Patent Application
20200054647
The present invention relates to the use of ruthenium complexes or of pharmaceutical compositions containing same for treating cancer comprising cancer stem cells.
It is known that metallic compounds containing platinum exhibit significant anti-tumor activities. The most well-known of these metallic compounds is cisplatin, which is used today in the clinical treatment of various cancers.
Nevertheless, anti-tumor treatment based on compounds of this type often leads to severe side effects. Therefore, there is a major interest in developing metallic compounds with anti-tumor activity that are more selective and less toxic than cisplatin. In this sense, ruthenium complexes are a promising alternative due to their kinetic stability and their redox and photochemical properties. Ruthenium complexes which recognize in a non-covalent manner double-stranded DNA and also form covalent adducts with the DNA have been described. Most of these complexes bind to the two strands of the DNA. However, due to the functional relevance of guanine quadruplexes (GQs), it would be of great interest to develop complexes capable of acting by means of binding to these structures. In this sense, Wu et al., Inorg. Chem. 2013, 2, 11332 describe ruthenium complexes capable of covalently metallating a GQ structure, however this reaction is rather non-selective.
In relation to tumor development and the onset of metastasis, a population of cells called “cancer stem cells” capable of differentiating into different cell types and self-renewal has recently been identified, so it is considered likely that these cells are related to relapse processes after anti-tumor treatment and to the onset of metastasis. Therefore, it is of great interest to provide new anti-tumor therapies capable of acting on this cell type.
Another challenge in the treatment of cancer consists of achieving targeted therapies which allow delivering the active compound selectively to the site of action, thereby reducing the required dose, and allow specifically acting on cancer cells, preventing normal cells from being damaged.
There is therefore a need to provide alternative methods for treating cancer which allow solving the deficiencies of the state of the art.
The inventors have discovered that ruthenium(II) complexes as defined herein are capable of selectively metallating unpaired guanines present in parallel guanine quadruplexes (GQs). As has been observed, this selective metallation causes an increase in the expression of the c-MYC oncogene, which is involved in many cellular processes. Additionally, the inventors have found that this increase in the proportion of c-MYC may promote the differentiation of cancer stem cells, which makes these ruthenium complexes an important tool in biology and medicine. It has furthermore been observed that metallation reaction increases by a greater or lesser degree depending on the type of complex when irradiated with light.
Taking this into account, in a first aspect the present invention relates to a ruthenium complex of formula (I)
wherein
N1—N1—N1 represents an N,N,N-tridentate aza-aromatic ligand;
N2—N2 represents an N,N-bidentate aza-aromatic ligand;
X is selected from OH2, Cl, Br, I, and SR1R2;
R1 and R2 are independently selected from optionally substituted C1-C12 alkyl;
Y− is a monovalent anion; and
n is 1 or 2;
for preparing a medicinal product for treating cancer comprising cancer stem cells.
In a second aspect, the present invention relates to a pharmaceutical composition or a medicinal product comprising a ruthenium complex of formula (I) as defined herein for use thereof in the treatment of cancer comprising cancer stem cells.
In a third aspect, the invention relates to a method for treating cancer comprising cancer stem cells, which method comprises administering a therapeutically effective amount of a ruthenium complex of formula (I) as defined herein and irradiating with light.
In another aspect, the invention relates to a conjugate comprising:
In another aspect, the invention relates to a conjugate comprising:
Other aspects of the invention relate to the conjugates of the invention for use thereof in medicine and for use thereof in the treatment of cancer comprising cancer stem cells. Finally, another aspect of the invention relates to a method for treating cancer comprising cancer stem cells, which method comprises administering a therapeutically effective amount of a conjugate as defined herein and irradiating with light.
Researchers have found that ruthenium complexes of formula (I) wherein X is Cl are capable of selectively metallating guanosine monophosphate (GMP) by means of prior transformation into the active aquo complex (X═H2O). This reaction can be accelerated by means of irradiation with light.
It has also been observed that these complexes cause an increase in the expression levels of the c-MYC oncogene, therefore acting as transcription activators. In the case of ruthenium complexes wherein X is selected from SR1R2, these complexes are stable in the dark, but the thioether ligand can be readily interchanged by means of irradiation with light, giving rise to the active aquo complex. In that case, it has been confirmed that the ruthenium complexes wherein X is selected from SR1R2 are completely inert in the absence of light, but cause an increase in c-MYC levels after irradiation with light. It has been furthermore confirmed that in the case of the ruthenium complexes of the invention wherein X is H2O (an aquo complex), it is possible to increase the c-MYC oncogene levels both in the dark and by means of irradiation with light.
In a first aspect, the invention relates to an Ru(II) complex of formula (I)
wherein
N1—N1—N1 represents an N,N,N-tridentate aza-aromatic ligand;
N2—N2 represents an N,N-bidentate aza-aromatic ligand;
X is selected from OH2, Cl, Br, I, and SR1R2;
R1 and R2 are independently selected from optionally substituted C1-C12 alkyl;
Y− is a monovalent anion; and
n is 1 or 2;
for preparing a medicinal product for treating cancer comprising cancer stem cells.
The term “alkyl” refers to a linear or branched alkane derivative which contains from 1 to 12 (“C1-C12 alkyl”), preferably from 1 to 6 (“C1-C6 alkyl”), more preferably from 1 to 3 (“C1-C3 alkyl”), carbon atoms and is bound to the rest of the molecule through a single bond. Illustrative examples of alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, pentyl, hexyl.
The term “alkenyl” refers to a linear or branched hydrocarbon chain radical which contains from 2 to 6 (“C2-C6 alkenyl”), more preferably from 2 to 3 (“C2-C3 alkenyl”), carbon atoms, contains at least one double bond, and is bound to the rest of the molecule by means of a single bond. Illustrative examples include ethenyl, propenyl, allyl, butenyl, 1-methyl-2-buten-1-yl, and the like.
The term “alkynyl” refers to a linear or branched hydrocarbon chain radical which contains from 2 to 6 (“C2-C6 alkynyl”), more preferably from 2 to 3 (“C2-C3” alkynyl), carbon atoms, contains at least one triple bond, and is bound to the rest of the molecule by means of a single bond. Illustrative examples include ethynyl, propinyl, butynyl, and the like.
The term “aryl” refers to an aromatic group having between 6 and 14, preferably between 6 and 10, carbon atoms, comprising 1 or 2 aromatic nuclei condensed with one another. Illustrative examples of aryl groups include phenyl, naphthyl, indenyl, phenanthryl, etc.
The term “heterocyclyl” refers to a monocyclic or bicyclic system which can be completely or partially saturated or aromatic (“heteroaryl”) containing from 3 to 10, preferably from 5 to 10, more preferably from 5 to 7, ring atoms containing one or more, specifically one, two, three, or four ring heteroatoms independently selected from N, O, and S, and with the remaining ring atoms being carbon.
The term “halogen” refers to bromine, chlorine, iodine, or fluorine.
The term N,N-bidentate or N,N,N-tridentate aza-aromatic ligand refers to aromatic molecules which can take up two (bidentates) or three (tridentates) coordination sites of the Ru(II) metal center by means of coordination through nitrogen atoms alone. Preferably, this aromatic molecule is a stable heteroaryl having from 10 to 32, preferably from 12 to 28, more preferably from 12 to 20, members (bidentate) or having from 15 to 32, preferably from 18 to 30, more preferably from 18 to 26, members (tridentate), formed by carbon atoms and from two (bidentate) or three (tridentate) up to six, preferably 2, 3, 4, or 5, nitrogen atoms. As it is used herein, the expression “heteroaryl having from 10 to 32 members or having from 15 to 32 members” means a heteroaryl group having a backbone from 10 to 32 atoms or from 15 to 32 atoms. For the purposes of this invention, the heterocycle can be a polycyclic ring system which can include fused or condensed ring systems. Examples of N,N-bidentate and N,N,N-tridentate aza-aromatic ligands include, but are not limited to, 2,2′-bipyridine, 2,2′-bipyrazine, 2,2′-bipyrimidine, 1,10-phenanthroline, bathophenanthroline, 2,2′-bisquinoline, 1,1′-bisisoquinoline, 2-pyridinyl-2-quinoline, 3-pyridinyl-2-quinoline, 1-pyridinyl-2-isoquinoline, 2-pyridinyl-2-[1,8]-naphthyridine, 2,2′:6′,2″-terpyridine, 2,6-bis(2′-benzimidazoly)pyridine, 2,6-bis(8′-quinolinyl)pyridine, 2,6-bis(2′-[1,8]-naphthyridinyl)pyridine, 2-pyridinyl-2-[1,10]-phenanthroline, 2-quinolinyl-8-[1,10]-phenanthroline, and the like. These aza-aromatic ligands can be optionally substituted.
