns on the left side of a black bar), with Fig. 32: Combination treatment with D,L-metha- doxorubicin alone or with D,L-methadone (as in- done and doxorubicin induced apoptosis de- dicated) in addition to doxorubicin (+Doxo, black pends on opioid-receptor triggering columns)) using the same concentration of Inhibition of opioid-receptor triggering inhibits apop- 15 0,1mg/mL doxorubicin for all treatments and dif- tosis induction mediated by combination treatment ferent concentrations of D,L-methadone as in- with D,L-methadone and doxorubicin. Glioblastoma dicated. 96h after stimulation, percentages of cells were incubated with mg/mL 60 naloxone apoptotic cells were measured by hypodiploid (Naloxone), 3mg/mL D,L-methadone (D,L-Metha- DNA analysis. done) and 0.1mg/mL doxorubicin (Doxo) alone or in 20 B) Leukemia cells (human B cell leukemia) were different combinations as indicated by the marks + treated with different concentrations of mor- and -.. After 120h and 144h, the percentages of ap- phine (as indicated) alone (-Doxo, white col- optotic cells were measured by FSC/SSC-analysis. umns on the left side of a black bar), with dox- The results of the different treatments which are in- orubicin alone or with morphine (as indicated) dicated concerning the given substances under the 25 in addition to doxorubicin (+Doxo, black col- single bars after 120 h (left side of the figure) and umns) using the same concentration of 144 h (right side of the figure) are shown in Fig. 32. 0,1mg/mL doxorubicin for all treatments and dif- ferent concentrations of D,L-methadone as in- Fig. 33: Different duration of effectiveness of dif- dicated. For the 96h after stimulation, percent- ferent opioids induces different rates of cell30 ages of apoptotic cells were measured by hy- death in glioblastoma cells podiploid DNA analysis.

A) Glioblastoma cells were treated with different The percentage of specific apoptosis was calculated concentrations of D,L-methadone (as indicated) as described in Fig.1B. Columns, mean of triplicates; alone (-Doxo, white columns, which are very low 35 bars, SD<10%. and on the left side of a black bar), with doxoru- bicinalone or with D,L-methadone (asindicated) Fig. 35: Combination treatment with D,L-metha- in addition to doxorubicin (+Doxo, black col- done and doxorubicin inhibits proliferation and umns) using the same lconcentration of induces S/G2-M cell cycle arrest in glioblasto- 0,1mg/mL doxorubicin for all treatments and dif- 40 mas cells. ferent concentrations of D,L-methadone as in- Flow cytometric analysis of glioblastoma cells treat- dicated. 144h after stimulation, percentages of ed with methadone and doxorubicin was shown. cell death and apoptotic cells were measured Flow cytometric analysis of untreated cells (Untreat- by hypodiploid DNA analysis. ed cells) (G1 peak is higher than G2 peak), cells B) Glioblastoma cells were treated with differ- 45 treated with 1mg/ml methadone (Methadone) (G1 ent concentrations of morphine (as indicated) peak is higher than G2 peak) and cells treated with alone (-Doxo, white columns which are very low methadone in addition to 0,1m g/ml doxorubicin and on the left side of a black bar), with doxoru- (Methadone + Doxo). Arrest of cell cycle progression bicin alone or with morphine (as indicated) in at the G2/M phase (G1 peak lower than in untreated addition to doxorubicin (+Doxo, black columns) 50 cells and G2 peak is higher than in untreated cells) using the same concentration of 0,1 mg/mL dox- was shown in glioblastoma cells treated with meth- orubicin for all treatments and different concen- adone in addition to doxorubicin after 96h (A). subG1 trations of D,L-methadone as indicated. 144h af- peak in front of G1 is the fragmentated DNA (per- ter stimulation, percentages of cell death and centage of cell death). Results are representative of apoptotic cells were measured by hypodiploid 55 3 independent experiments. DNA analysis.