The aforementioned groups can be optionally substituted in one or more available positions with one or more suitable groups such as OR, SR, SOR, SO2R, OSO2R, SO3R, SO3−Z+, NO2, N(R)2, N(R) COR, N(R) SO2R, CN, halogen, COR, CO2R, CO2−Z+, OCOR, OCO2R, OCONHR, OCON(R)2, CONHR, CON(R)2, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C6-C14 aryl, and 3- to 10-membered heterocyclyl, wherein each of the groups R is independently selected from the group consisting of hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C6-C14 aryl, and 3- to 10-membered heterocyclyl, and where Z is preferably an inorganic monovalent cation, such as a sodium or potassium cation, for example. In a particular embodiment, between 1 to 5 aza-aromatic ligands in a compound of formula (I), preferably 2 or 3, are substituted in the para position by a sulfonate (SO3−Z+) or carboxylate (CO2−Z+) group.
In the present document, “monovalent anion” refers to an inorganic or organic anion with a single negative charge which can form an ionic bond with a cation of a Ru(II) complex. Preferably, the monovalent anion is inorganic. Examples of monovalent anions include PF6−, Cl−, Br, I−, F−, BF4−, CF3SO3−, CH3SO3−, CH3C6H4SO3NO3−, NO2−, SCN−, BrO3−, IO3−, HCO3−, HCOO−, CH3COO−, CF3CO2−, HSO4−, HSO3−, and H2PO3−.
In a particular embodiment, N1—N1—N1 is an N,N,N-tridentate aza-aromatic ligand selected from the group consisting of optionally substituted 2,2′:6′,2″-terpyridine, 2,6-bis(2′-benzimidazoly)pyridine, 2,6-bis(8′-quinolinyl)pyridine, 2,6-bis(2′-[1,8]-naphthyridinyl)pyridine, 2-pyridinyl-2-[1,10]-phenanthroline, and 2-quinolinyl-8-[1,10]-phenanthroline.
In one embodiment, N1—N1—N1 is selected from the group consisting of:
wherein each group R′ is independently selected from hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C6-C14 aryl, optionally substituted 5- to 10-membered heteroaryl, and halogen. Preferably, each group R′ is independently selected from hydrogen, halogen, sulfonate, carboxylate, unsubstituted C1-C6 alkyl, unsubstituted C6-C14 aryl, and unsubstituted 5- to a 10-membered heteroaryl. More preferably, each group R′ is independently selected from hydrogen, Cl, Br, methyl, ethyl, n-propyl, i-propyl, n-butyl, and t-butyl. In a particular embodiment, each group R′ is hydrogen.
According to a particular embodiment, N1—N1—N1 is selected from the group consisting of:
In another embodiment, N1—N1—N1 represents optionally substituted 2,2′:6′,2″-terpyridine. Preferably, N1—N1—N1 is selected from 2,2′:6′,2″-terpyridine optionally substituted with C1-C6 alkyl, C6-C14 aryl, 5- to 10-membered heteroaryl, sulfonate, carboxylate, or halogen; more preferably 2,2′:6′,2″-terpyridine optionally substituted with Cl, Br, sulfonate, carboxylate, methyl, ethyl, n-propyl, i-propyl, n-butyl, and t-butyl. In a particular embodiment, N1—N1—N1 represents 2,2′:6′,2″-terpyridine.
According to a particular embodiment of the invention, N2—N2 is an N,N-bidentate aza-aromatic ligand selected from the group consisting of optionally substituted 2,2′-bipyridine, 2,2′-bipyrazine, 2,2′-bipyrimidine, 1,10-phenanthroline, bathophenanthroline, 2,2′-bisquinoline, 1,1′-bisisoquinoline, 2-pyridinyl-2-quinoline, 3-pyridinyl-2-quinoline, 1-pyridinyl-2-isoquinoline, and 2-pyridinyl-2-[1,8]-naphthyridine.
In one embodiment, N2—N2 is selected from the group consisting of:
wherein each group R′ is independently selected from hydrogen, sulfonate, carboxylate, optionally substituted C1-C6 alkyl, optionally substituted C6-C14 aryl, optionally substituted 5- to 10-membered heteroaryl, and halogen. Preferably, each group R′ is independently selected from hydrogen, halogen, unsubstituted C1-C6 alkyl, unsubstituted C6-C14 aryl, and unsubstituted 5- to 10-membered heteroaryl. More preferably, each group R′ is independently selected from hydrogen, Cl, Br, sulfonate, carboxylate, methyl, ethyl, n-propyl, i-propyl, n-butyl, and t-butyl. In a particular embodiment, each group R′ is hydrogen.
According to a particular embodiment, N2—N2 is selected from the group consisting of:
In another embodiment, N2—N2 represents optionally substituted 2,2′-bipyridine. Preferably, N2—N2 is selected from 2,2′-bipyridine optionally substituted with C1-C6 alkyl, C6-C14 aryl, 5- to 10-membered heteroaryl, sulfonate, carboxylate, or halogen; more preferably 2,2′-bipyridine optionally substituted with Cl, Br, sulfonate, carboxylate, methyl, ethyl, n-propyl, i-propyl, n-butyl, and t-butyl. In a particular embodiment, N2—N2 represents 2,2′-bipyridine.
According to an embodiment of the invention, N1—N1—N1 represents
and N2—N2 represents
where each group R′ is independently selected from hydrogen, sulfonate, carboxylate, optionally substituted C1-C6 alkyl, optionally substituted C6-C14 aryl, optionally substituted 5- to 10-membered heteroaryl, and halogen.
In a particular embodiment, the Ru(II) complex has the following formula:
wherein X, Y−, and n are as defined herein.
The value of n is determined by the chemical structure and electric charge of the selected ligands and the 2+ electric charge of the central Ru(II) atom. Therefore, when X represents OH2 or SR1R2, then n is 2. When X represents Cl, Br, or I, then the value of n is 1. The complex of formula (I) is neutral, i.e., it has a zero overall charge.
In a particular embodiment, X represents OH2. Preferably, N1—N1—N1 represents optionally substituted 2,2′:6′,2″-terpyridine as defined above, N2—N2 represents optionally substituted 2,2′-bipyridine as defined above, and X represents OH2.
In another particular embodiment, X represents Cl, Br, or I, preferably Cl. In a particular embodiment, N1—N1—N1 represents optionally substituted 2,2′:6′,2″-terpyridine as defined above, N2—N2 represents optionally substituted 2,2′-bipyridine as defined above, and X represents Cl, Br, or I, preferably Cl.
In one embodiment, X represents SR1R2, where R1 and R2 are independently selected from optionally substituted C1-C12 alkyl. Preferably, R1 and R2 are independently selected from C1-C12 alkyl, preferably C1-C6 alkyl optionally substituted with one or more groups selected from halogen, OR″, N(R″)2, N(R″)COR″, CN, NO2, COR″, CO2R″, OCOR″, OCO2R″, OCONHR″, OCON(R″)2, CONHR″, CON(R″)2, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C6-C14 aryl, and 3- to 10-membered heterocyclyl, where each group R″ is independently selected from the group consisting of hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C6-C14 aryl, and 3- to 10-membered heterocyclyl. More preferably, R1 and R2 are independently selected from C1-C6 alkyl optionally substituted with one or more groups selected from OR″, N(R″)COR″, and CO2R″, where each group R″ is independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C6-C14 aryl, and 3- to 10-membered heterocyclyl. More preferably, R1 and R2 are independently selected from C1-C6 alkyl optionally substituted with one or more groups selected from OH, N(H)COCH3, and CO2H. In one embodiment, R1 and R2 are independently selected from CH3, CH2CH2OH, and CH2CH2CH(COOH)N(H)COCH3.
In an embodiment of the invention, the ruthenium complex is selected from the group consisting of:
wherein Y− is as defined herein.
In a particular embodiment, Y− is selected from the group consisting of PF6−, Cl−, Br−, I−, F−, BF4−, CF3SO3—, CH3SO3—, CH3C6H4SO3NO3—, NO2−, SCN−, BrO3−, IO3−, HCO3−, HCOO−, CH3COO−, HSO4−, HSO3−, and H2PO3−. Preferably, Y− is selected from the group consisting of PF6−, Cl−, Br, BF4−, CF3SO3—, CH3SO3—, and CH3C6H4SO3−. In one embodiment, Y— is Cl− or PF6−, preferably PF6−.
The ruthenium complexes of the present invention can be obtained by means of methods known by one skilled in the art, for example, by means of the methods mentioned in the example of this document or modifications thereof known by one skilled in the art.
The inventors have observed that the binding of the ruthenium complex to an ABCG2 substrate, such as riboflavin, for example, allows targeting the ruthenium complexes to cancer stem cells.
Therefore, in another aspect the invention relates to a conjugate comprising:
a ruthenium complex of formula (I), and
an ABCG2 substrate.
As it is used herein, “ABCG2” refers to a member of the superfamily of ATP transporters which is known to contribute to multiple drug resistance (MDR) in cancer chemotherapy. The protein sequence encoded by the ABCG2 gene in humans corresponds with the sequence with accession number Q9UNQ0 in the Uniprot database dated 5 Nov. 2016.
As it is used herein, “ABCG2 substrate” refers to a compound which can be transported by the ABCG2 transporter, particularly hydrophilic conjugated anions, more particularly sulfated anions or hydrophobic molecules. One skilled in the art will be able to identify if a compound is an ABCG2 substrate, for example, by means of using an ATPase assay using Sf9 membranes or in an essay shown in Glavinas H. et al., Drug Metab Dispos. 2007 September; 35(9):1533-42
In a particular embodiment, the ABCG2 substrate is a tyrosine kinase inhibitor such as imatinib and gefitinib, flavopiridol and the camptothecins topotecan, irinotecan, and the active metabolite SN-38 thereof [7-ethyl-10-hydroxy-camptothecin], or mitoxantrone. Other substrates include drugs such as cimetidine, prazosin, statins, and zidovudine. Additionally, other ABCG2 substrates are estrone, 17β estradiol, porphyrins such as heme, protoporphyrin IX, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. In another embodiment, the ABCG2 substrate is a chemotherapeutic agent such as amsacrine, asparaginase, azathioprine, bisantrene, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, flavopiridol, fludarabine, fluorouracil, gemcitabine, idarubicin, ifosfamide, irinotecan, hydroxyurea, leucovorin, liposomal daunorubicin, liposomal doxorubicin, lomustine, chlormethine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, satraplatin, streptozotocin, tegafur-uracil, temozolomide, teniposide, thioguanine, thiotepa, treosulfan, topotecan, vinblastine, vincristine, vindesine, SN-38, and vinorelbine.
In a particular embodiment, the ABCG2 substrate is a fluorescent compound. By way of non-limiting illustrative example, the fluorescent compound can be riboflavin, D-luciferin, rhodamine 123, pheophorbide a, BODIPY-prazosin, Hoechst 33342. In an even more preferred embodiment, the fluorescent ABCG2 substrate is riboflavin. As it is used herein, “riboflavin” refers to vitamin B2 with CAS number 83-88-5.
Preferably, the ruthenium complex and the ABCG2 substrate bind covalently to one another. The ruthenium complex and the ABCG2 substrate can be bound to one another directly, or alternatively through a linker or spacer. Therefore, in a particular embodiment the conjugate comprises a ruthenium complex of formula (I), an ABCG2 substrate, and a linker.
As it is used herein, the term “linker,” also referred to as “spacer,” refers to a chemical group or moiety which covalently binds to the ruthenium complex and ABCG2 substrate. It includes compounds comprising or deriving from divalent radicals, such as alkylene, arylene, or heteroarylene.
In a particular embodiment, the ruthenium complex and the ABCG2 substrate are bound together through a linker. In a particular embodiment, the linker is a hydrocarbon chain between 1 and 20, preferably between 1 and 10, more preferably between 1 and 6 atoms long, where one or more of the carbon atoms of the chain can be replaced with a heteroatom selected from NR′, O, and S, where R′ is selected from H, C1-C6 alkyl, and acetyl; and where one or more of the carbon atoms of the chain can be substituted by a substituent selected from ═O, OH, SH, NH2, COOH, CH3, and Ph.
In a particular embodiment, the linker is an amino acid or a sequence of amino acids, preferably between 1 and 5 amino acids bound through peptide bonds. The term “amino acid” refers to compounds having an amino group and a carboxylic acid group and includes both natural amino acids and non-natural amino acids. The configuration of the amino acid can be L, D, or a racemic mixture.
One skilled in the art can determine the functional groups present in the linker and the way in which it binds to the ruthenium complex and the ABCG2 substrate, depending on the ruthenium complex and the ABCG2 substrate used. Preferably, the linker is bound to the ruthenium complex and the ABCG2 substrate by means of amide-, amine-, ether-, or ester-type bonds.
Preferably, the ruthenium complex in the conjugate is a complex of formula (I), wherein X is SR1R2, where R1 and R2 are as defined above. Once the conjugate is at the site of action or close to same, the active aquo complex (X═OH2) can be released by means of irradiation with light as described herein. An inactive derivative that can be handled with ease and activated in a controlled manner at the desired actuation time and place can thereby be provided. In a particular embodiment, SR1R2 is methionine or a derivative thereof.
The term “methionine derivative” refers fundamentally to derivatives preferably substituted in the amino and/or carboxylic acid group of the methionine. Methionine derivatives include C1-C6 alkyl esters, acetates, amides, C1-C6 alkyl amines, and C1-C6 dialkyl amines of methionine.
In a preferred embodiment, the methionine derivative is a compound of formula:
where
Ra is selected from OH, C1-C6 O-alkyl, OAc, NH2, NH— C1-C6 alkyl, N(C1-C6 alkyl)2, and NHAc; and
each Rb is independently selected from H, C1-C6 alkyl, and Ac.
In one embodiment, the ruthenium complex is a complex of formula (I′)
where X is SR1R2 as defined above, preferably X is methionine or a derivative thereof.
In a particular embodiment, the ruthenium complex is a complex of formula (I′) where SR1R2 is methionine or a derivative thereof, preferably methionine, and is bound directly to the ABCG2 substrate, preferably through the amino group or the carboxylic acid group.
In another embodiment, the ruthenium complex is a complex of formula (I′) where SR1R2 is methionine or a derivative thereof, preferably methionine, and is bound to the ABCG2 substrate by means of a linker, preferably through the amino group or the carboxylic acid group.
In a particular embodiment, the conjugate comprises:
In one embodiment, the conjugate is:
where Y is as defined above, preferably Y is CF3CO2−.
The conjugate of the invention has various advantages. It allows precise transport of the active complex to the site of action, reducing the required dose, and allows selectively acting on cancer cells, preventing the attack on normal cells.
These conjugates can be obtained by means of conventional techniques known by one skilled in the art. If the ruthenium complex and the ABCG2 substrate do not contain functional groups suitable for chemically binding to one another, they can be derivatized to achieve the desired binding. Alternatively, the ruthenium complex and the ABCG2 substrate can be bound to one another (directly or after derivatization) through a bifunctional linker by means of reactions known by one skilled in the art.
The Ru complexes and the conjugates of the invention can be used for preparing a pharmaceutical composition for treating cancer comprising cancer stem cells. Therefore, another aspect of the invention is a pharmaceutical composition comprising a ruthenium complex of formula (I) or a conjugate as defined herein, for use thereof in the treatment of cancer comprising cancer stem cells.
As it is used herein, the expression “pharmaceutical composition” refers to a formulation which has been adapted for administering a predetermined dose of one or more useful therapeutic agents to a cell, a group of cells, an organ, a tissue, or an organism.
The ruthenium complexes or the conjugates are administered in a therapeutically effective amount. A “therapeutically effective amount” is understood to be an amount which can provide a therapeutic effect and can be determined by one skilled in the art using means that are commonly used. The effective amount will vary with the particular disorder being treated, the age and physical condition of the subject being treated, the acuteness of the disorder, the duration of treatment, the nature of the simultaneous or combination therapy (if any), the specific route of administration, and similar factors within the knowledge and experience of the health professional. Generally, a maximum dose, i.e., the maximum safe dose according to reasonable medical judgment, is preferably used. For example, if the subject has a tumor, an effective amount can be the amount which reduces tumor load or volume (as determined, for example, by means of obtaining images of the tumor). Effective amounts can also be evaluated by means of the presence and/or frequency of cancer cells in blood or another body liquid or tissue (for example, a biopsy). If the tumor affects normal tissue or organ operation, then the effective amount can be evaluated by measuring the normal tissue or organ operation. Those skilled in the art will see that dosages can also be determined with the guidelines found in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 9th edition (1996), Annex II, pages 1707-1711 and in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th edition (2001), Annex II, pages 475-493.