The percentage of specific cell death was calculated

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LITERATURE Friesen C, Kiess Y, Debatin KM. A critical role of glutathione in determining apoptosis sensitivity and [0178] resistance in leukaemia cells. Cell Death Differ 2004; 11 (Suppl 1):S73-85. Addeo, R., Caraglia, M., Baldi, A., D’Angelo, V., Ca- 5 sale, F., Crisci, S., Abbruzzese, A., Vincenze, B., Fulda, S. (2009a). Cell death in hematological tu- Campioni, M., Di Tullio, M. T., and Indolfi, P. (2005). mors. Apoptosis 14, 409-423. Prognostic role of bcl-xL and p53 in childhood acute lymphoblastic leukemia (ALL). Cancer Biol Ther 4, Fulda, S. (2009b). Therapeutic opportunities for 32-38. 10 counteracting apoptosis resistance in childhood leu- kaemia. Br J Haematol 145, 441-454. Bergmann JP, Harris D. Radioresistance, chemore- sistance and apoptosis resistance. Radiation Oncol- Fulda, S. (2009c). Tumor resistance to apoptosis. ogy 1997; 27:47-57. Int J Cancer 124, 511-515. 15 Boecker W et al., eds., chapter 6: "Tumorerkrankun- Hilger, R. A., Richly, H., Grubert, M., Oberhoff, C., gen" in Pathologie, Urban &Fischer, Elsevier, 4th Strumberg, D., Scheulen, M. E., and Seeber, S. edition, 2008. (2005). Pharmacokinetics (PK) of a liposomal en- capsulated fraction containing doxorubicin and of Borgmann, A., Baldy, C., von Stackelberg, A., Beyer- 20 doxorubicin released from the liposomal capsule af- mann, B., Fichtner, I., Nurnberg, P., and Henze, G. ter intravenous infusion of Caelyx/Doxil. Int J Clin (2000). Childhood all blasts retain phenotypic and Pharmacol Ther 43, 588-589. genotypic characteristics upon long-term serial pas- sage in NOD/SCID mice. Pediatr Hematol Oncol 17, Law, P. Y., Wong, Y. H., and Loh, H. H. (2000). Mo- 635-650. 25 lecular mechanisms and regulation of opioid recep- tor signaling. Annu Rev Pharmacol Toxicol 40, Carbonari M, Cibati M, Cherchi M, et al. Detection 389-430. and characterization of apoptotic peripheral blood Law 2000. lymphocytes in human immunodeficiency virus-in- fection and cancer chemotherapy by a novel flow 30 Naderi, E. H., Findley, H. W., Ruud, E., Blomhoff, H. immunocytometric method. Blood 1994;K., and Naderi, S. (2009). Activation of cAMP sign- 83:1268-77. aling inhibits DNA damage-induced apoptosis in BCP-ALL cells through abrogation of p53 accumu- Classen, C. F., Falk, C. S., Friesen, C., Fulda, S., lation. Blood 114, 608-618. Herr, I., and Debatin, K. M. (2003). Natural k iller re- 35 sistance of a drug-resistant leukemia cell line, me- Nicoletti, I., Migliorati, G., Pagliacci, M. C., Grignani, diated by up-regulation of HLA class I expression. F., and Riccardi, C. (1991). A rapid and simple meth- Haematologica 88, 509-521. od for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Meth Crettol, S., Digon, P., Golay, K. P., Brawand, M., and 40 139, 271-279. Eap, C. B. (2007). In vitro P-glycoprotein-mediated transport of (R)-, (S)-, (R,S)-methadone, LAAM and Posovszky, C., Friesen, C., Herr, I., and Debatin, K. their main metabolites. Pharmacology 80, 304-311. M. (1999). Chemotherapeutic drugs sensitize pre-B ALL cells for CD95- and cytotoxic T-lymphocyte-me- Diestra, J. E., Condom, E., Del Muro, X. G., Scheffer, 45 diated apoptosis. Leukemia 13, 400-409. G. L., Perez, J., Zurita, A. J., Munoz-Segui, J., Vigues, F., Scheper, R. J., Capella, G., et al. (2003). Richly, H., Henning, B. F., Kupsch, P., Passarge, K., Expression of multidrug resistance proteins P-glyc- Grubert, M., Hilger, R. A., Christensen, O., Brendel, oprotein, multidrug resistance protein 1, breast can- E., Schwartz, B., Ludwig, M., et al. (2006). Results cer resistance protein and lung resistance related 50 of a Phase I trial of sorafenib (BAY 43-9006) in com- protein in locally advanced bladder cancer treated bination with doxorubicin in patients with refractory with neoadjuvant chemotherapy: biological and clin- solid tumors. Ann Oncol 17, 866-873. ical implications. J Urol 170, 1383-1387. Safa, M., Kazemi, A., Zand, H., Azarkeivan, A., Za- Friesen C, Glatting G, Koop B, et al. Breaking chemo- 55 ker,F., and Hayat, P. (2010a). Inhibitoryrole ofcAMP and radioresistance with [213Bi]anti-CD45 antibod- on doxorubicin-induced apoptosis in pre-B ALL cells ies in leukaemia cells. Cancer Res 2007; through dephosphorylation of p53 serine residues. 67(5):1950-8. Apoptosis 15, 196-203.