The pharmaceutical compositions of the invention may include at least one pharmaceutically acceptable vehicle. As it is used herein, the term “pharmaceutically acceptable vehicle” means a filler, diluent, encapsulating material, or adjuvant for solid, semisolid, or liquid formulation, that is of any inert non-toxic type which is acceptable to the patient from a pharmacological/toxicological viewpoint and to the manufacturing pharmaceutical chemist from a physical/chemical viewpoint regarding composition, formulation, stability, patient acceptance, and bioavailability. Remington's Pharmaceutical Sciences, edited by Gennaro, Mack Publishing, Easton, Pa., 1995 describes various vehicles used in the formulation of pharmaceutical compositions and known techniques for preparing same. Some examples of materials that can be used as pharmaceutically acceptable vehicles include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; gum tragacanth powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propyleneglycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as TWEEN™ 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline solution; Ringer's solution; ethyl alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate; furthermore, coloring agents, release agents, coating agents, sweetening agents, flavoring agents and perfumes, preservatives, and antioxidants can also be present in the composition, according to the criterion of the one preparing the formulation. If filtration or other terminal sterilization methods are not viable, the formulations can be manufactured under aseptic conditions.
Pharmaceutical compositions of the invention include any solid composition (tablets, pills, capsules, granules, etc.), semi-solid composition (creams, salves, etc.), or liquid composition (solution, suspension, or emulsion).
The pharmaceutical compositions of this invention can be administered to a patient using any means known in the art including oral and parenteral routes. According to such embodiments, the compositions of the invention can be administered by means of injection (for example, intravenous, subcutaneous or intramuscular, intraperitoneal injection). In a particular embodiment, the ruthenium complexes or the conjugates of the invention are administered systemically, for example by means of i.v. infusion or injection. Injectable preparations, for example, sterile aqueous or oily injectable suspensions, can be formulated according to the known technique using suitable suspending agents and dispersing agents or wetting agents. The sterile injectable preparation can also be a sterile injectable solution, suspension, or emulsion in a non-toxic diluent or solvent, acceptable by means of parenteral route, for example, as a solution in 1,3-butanediol. The acceptable vehicles and solvents that can be used include, among others, water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. Furthermore, sterile fixed oils are conventionally used as solvent or suspension medium. Any light fixed oil including synthetic mono- or diglycerides can be used for this purpose. Furthermore, fatty acids such as oleic acid are used in the preparation of injectable products. The injectable formulations can be sterilized, for example, by means of filtering through a bacteria-retaining filter, or by means of incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or another sterile injectable medium before use.
The compositions may comprise the ruthenium complex or the conjugate as a single agent or in combination with another therapeutic agent, such as an anti-tumor drug. In one embodiment, the pharmaceutical composition or the medicinal product of the invention comprises the combination of a ruthenium complex of formula (I) or a conjugate and an anti-tumor drug formulated for simultaneous, separate, or sequential administration. This means that the combination of the two compounds can be administered:
In a particular embodiment, the ruthenium complex of formula (I) or the conjugate and the anti-tumor drug are administered independently (i.e., in two dosage forms) but at the same time. In another particular embodiment, the ruthenium complex of formula (I) or the conjugate is administered first, and the other anti-tumor drug is then administered separately or sequentially. In an additional particular embodiment, the other anti-tumor drug is administered first and the compound of formula (I) or the conjugate is then administered separately or sequentially, as defined.
As it is used herein, the term “anti-cancer drug” or “anti-tumor drug,” also referred to as “anti-cancer agent,” “anti-tumor agent,” or “antineoplastic agent,” refers to an agent which is useful in the treatment of cancer. Anti-tumor agents according to the present invention include, without limitation, alkylating agents, antimetabolites, topoisomerase inhibitors, and anthracyclines.
As it is used herein, the term “alkylating agent” also referred to as “antineoplastic alkylating agent,” refers to an agent mediating the transfer of an alkyl group from a molecule to DNA. The alkyl group can be transferred as an alkyl carbocation, a free radical, a carbanion, or a carbene (or the equivalent thereof). Alkylating agents are used in chemotherapy to cause damage to the DNA of cancer cells. Alkylating agents are generally divided into six classes:
As it is used herein, the term “antimetabolite” refers to a chemical which inhibits the use of a metabolite, which is another chemical that is part of normal metabolism. Such substances often have a structure similar to the structure of the metabolite with which they interfere, such as antifolates which interfere with the use of folic acid. The presence of antimetabolites may have toxic effects on cells, such as stopping cell growth and cell division, such that these compounds are used for cancer chemotherapy. The antimetabolites pose as a purine or a pyrimidine, preventing the incorporation thereof into DNA during the S phase (of the cell cycle), stopping normal development and division. They also affect RNA synthesis. However, given that thymidine is used in DNA but not in RNA (in which uracil is used instead), the inhibition of thymidine synthesis by thymidylate synthase selectively inhibits DNA synthesis with respect to RNA synthesis. The antimetabolites can be selected from:
As it is used herein, term “topoisomerase inhibitor” refers to an agent designed for interfering with the action of topoisomerase enzymes (topoisomerases I and II). It is believed that topoisomerase inhibitors block the ligation step of the cell cycle, generating single- and double-stranded breaks which damage genome integrity. The introduction of these breaks subsequently leads to apoptosis and cell death. Non-limiting illustrative examples of topoisomerase inhibitors include etoposide, teniposide, topotecan, irinotecan, diflomotecan, or elomotecan.
As it is used herein, the term “anthracycline” refers to a class of (CCNS or cell cycle non-specific) drugs derived from Streptomyces bacteria strains and used in cancer chemotherapy. Non-limiting illustrative examples of anthracyclines include daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, mitoxantrone, etc.
Other anti-cancer or anti-tumor agents according to the present invention include, without limitation, the following agents:
Alternatively, the ruthenium complex and the anti-tumor drug can be bound to one another forming a conjugate. Therefore, in another aspect the invention relates to a conjugate comprising:
Preferably, the ruthenium complex and the anti-tumor drug are covalently bound to one another directly or through a linker or spacer. Therefore, in a particular embodiment, the conjugate comprises a ruthenium complex of formula (I), an anti-tumor drug, and a linker.
Particular and preferred embodiments for the linker, the ruthenium complex, and the way of binding, are defined above in relation to the ruthenium complex-ABCG2 substrate conjugate.
One aspect of the invention is the use of a ruthenium complex or a conjugate as defined herein for preparing a medicinal product for treating cancer comprising cancer stem cells.
Another aspect of the invention relates to a ruthenium complex or a conjugate as defined herein for use thereof in the treatment of cancer comprising cancer stem cells.
Another aspect of the invention relates to a method for treating cancer comprising cancer stem cells, which method comprises administering to a patient in need of said treatment a therapeutically effective amount of a ruthenium complex or a conjugate as defined herein.
As they are used herein, the terms “treat,” “treating,” and “treatment” generally include the eradication, elimination, reversion, relief, modification, or control of cancer after its onset.
As it is used herein, the term “cancer,” also referred to as “carcinoma,” refers to a disease characterized by an uncontrolled proliferation of abnormal cells capable of invading adjacent tissues and spreading to distant organs. Within the context of the present invention, this term includes any type of cancer or tumor. Non-limiting illustrative examples of said cancers or tumors include blood cancers (e.g., leukemias or lymphomas), neurological tumors (e.g., astrocytomas or glioblastomas), melanoma, breast cancer, lung cancer, head and neck cancer, gastrointestinal tumors (e.g., stomach cancer, pancreatic cancer, or colorectal cancer (CRC)), liver cancer (e.g., hepatocellular carcinoma), renal cell cancer, genitourinary tumors (e.g., ovarian cancer, vaginal cancer, cervical cancer, bladder cancer, testicular cancer, prostate cancer), bone tumors, vascular tumors, etc.
As it is used herein, the term “cancer stem cell,” also known as “CSC”, refers to a type of stem cell which conserves self-renewal and differentiation capacities and is furthermore capable of causing tumors. Cancer stem cells are highly tumorigenic and chemoresistant. It is believed that these cells may be related to tumor relapse processes and/or metastasis after chemotherapy. The presence of cells of this type has been described in various types of tumors including, without limitation, hematologic neoplasm (Lapidot T et al. 1994 Nature 367(6464): 645-648), breast cancer (Al-Hajj M et al. 2003 Proc Natl Acad Sci USA 100 (7): 3983-3988), lung cancer (Kim C F et al. 2005 Cell 121(6): 823-835), colon cancer (O'Brien C A et al. 2007 Nature 445(7123): 106-110), prostate cancer (Collins A T et al. 2005 Cancer Res 65(23): 10946-10951), ovarian cancer (Szotec P P et al. 2006 Proc Natl Acad Sci USA 103(30): 11154-11159), and pancreatic cancer (Li C et al. 2007 Cancer Res 67(3): 1030-1037).
In a particular embodiment, the cancer comprising cancer stem cells is selected from hematologic neoplasm, breast cancer, lung cancer, colon cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, and kidney cancer. In an even more particular embodiment, the cancer is pancreatic cancer, preferably pancreatic adenocarcinoma.
As it is used herein, the term “hematologic neoplasm” refers to a heterogeneous group of cancers affecting the blood, the bone marrow, and lymph nodes. This term includes leukemias (affecting the bone marrow and spreading to peripheral blood) and lymphomas (originated in different lymphoid tissues: lymph nodes, spleen, and mucosa-associated lymphoid tissue). Hematologic neoplasms can be myeloid or lymphoid neoplasms depending on their origin.
Myeloid hematologic neoplasms include:
Lymphoid hematologic neoplasms include:
As it is used herein, the term “breast cancer,” also known as “malignant breast neoplasm” or “breast tumor,” refers to cancer originated in the breast tissue, usually in the inner lining of milk ducts or lobules that supply milk to the ducts. Depending on the receptor state detected by means of immunohistochemistry, particularly on the presence or absence of estrogen receptor (ER), progesterone receptor (PR), and on the expression level of HER2/neu (normal expression/underexpression with respect to overexpression), breast cancers can be divided into ER-positive (ER+) breast cancer, ER-negative (ER−) breast cancer, PR-positive (PR+) breast cancer, PR-negative (PR−) breast cancer, HER2-positive (HER2+) breast cancer (cancer overexpressing HER2), HER2-negative (HER2−) breast cancer (cancer expressing normal levels of HER2, or underexpressing HER2, or not expressing a detectable level of HER2), hormone receptor-negative breast cancer, i.e., breast cancer without estrogen or progesterone receptors (abbreviated as ER−/PR− breast cancer); and triple negative breast cancer, i.e., breast cancer without estrogen or progesterone receptors and with normal expression/underexpression (or with the absence of a detectable expression level) of HER2 (abbreviated as ER-/PR-/HER2-breast cancer).
As it is used herein, the term “lung cancer,” also referred to as “pulmonary cancer” or “pulmonary tumor,” refers to any uncontrolled cell growth in lung tissues, including, but not limited to, microcytic lung carcinoma, combined microcytic carcinoma, non-microcytic lung carcinoma, sarcomatoid carcinoma, salivary gland tumor, carcinoid tumor, adenosquamous carcinoma, pleuropulmonary blastoma, and carcinoid tumor.
As it is used herein, the term “prostate cancer” refers to an uncontrolled (malignant) growth of cells originating from the prostate gland.
As it is used herein, the term “ovarian cancer,” also referred to as “ovarian tumor,” refers to a group of tumors originating in the ovaries and includes, without limitation, serous ovarian cancer, non-invasive ovarian cancer, mixed phenotype ovarian cancer, mucinous ovarian cancer, endometrioid ovarian cancer, ovarian clear cell cancer, ovarian serous papillary cancer, Brenner tumor of ovary, and undifferentiated adenocarcinoma.
As it is used herein, the term “pancreatic cancer,” also referred to as “pancreatic tumor,” refers to cancer derived from pancreatic cells including, but not limited to, adenocarcinomas, adenosquamous carcinomas, signet ring cell carcinomas, hepatoid carcinomas, colloid carcinomas, undifferentiated carcinomas, undifferentiated carcinomas with osteoclast-like giant cells, and islet cell carcinomas.
As it is used herein, the term “cervical cancer,” also referred to as “cervical carcinoma,” refers to a malignant tumor developed in the lower fibromuscular portion of the uterus which projects into the vagina.
As it is used herein, the term “kidney cancer,” also referred to as “renal cancer,” “renal adenocarcinoma,” or “renal cell cancer,” refers to any malignant proliferative disorder of kidney cells, particularly to a cancer formed by malignant cells in the tubules of the kidney.
In a particular embodiment, cancer stem cells have low c-MYC expression levels.
As it is used herein, the term “c-MYC” refers to the gene encoding a multifunctional nuclear phosphoprotein which participates in cell cycle progression, apoptosis, and cell transformation. The c-MYC protein in humans has the sequence shown with accession number P01106 in the Uniprot database dated 2 Nov. 2016.
As it is used herein, the term “expression level” of a gene refers to the amount of the gene product that can be measured in cells, in which the gene product can be a transcription product or a translation product. Accordingly, the expression level may correspond to a nucleic acid product of the gene such as an mRNA or a cDNA or a polypeptide product of the gene.
As understood by one skilled in the art, the expression level of the c-MYC gene can be quantified by means of quantifying the expression levels of the protein encoded by said gene, i.e., c-MYC, or by means of determining the activity of said protein. c-MYC activity can be determined using various assays known by one skilled in the art, by way of non-limiting illustrative example, by determining transcriptional activity.
The expression level of the protein can be quantified by means of any conventional method which allows detecting and quantifying said protein in cells, such as Western blot Western, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemical and immunohistochemical techniques, techniques based on the use of protein microarrays or biochips including specific antibodies or assays based on colloidal precipitation in formats such as dipsticks.
Moreover, the quantification of the expression levels of a gene can be determined by measuring the levels of the RNA resulting from the transcription of said gene (messenger RNA or mRNA), or alternatively, from the complementary DNA (cDNA) of said gene. Virtually any conventional method can be used within the framework of the invention for detecting and quantifying the levels of mRNA or its corresponding cDNA. By way of non-limiting illustration, the mRNA levels encoded by said gene can be quantified by means of using conventional methods, for example, methods which comprise amplifying the mRNA and quantifying the amplification product of said mRNA, such as electrophoresis and staining, or alternatively, by means of Southern blot and the use of suitable probes, Northern blot and the use of probes specific for the mRNA of the gene of interest or its corresponding cDNA, nuclease S1 mapping, RT-LCR, hybridization, microarrays, RT-PCR, etc.
As it is used herein, “low expression levels” refers to the expression level of a gene that is lower than the reference value. In particular, cells can be considered to have low c-MYC expression levels when the expression levels in the cells are at least 1.1 times, 1.5 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times lower, or even lower, with respect to the reference value.
As it is used herein, “reference value” refers to a value obtained in the laboratory and used as a reference for the expression values of c-MYC. The reference value or reference level can be an absolute value, a relative value, a value a value having an upper and/or lower limit, a range of values, an average value, a median value, a mean value, or a value as compared to a specific control or reference value. The reference value can be based on an individual sample value, such as a value obtained from a cancer stem cell sample of a subject, for example. The reference value can be based on a large number of samples, such as the expression values of c-MYC in cancer stem cell samples of a population of subjects. In a particular embodiment, the reference value corresponds to the expression level in a cancer not having cancer stem cells.
The inventors have observed that the metallation reaction increases upon irradiating with light. Therefore, in a preferred embodiment the ruthenium complexes or the conjugates of the invention are used in the treatment of cancer in the presence of irradiation with light. In one embodiment, the irradiation is performed with ultraviolet (UV), visible, or near infrared (IR) light. In a particular embodiment, irradiation is performed with light having a wavelength between 200 and 1000 nm, preferably between 300 and 800 nm, more preferably between 400 and 600 nm. Preferably, irradiation is performed with light having a wavelength between 400 and 500 nm.
Therefore, in one embodiment the invention relates to a ruthenium complex or a conjugate as defined herein for use thereof in the treatment of cancer comprising cancer stem cells by means of irradiation with light, preferably with light having a wavelength between 200 and 1000 nm, more preferably between 300 and 800 nm, more preferably between 400 and 600 nm. In a particular embodiment, irradiation is performed with light having a wavelength between 400 and 500 nm.
Another aspect of the invention relates to a method for treating cancer comprising cancer stem cells, which method comprises administering to a patient in need of said treatment a therapeutically effective amount of a ruthenium complex or a conjugate as defined herein, and where said method comprises irradiating said ruthenium complex or conjugate with light, preferably with light having a wavelength between 200 and 1000 nm, more preferably between 300 and 800 nm, more preferably between 400 and 600 nm. In a particular embodiment, irradiation is performed with light having a wavelength of between 400 and 500 nm.
Methods suitable for performing irradiation at the desired wavelength are known by one skilled in the art.
Additionally, it has been observed that the ruthenium complexes of formula (I) and the corresponding conjugates, wherein X is selected from SR1R2 are kinematically stable and only react with c-MYC by means of irradiation with light. Therefore, in these cases it is possible to provide an inactive derivative that can be handled with ease and activated in a controlled manner at the desired actuation time and place by means of irradiation with light. This represents a significant advantage in cancer treatment. Therefore, a preferred embodiment of the invention relates to the ruthenium complexes as defined herein, wherein X is selected from SR1R2.