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Workman, P., Aboagye, E. O., Balkwill, F., Balmain, nol,methorphane, morphanol, oxilorphan, phe- A., Bruder, G., Chaplin, D. J., Double, J. A., Everitt, nomorphan, and xorphanol, J., Farningham, D. A., Glennie, M. J., et al. (2010), iv. benzomorphane derivatives such as 5,9-DE- Guidelines for the welfare and use of animals in can- HB,alazocine, , bremazocine, butina- cer research. Br J Cancer 102, 1555-1577 5 zocine, carbazocine, , cyclazocine, dezocine, eptazocine, etazocine, ethylketocy- clazocine, fluorophen, gemazocine, , Claims ketazocine, metazocine, , pentazo- cine, phenazocine, quadazocine, thiazocine, 1. A combination of an opioid receptor agonist and at 10 tonazocine, volazocine and 8-CAC; least one anticancer agent for use in the treatment v. 4-phenylpiperidine derivatives such as pethi- of cancer, wherein dine, ketobemidone, anileridine, piminodine, phenoperidine, furethidine, alpha-prodin, tri- (a) said opioid receptor agonist is administered meperidine, including 4-phenylpyrrolidine deriv- to a patient in one or more doses to establish a 15 atives such as profadol and 4-phenylazepan- therapeutically effective plasma level for a peri- derivates such as meptazinol, od of at least one week,and vi. cyclohexane derivatives such as tilidine, U- (b) at least one anticancer agent selected from 50488, tramadol and tapentadol the group consisting of chemotherapeutical vii. endogenous opioids such as endorphins, agents, cytotoxic agents, cytostatic agents, im- 20 enkephalins, dynorphins, nociceptin, dermor- munotoxic agents and/or radiotherapy is admin- phins, morphiceptin, endomorphines and frag- istered to establish a period with a therapeuti- ments derived from the protein proopiomelano- cally effective plasma level, and cortin (POMC). (c) said periods of a) and b) overlap. 25 7. The combination according to any of the above 2. A combination according claim 1, wherein said anti- claims wherein the opioid receptor agonist belongs cancer agent and said opioid receptor agonist are to the methadone group. administered simultaneously or successively. 8. The combination according to claim 7, wherein the 3. A combination according claim 1 or 2, wherein said 30 opioid receptor agonist is D/L methadone and pref- opioid receptor agonist is capable of inhibiting cell erably the hydrochloride form thereof. proliferation. 9. The combination according to any of the above 4. A combination according to any of claims 1 to 3, claims wherein the anticancer agent is selected from wherein the patient has received a pre-treatment 35 the list consisting of: comprising an anticancer agent. i. intercalating substances such as anthracy- 5. The combination according to any of the above cline, doxorubicin, idarubizin, epirubizin,and claims, wherein the administration period for the opi- daunorubizin; oid receptor agonist providing a therapeutically rel- 40 ii. topoisomerase inhibitors such as irinotecan, evant dose is at least two weeks, more preferably topotecan, camptothecin, lamellarin D, etopo- four weeks and even more preferably represents a side, teniposide, mitoxantrone, amsacrine, ellip- chronic treatment. ticines and aurintricarboxylic acid; iii. nitrosourea compounds such as carmustine 6. The combination according to any of the above45 (BCNU), lomustine (CCNU), streptozocin; claims wherein the opioid receptor agonist is select- iv. nitrogen mustards such as cyclophospha- ed from the list consisting of mide, mechlorethamine, uramustine, benda- mustine, melphalan, chlorambucil, mafosfa- i. compounds of the methadone group such as mide, trofosfamid and ifosfamide; D/L-methadone, D-methadone, L-methadone, 50 v. alkyl sulfonates such as busulfan and treosul- normethadone, fan; ii. fentanyl derivatives such as fentanyl, sufen- vi. alkylating agents such as procarbazin, dacar- tanyl and carfentanyl; bazin, temozolomid and thiotepa; iii. morphinane compounds such as morphine, vii. platinum analogues such as cisplatin, carbo- codeine, heroine, dextrallorphane, dextrometh- 55 platin, nedaplatin, oxaliplatin, satraplatin, and orphan, dextrophanol, dimemorfan, levalor- triplatin tetranitrate; phan, levofurethylnormorphanol, levomethor- viii. Microtubule disruptive drugs such as vin- phane, levophenacylmorphane, levorpha- blastine, colcemid and nocodazole;