The following non-limiting examples seek to illustrate the present invention and must not be interpreted as limiting the scope thereof.
High-performance liquid chromatography (HPLC) was carried out using Agilent 1100 liquid chromatography-mass spectrometer equipment. Analytical HPLC was carried out using a Phenomenex Luna-C18 reversed-phase analytical column (10×250 mm, 5 μm), 1 mL/min, with different gradients (see below). The adducts were purified in a Phenomenex Luna-C18 reversed-phase analytical column (250×10 mm). Electrospray ionization mass spectrometry (ESI/MS) was performed with an Agilent 1100 Series LC/MSD VL G1956A model in positive polarity mode.
a) A: H2O containing 0.1% TFA, B: ACN containing 0.1% TFA.
b) A: 95:5 H2O:ACN containing 100 mM TEAA, B: 70:30 ACN:H2O containing 100 mM TEAA.
Gradient 2: 0% of B for 5 min followed by 0%→100% of B for 55 min.
Ruthenium complex 1 was prepared following the method described in Kaveevivitchai et al., Inorg. Chem. 2012, 51, 2930. RuCl3.3H2O (500 mg, 2 mmol) and 2,2′:6′,2″ terpyridine (460 mg, 1 equiv) was dissolved in a deoxygenated H2O:EtOH 1:1 mixture (20 mL), and heated at reflux for 4 h in the dark. The resulting precipitate was washed with EtOH (×3) and Et2O and used directly in a second step involving treatment with 2,2′-bipyridine (312 mg, 1 equiv) in a deoxygenated H2O:EtOH 1:1 mixture (20 mL) and heated at reflux overnight. Precipitation with excess KPF6 gave rise to the salt [Ru(terpy) (bpy)Cl]PF6 1 as a brown powder with an overall yield of 60% (800 mg). EM-ESI+ (m/z): Calculated for C25H19ClN5Ru: 525.0. Found: 526.0 [M+1H]+.
Ruthenium complex 2 was prepared following a modified method of the literature (Takeuchi et al., Inorg. Chem. 1984, 23, 1845). Complex 1 (150 mg, 0.22 mmol) was dissolved in a deoxygenated H2O:EtOH 1:1 mixture (20 mL) and heated at reflux for 1 h. The crude was concentrated, and the product was purified by means of reversed-phase HPLC (HPLC-FR), 4 mL/min, gradient of 5 to 95% of B for 35 min (A: H2O containing 0.1% TFA, B: ACN with 0.1% TFA), and identified as the desired product by means of mass spectrometry (78% yield). EM-ESI+ (m/z): Calculated for C25H21N5ORu: 509.1. Found: 508.0 [M−1H]+.
Ruthenium complex 4 was prepared following a modified method of the literature (Bahreman et al., Eur. J. 2012, 18, 10271). Complex 1 (50 mg, 0.07 mmol) was dissolved in thiodiethanol (10 μL, 1.3 equiv) and heated at reflux overnight in the dark. The product was purified by means of HPLC-FR at 4 mL/min with a gradient of 5 to 95% of B for 35 min (A: H2O containing 0.1% TFA, B: ACN containing 0.1% TFA) and identified as the desired product by means of mass spectrometry and NMR (33 mg, 65% yield). 1H NMR (500 MHz, D2O) 9.70 (m, 1H), 8.55 (m, 3H), 8.38 (m, 2H), 8.31 (m, 1H), 8.23 (m, 2H), 7.90 (m, 3H), 7.72 (m, 3H), 7.24 (ddd, J=7.6, 5.6, 1.3 Hz, 2H), 7.13 (dt, J=4.1, 2.0 Hz, 1H), 6.98 (ddd, J=7.2, 4.8, 1.3 Hz, 1H), 3.20 (t, J=5.9 Hz, 4H), 1.87 (m, 4H). 13C NMR (500 MHz, D2O) δ 157.68 (C), 157.15 (C), 156.62 (C), 152.96 (CH), 151.72 (CH), 149.42 (CH), 138.62 (CH), 138.02 (C), 137.56 (CH), 136.56 (CH), 128.01 (CH), 127.29 (CH), 126.52 (CH), 124.63 (C), 124.25 (CH), 123.77 (CH), 123.37 (CH), 57.57 (CH2), 35.01 (CH2). EM-ESI+ (m/z): Calculated for C29H29N5O2RuS: 613.1. Found: 612.0 [M−1H]+.
Ruthenium complex 5 was prepared following a modified method of the literature (Goldbach et al., Chem. Eur. J. 2011, 17, 9924). Complex 1 (50 mg, 0.07 mmol) and N-acetyl-L-methionine (19 mg, 5 equiv) were dissolved in a deoxygenated H2O:EtOH 1:1 mixture (2 mL) and heated at reflux overnight in the dark. The product was purified by means of HPLC-FR at 4 mL/min with a gradient of 5 to 95% of B for 35 min (A: H2O containing 0.1% TFA, B: ACN containing 0.1% TFA) and identified as the desired product by means of mass spectrometry (24 mg, 45% yield). EM-ESI+ (m/z): Calculated for C32H32N6O3RuS: 682.1. Found: 681.0 [M−1H]+.
This synthesis requires mixing aquo complex 2 and 1 thioether ligand equivalent in water at 80° C. in the dark. The course of the reaction can be monitored using RP-HPLC. After purification by means of RP-HPLC, compound [10].TFA2 was obtained as a brown powder.
Compound 7. A suspension of riboflavin 6 (2 g, 5.3 mmol, 1.0 equiv), potassium carbonate (1.5 equiv, 8 mmol, 1.1 g), in 12 mL of dry DMF at room temperature was stirred for 45 min under inert atmosphere. An ethyl bromoacetate solution (2 equiv, 10.6 mmol, 1.1 mL) in 4 mL of dry DMF was then added dropwise and stirring was maintained until the reaction (24 h) was completed. The solvent was evaporated and the resulting residue was purified by means of column chromatography (10% MeOH/CH2Cl2) to obtain 7 as a yellow solid (1.2 g, 50%). 1H NMR (500 MHz, DC3OD) δ 8.08 (s, 1H), 4.82 (s, 2H), 4.47 (i, 1H), 4.26 (q, J=6.8 Hz, 2H), 3.85 (m, 4H), 3.69 (dd, J=11.4, 6.0 Hz, 2H), 2.60 (s, 3H), 2.49 (s, 3H), 1.31 (t, J=6.8 Hz, 3H). EMAR (ESI): Calculated for CH6N4O8: 462.18. Found: 463.2 [M+1H]+.
Compound 8. Compound 7 (400 mg, 0.86 mmol) was suspended in 6 M HCl (15 mL) and heated at reflux for 1 h. The reaction was neutralized with NaOH until about pH 7, diluted with water, and purified by means of preparative RP-Büchi Sepacore (gradient: 5% of B, 5 min; 15%→95% of B, 40 min). The solvent was eliminated at reduced pressure, giving rise to a yellow solid (260 mg, 69%). 1H NMR (500 MHz, D2O) δ 7.71 (s, 1H), 7.64 (s, 1H), 4.87 (m, 1H), 4.59 (s, 2H), 4.23 (m, 1H), 3.78 (m, 4H), 3.60 (m, 1H), 2.40 (s, 3H), 2.28 (s, 3H). EMAR (ESI): Calculated for C19H22N4O8: 434.14. Found: 435.2 [M+1H]+, 457.1 [M+1Na]+.
Compound 9. A mixture of Fmoc-Met-OH (371 mg, 1 mmol) and DIEA (695 μL, 4 mmol, 4 equiv) was added to a suspension of 2-chlorotrityl resin (0.25 mmol) in 2.5 mL of CH2Cl2, and the mixture was stirred for 50 min. The resin was washed with CH2C12/MeOH/DIEA (17:2:1) (3×5 mL, 2 min), CH2Cl2 (3×5 mL, 2 min), and treated with 20% piperidine in DMF (1×5 mL, 10 min).
A solution of compound 8 (170 mg, 0.4 mmol), DIEA/DMF 0.2 M (8.2 mL, 1.6 mmol, 4 equiv), and HATU (152 mg, 0.4 mmol, 1 equiv) was added to the preceding resin (0.4 mmol) and the suspension was stirred for 50 min. The resin was washed with DMF (3×10 mL, 2 min) and CH2Cl2 (2×10 mL, 2 min) and then treated with the deprotection cocktail (900 μL TFA, 50 μL CH2Cl2, 25 μL H2O and 25 μL TIS; 1 mL of cocktail/40 mg resin) for 1.5-2 h. After filtration, the crude was concentrated at reduced pressure.