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ix. antifolates like methotrexate, aminopterin, i. apoptosis resistance dichloromethotrexat, pemetrexed, raltitrexed ii. multi-drug resistance and pralatrexate: iii. anticancer drug resistance x. purine analogues like azathioprine, mercap- iv. cytotoxic drug resistance topurine, thioguanine, fludarabine, fludarabine 5 v. resistance to reactive oxygen species phosphate, pentostatin and cladribine; vi. resistance to DNA damaging agents xi. pyrimidine analogues like 5-fluorouracil, vii. resistance to toxic antibodies floxuridine, cytarabine, 6-azauracil, gemcitab- viii. doxorubicin or rituximab resistance ine; ix. single or cross resistance, in particular to one xii. steroid hormones like gestagene, andro-10 or more of the following drug substances: meth- gene, glucocorticoids, dexamethasone, pred- otrexate, cytarabine, cisplatine, etoposide, vin- nisolone, and prednisone; cristine, paclitaxel, carboplatin, oxaliplatin, ten- xiii. anti-cancer antibodies such as monoclonal iposide, dexamethasone, prednisolone, cyclo- antibodies, radioactively labeled antibodies and phosphamide, diphosphamide, doxorubicin, antibody-drug conjugates; 15 epirubicin, idarubicin, daunorubicin, mercaptop- xiv. anti-cancer peptides such as radioactively urine and fludarabine. labeled peptides and peptide-drug conjugates; x. Irradiation resistance (e.g. alpha, beta, gam- xv. alpha, beta or gamma irradiation, electron ma or Auger electrons). particles, or radioactively labeled chemical compounds . 20 15. The combination according to any of the above claims, wherein the anticancer agent is given at a 10. The combination according to any of the above dose, which is equal to or lower than the recommend- claims wherein the anticancer agent is methotrexate, ed dose for the treatment of cancer using the anti- cytarabine, cisplatine, etoposide, vincristine, beta ir- cancer agent only. radiation or gamma irradiation, and especially dox- 25 orubicin or rituximab. 16. Method for selection of the combination according to any of the above claims and/or the doses of the 11. The combination according to any of the above drugs used within said combination comprising the claims wherein patients suffer from a neoplasm as following steps: classified according the International statistical clas- 30 sification of Diseases and related health problems (a) providing an vitro culture of cancer cells, cell 10th Revision (ICD-10), and wherein the neoplasm lines or primary cells, preferably isolated from a is from the group consisting of malignant neoplasms cancer biopsy or from a liquid sample (such as of classes C00 to C97, in situ neoplasms of classes e.g. blood, amniotic fluid, pleural fluid, cerebro- D00 to D09, benign neoplasms of classes D10 to 35 spinal fluid or peritoneal fluid); D36, and neoplasms of uncertain or unknown be- (b) optionally testing the cells from step (a) for haviour of classes D37 to D48. expression of opioid receptors; (c) treating the cells from step (a) with an opioid 12. The combination according to claim 11 wherein pa- agonist, or at least one anticancer drug or a com- tients suffer from neoplasm from the group consist- 40 bination thereof; ing of pancreatic carcinoma, hepatoblastoma, colon (d) analysing the cells for cell death/viability carcinoma, (small cell lung cancer, melanoma, and/or expression of opioid receptors mamma carcinoma, ovarian carcinoma, prostate (e) selecting the opioid receptor/anticancer drug carcinoma, glioblastoma, acute lymphoblastic leu- combination and preferably a dose for said com- kaemia, acute myeloid leukaemia, chronic myeloid 45 bination based on the desired extent of cell leukaemia, chronic lymphocytic leukaemia, pro- death/viability or inhibition of proliferation; forms of leukaemia, hairy cell leukaemia, Hodgkin’s and/or disease, Non-Hodgkin lymphoma, glioblastoma-ini- (f) selecting the anticancer agent and preferably tiating stem cells, tumor stem cells and multiple my- a dose for said anticancer agent which shows eloma. 50 the desired extent of induction of opioid recep- tors. 13. The combination according to any of the preceding claims, wherein the patient exhibits either an intrinsic 17. Method for selection of an opioid receptor agonist or an acquired resistance. for the treatment of cancer and preferably a treat- 55 ment dose for said opioid receptor agonist compris- 14. The combination according to claim 13, wherein the ing the following steps: patient exhibits one or more of the subsequent re- sistances: (a) providing an vitro culture of cancer cells, cell