The residue obtained after the concentration was purified by means of preparative RP-Büchi Sepacore, gradient: 5% of B, 5 min; 15%-95% of B, 40 min (A: H2O containing 0.1% TFA, B: MeOH containing 0.1% TFA). The suitable fractions were collected, concentrated, and lyophilized to obtain the desired product 9 as a pale yellow powder (50 mg, 23%). 1H NMR (500 MHz, D2O) δ 8.00 (s, 1H), 7.97 (s, 1H), 5.13 (m, 1H), 4.97 (d, J=14.0 Hz, 1H), 4.86 (m, 2H), 4.46 (m, 1H), 3.97 (m, 2H), 3.89 (d, J=11.9 Hz, 1H), 3.75 (m, 1H), 3.01 (m, 2H), 2.76 (d, J=2.4 Hz, 1H), 2.61 (s, 3H), 2.50 (s, 3H), 2.26 (m, 2H), 2.09 (s, 3H). EMAR (ESI): Calculated for C24H31N5O9S: 565.18. Found: 566.19.
Compound 10. Complex 2 (35 mg, 0.07 mmol) and compound 9 (40 mg, 1 equiv) were dissolved in deoxygenated H2O:EtOH 1:1 (2 mL) and heated at reflux overnight in the dark. The product was purified by means of RP-HPLC at 4 mL/min with a gradient of 5 to 95% of B for 35 min (A: H2O containing 0.1% TFA, B: ACN containing 0.1% TFA). Suitable fractions were collected, concentrated and lyophilized to obtain the desired product 10 as a brown powder (10 mg, 13%). 1H NMR (500 MHz, D2O) δ 8.64 (dd, J=19.0, 8.2 Hz, 2H), 8.48 (dd, J=24.0, 8.1 Hz, 2H), 8.32 (m, 2H), 8.09 (m, 3H), 7.98 (m, 2H), 7.81 (m, 3H), 7.62 (t, J=5.4 Hz, 1H), 7.54 (m, 3H), 7.26 (m, 2H), 7.09 (t, J=6 Hz, 1H), 5.15 (m, 1H), 4.91 (m, 2H), 4.60 (m, 1H), 4.50 (m, 2H), 3.97 (m, 2H), 3.86 (m, 1H), 3.73 (dd, J=11.8, 5.8 Hz, 1H), 2.60 (s, 3H), 2.36 (s, 3H), 1.92 (m, 1H), 1.79 (m, 2H), 1.69 (m, 1H), 1.24 (s, 3H). EMAR (ESI): Calculated for C49H50N10O9RuS: 1056.25. Found: 1055.25 [M−1H]+.
The oligonucleotides (10 μM) were treated with ruthenium complexes in 10 mM potassium phosphate buffer with 100 mM KCl, pH=7.5, for 30 min at room temperature. The solutions were incubated in the dark or irradiated with a high-power LED. For irradiation, the samples were placed in a standard 10 mm cuvette in a sample support and irradiated with a high-power LED unit at 455 nm with 900 mW for 30 min (Thorlabs, Inc., Catalog No.: M455L3). The light of the LED is collimated by a planar-convex lens with a short focal length for the purpose of maximizing irradiation power.
First, guanosine monophosphate (GMP) metallating capacity of complex 1 was studied. To that end, complex 1 (250 μM) was mixed with 3 equivalents of GMP (750 μM) in 10 mM phosphate buffer (pH=7.5) and 100 mM NaCl. After 30 min at room temperature, partial formation (more than 50%) of the aquo [Ru(terpy) (bpy)H2O]2+ complex (2) was observed, whereas the GMP remained essentially unreacted (
To confirm that GMP metallation is completely orthogonal, competitive experiments were carried out with the other three nucleotides (AMP, CMP, and TMP). Specifically, complex 1 (250 μM) was mixed with GMP (3 equiv, 750 μM), AMP (3 equiv, 750 μM), TMP (3 equiv, 750 μM), and CMP (3 equiv, 750 μM) in 10 mM phosphate buffer (pH=7.5) and 100 mM NaCl. After irradiating at 455 nm for 30 min, the formation of the aquo [Ru(terpy) (bpy)H2O]2 complex (2) and the exclusive modification of the GMP was observed, giving rise to monoadduct 3 (
The selectivity of GMP metallation in the presence of lysine and cysteine derivatives was also confirmed. To that end, complex 1 (250 μM) was mixed with GMP (3 equiv, 750 μM) in the presence of H-Lys(Boc)-OH (3 equiv, 750 μM) or Ac-Cys-OH (3 equiv, 750 μM) in 10 mM phosphate buffer (pH=7.5) and 100 mM NaCl. After irradiating at 455 nm for 30 min, the exclusive modification of the GMP was observed, giving rise to monoadduct 3 (
A 10 μM solution of parallel c-MYC quadruplex d[TTGAG3TG3TAG3TG3TA3] (SEQ ID NO: 1) was treated with 5 equivalents of ruthenium complex 1 in 10 mM phosphate buffer (pH=7.5) and 100 mM KCl and the mixture was irradiated for 30 min (λ=455 nm) at room temperature. The formation of a product with mass corresponding to the monoadduct derivative MYC-[Ru] with a conversion of 81% was observed (
Complex 4 (250 μM) was mixed with 3 equivalents of GMP (750 μM) in 10 mM phosphate buffer (pH=7.5) and 100 mM NaCl. After 30 min at room temperature in the dark, no new product formation was observed (
A 10 μM solution of quadruplex c-MYC was treated with 5 equivalents of ruthenium complex 4 in 10 mM phosphate buffer (pH=7.5) and 100 mM KCl. The irradiation of the mixture for 30 min (λ=455 nm) at room temperature gave rise to the formation of the product MYC-[Ru] (
Complex 5 (250 μM) was mixed with 3 equivalents of GMP (750 μM) in 10 mM phosphate buffer (pH=7.5) and 100 mM NaCl. After 30 min at room temperature in the dark, no new product formation was observed (
A 10 μM solution of quadruplex c-MYC was treated with 5 equivalents of ruthenium complex 5 in 10 mM phosphate buffer (pH=7.5) and 100 mM KCl. The irradiation of the mixture for 30 min (λ=455 nm) at room temperature gave rise to the formation of the product MYC-[Ru] (
It was observed that complex 1 (250 μM) in 10 mM phosphate buffer (pH=7.5) and 100 mM NaCl evolved slowly to the aquo 2 derivative in the dark. This hydrolysis is significantly accelerated by means of irradiation (λ=455 nm), given that complete conversion was observed after 30 min (
The cell lines were cultured in DMEM supplemented with 10% (v/v) of FCS (fetal calf serum, Gibco), penicillin (100 U/mL), and streptomycin (100 U/mL). The cell cultures were kept in 5% CO2, humidity, and at 37° C.
Real Time Quantitative PCR (qRT-PCR)
3×105 HeLa cells (cervical cancer cell line) or Vero cells (renal epithelial cell line) per well were seeded in 6-well plates. After one day, the compounds being assayed were added to the cell culture medium and the cells were incubated for 16 h or 48 h. The total RNA was extracted with a commercial kit (Qiagen RNeasy Mini Kit) following manufacturer's recommendations. The RNA was quantified in a Nanodrop ND-1000. Identical amounts of RNA were used in qRT-PCR (Promega GoTaq 1-Step RT-qPCR System) in CFX96 Real Time System equipment (Bio-Rad). The primers used were: c-MYC: sense: 5′-CTG AGG AGG AAC AAG AAG ATG AG-3′ (SEQ ID NO:2), antisense: 5′-TGT GAG GA GGT TTG CTG TG-3′ (SEQ ID NO:3); ALAS: sense: 5′-GTT TGG AGC AAT CAC CTT CG-3′ (SEQ ID NO:4), antisense: 5′-ACC CTC CAA CAC AAC AAC AG-3′ (SEQ ID NO:5); GAPDH: sense: 5′-GGT GTG AAC CAT GAG AAG TAT GA-3′ (SEQ ID NO:6), antisense: 5′-GAG TCC TTC GAT CAC CAC AAA G-3′ (SEQ ID NO:7)
HeLa cells were incubated to a confluence of 50% in the presence of the indicated concentrations of the assayed agents for 16 h. The cells were lysed in SDS-PAGE Laemmli buffer and subjected to SDS-PAGE and Western blot according to standard methods. The c-MYC protein was detected by means of an anti-MYC antibody (Santa-Cruz Biotechnologies). To detect the signal of the immunoreactive protein, a chemiluminescence method was followed (ECL, Amersham Biosciences).