28 55 EP 2 716 291 A1 56 lines or primary cells, preferably isolated from a cancer biopsy or from a liquid sample (such as e.g. blood, amniotic fluid, pleural fluid, cerebro- spinal fluid or peritoneal fluid); (b) optionally testing the cells from step (a) for 5 expression of opioid receptors; (c) treating the cells from step (a); (d) analysing the cells for cell death/viability; (e) selecting the opioid receptor/anticancer drug combination and preferably a dose for said com- 10 bination based on the desired extent of cell death/viability or inhibition of proliferation; and/or (f) selecting the opioid receptor agonist and pref- erably a dose for said opioid receptor agonist 15 which shows the desired extent of induction of cell death.

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Non-patent literature cited in the description

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• SAFA, M. ; KAZEMI, A. ; ZAND, H. ; AZARKEIVAN, • WORKMAN, P. ; ABOAGYE, E. O. ; BALKWILL, A. ; ZAKER, F. ; HAYAT, P. Inhibitory role of cAMP F. ; BALMAIN, A. ; BRUDER, G. ; CHAPLIN, D. J. ; on doxorubicin-induced apoptosis in pre-B ALL cells DOUBLE, J. A. ; EVERITT, J. ; FARNINGHAM, D. through dephosphorylation of p53 serine residues. A. ; GLENNIE, M. J. et al. Guidelines for the welfare Apoptosis, 2010, vol. 15, 196-203 [0178] and use of animals in cancer research. Br J Cancer, 2010, vol. 102, 1555-1577 [0178]

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