HeLa cells were seeded in 6-well plates at a density of 3×105 cells per well. On the next day, complex 1 was added to the cell culture medium in a concentration of 100 μM and the cells were incubated for 16 h. The cells were washed two times with fresh medium and lysed in 70% HNO3/H2O. The samples were diluted to a total volume of 3 mL of water and analyzed by means of ICP-MS (7700x Agilent).
Four thousand cells per well were seeded in 96-well plates and incubated for 24 h. Different concentrations of the assayed agents were added to the culture medium and the cells were left in the incubator for another 72 h. MTT was then added at a final concentration of 0.5 mg/ml and incubated for 4 h. The cells were lysed and the formazan precipitates were solubilized by means of adding a volume of a solubilization solution containing 0.1 M HCl and 10% SDS in water. The absorbance was determined at 570 nm in a Tecan Infinito F200 Pro plate reader.
HeLa cells were seeded in 6-well plates at a density of 3×105 cells per well. On the next day, complex 1 was added to the cell culture medium at a concentration of 100 μM and the cells were incubated for 16 h. The following protocol was followed for nuclear isolation: the cells were washed with PBS and caused to settle by means of centrifugation. They were then resuspended in a cell lysis buffer [HEPES 10 mM; pH=7.5, KCl 10 mM, EDTA 0.1 mM, dithiothreitol 1 mM (DTT), and 0.5% Nonidet-40] and left on ice for 15-20 min, being mixed intermittently. The tubes were vigorously stirred to break the cell membranes and then centrifuged at 12,000 g at 4° C. for 10 min. The integrity of the deposited nuclei was confirmed by means of phase contrast microscopy in Nikon Eclipse TiE equipment. The supernatant fraction containing the cytoplasm and the sediment containing the nuclei were homogenized in 70% HNO3/H2O and analyzed by means of ICP-MS as described above. For chromatin extraction, a commercial kit was used (Chromatin Extraction kit, Abcam). The isolated chromatin was resuspended in 70% HNO3/H2O and analyzed by means of ICP-MS.
The inventors analyzed the effect of the complexes of the invention on the expression levels of the c-MYC oncogene, both on the messenger expression level and on the protein expression level.
As shown in
This data confirms that, unlike most quadruplex-targeting agents, complex 1 promotes an increase instead of a decrease in gene expression level, thereby acting as a transcriptional activator.
ICP-MS analysis of the isolated nuclei and chromatin obtained after treatment with complex 1 confirmed the presence of relatively significant amounts of ruthenium, in accordance with an efficient cell uptake and nuclear transport of the complexes, as can be seen in Tables 1 and 2 below.
Moreover, the cell viability assays confirmed that complex 1 is essentially non-cytotoxic (
In relation to the light-activated complexes (complex 5), qRT-PCR analysis of the expression of messenger RNA of the c-MYC gene showed that RNA levels increased after irradiation, being similar to those observed with complex 2 (aquo). The Western blot protein expression analysis confirmed that the increase of c-MYC protein expression took place in the presence of light, and that the protein levels did not change in the dark (
Pancreatic adenocarcinoma is a pancreatic cancer with a heterogeneous population of tumor cells, including a subpopulation of cancer stem cells (CSCs).
The inventors isolated CSCs and non-CSCs from 6 patient-derived primary tumors by means of FACS (fluorescence-activated cell sorting”). High throughput RNA sequencing (RNAseq) was carried out. RNAseq analysis for the CSC population in comparison with the corresponding non-CSC population generated a list of target genes based on common elements and their differential expression. Among negatively regulated genes, c-MYC was identified as being considerably downregulated in the subCSC population in 6 tumors. It must be highlighted that a downregulation of c-MYC is contrary to what has been commonly characterized for c-MYC in various tumors. However, the inventors have discovered that the expression of c-MYC is differentially regulated in CSCs in comparison with the more differentiated equivalents thereof.
On one hand, the inventors analyzed if complex 2 may increase c-MYC expression in pancreatic CSCs, and on the other hand, if the increase in c-MYC expression would negatively affect the CSC phenotype, for example, CSC self-renewal capacity. For this purpose, pancreatic ductal adenocarcinoma (PDAC) cells established from patient-derived xenografts with a low number of passes were cultured, treated with ruthenium complex 2 (RuH2O) at a concentration of 100 and 250 μM for 6, 12, or 24 hours, and c-MYC protein expression was evaluated by means of Western blot.
Using the complexes of the invention, it was tested if RuH2O complex 2 increases c-MYC expression in PDAC adherent cultures established based on patient-derived xenografts with a low number of passes in order to confirm the observations made in HeLa cells. In line with previous observations, treatment of Panc185 and PancA6L cells with 100 μM of RuH2O increased c-MYC protein expression 24 hours after treatment (
Furthermore, a reduction in PGC1alpha expression, a known c-MYC target, and a reduction in pluripotency-associated genes Oct3/4 and Sox2 were observed after treatment with 100 μM of RuH2O for 24 hours (
It was then studied if the increased c-MYC expression and the phenotype and transcriptional changes observed were cytotoxic. For this analysis, the ToxiLight™ BioAssay kit, a bioluminescent, non-destructive cytolysis assay kit designed for measuring the adenylate kinase (AK) enzyme released from damaged cells (
The effect of 100 μM of RuH2O on cultures rich in CSC was confirmed at the microscopic level and by measuring annexin V staining. As shown in
It was then evaluated if ruthenium complex 2 may negatively affect CSC phenotypes, such as self-renewal and tumorigenicity. To that end, PDAC adherent cultures established based on patient-derived xenografts with a low number of passes were treated with 250 μM of ruthenium complex 2 (RuH2O) for 24 hours. Treated and untreated cells were trypsinized and sphere cultures were established following the standard protocols (Cioffi M et al. 2015 Gut 12: 1936-1948). The cells were cultured in ultra-low adherent plates for 7 days in serum-free DMEM/F12 supplemented with B27. During these 7 days, the samples were treated again with 250 μM of RuH2O on days 1, 3, and 5 after initiation of the spheres.
The spheres were then disassociated and equal numbers of control-treated cells and RuH2O-treated cells were injected subcutaneously into immunocompromised NOD-SCID mice to evaluate the tumorigenicity thereof. Twelve weeks after the injection, the tumors were extracted and weighed.
To rule out the possibility that the effects mentioned above arise as a result of unspecific cytotoxic effects, adherent cultures (low in CSC) were treated for 48 hours with a single dose of RuH2O (100 or 250 μM) and their subsequent clonogenicity, self-renewal, and tumorigenicity was checked. Under these experimental conditions, no toxicity was observed using the present ToxiLight assay (
The trypsinized cells were then: 1) placed in a plate at a low confluence to evaluate their clonogenicity (
Finally, to investigate the capacity of the complexes of the invention to specifically target CSCs, RuH2O was bound to riboflavin through methionine sulfur (conjugate 10) and the cells were treated with this modified compound as described in detail. Again, the PDAC adherent cultures established based on patient-derived xenografts with a low number of passes were treated with 250 μM of Ru-riboflavin. To activate the ruthenium complex, the cells were treated with visible light to release and activate RuH2O. Twenty-four hours after treatment, the treated and untreated cells were trypsinized and sphere cultures were established following the standard protocols (Cioffi M et al. 2015 Gut 12: 1936-1948). The cells were cultured in ultra-low adherent plates for 7 days in serum-free DMEM/F12 supplemented with B27. On day 3 after initiation of the spheres, the cells were again treated with 250 μM of Ru-riboflavin.
Furthermore, the cells were also treated with fumitremorgin C (FTC), a specific ABCG2 inhibitor. After the first treatment and activation with Ru-riboflavin, a qRT-PCR analysis was performed to evaluate cMYC mRNA levels, as well as the expression of known CSC-related genes, such as the pluripotency-associated genes Klf4 and Oct3/4. Cells treated with Ru-riboflavin in the presence or absence of FTC showed an increase in cMYC RNA levels and a decrease in Klf4 and Oct3/4 CSC-related transcripts (
Finally, the tumorigenicity of sphere-rich CSCs was studied as described above. The analysis of the extracted tumors showed a trend towards tumor reduction and proliferation for Ru-riboflavin-treated cells in comparison with the control- and Ru-riboflavin/FTC-treated cells (
| Number | Date | Country | Kind |
|---|---|---|---|
| P201631426 | Nov 2016 | ES | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/ES2017/070745 | 11/10/2017 | WO | 00 |
