HK1151971B - Compounds for use in the treatment of cancer - Google Patents

Description

Compounds for the treatment of cancer

Technical Field

The present invention relates to compounds useful for the treatment of cancer. The invention also relates to the use of said compounds for the preparation of a medicament for the treatment of cancer. Also disclosed is a method of treating cancer in a human or non-human body, wherein said method comprises administering to said body a compound as described above. The invention further relates to pharmaceutical compositions comprising said compounds and compounds having cytoprotective activity. The invention also relates to the use of the pharmaceutical composition in the manufacture of a medicament for the treatment of cancer.

Background

EP0910360, US6147094, EP0936915, US6258828, EP1054670, US6310051, EP1060174, US6391895 disclose the use of dipyridoxyl (dipyridoxyl) -based chelators and metal chelates thereof in medicine and the use of certain manganese-containing compounds, especially manganese chelates, in medicine. Also disclosed is the use of the compounds as cytoprotective agents in the treatment of cancer. The above references disclose that certain chelating agents, especially those based on pyridoxine and aminopolycarboxylic acids, and their metal chelates are effective in treating or preventing cardiotoxicity due to anthracyclines, damage due to ischemia-reperfusion, and atherosclerosis. Pyridoxyl-based chelators and their chelates with trivalent metals have been previously described by Tafferro (Inorg. chem. 1984; 23: 1183-1192).

DPDP (N, N '-bis- (pyridoxal-5-phosphate) -ethylenediamine-N, N' -diacetic acid), and the dephosphorylated compound PLED (N, N '-bipyridoylethylenediamine-N, N' -diacetic acid) are bipyridoxyl compounds capable of chelating metals. The manganese chelate of these compounds, MnDPDP, and its dephosphorylated compound, MnPLED, have been previously described as having catalytic antioxidant activity, i.e., superoxide dismutase (SOD) -like activity. These compounds have been shown to have protective effects on normal cells, for example against the cytostatic drug doxorubicin and ischemia reperfusion. It is SOD-like active, a redox active manganese (Mn) bound to DPDP/PLED²⁺/Mn³⁺) Intrinsic properties of (Brurok et al, Biochem Biophys Res Commun.1999; 254: 768-721), which explains the protective effect. Thus, Brurok and co-workers (1999) have shown to be using non-redox active zinc (zink) (Zn)²⁺) The PLED metal complex loses its catalytic activity after substitution of the redox active manganese.

Laurent et al (Cancer Res.2005; 6: 948-56) and Alexandre et al (Jnat Cancer Inst.2006; 98: 236-44) have recently been described inMnDPDP (equivalent to the ready-to-use MRI contrast agent tylosin (teslas) not only increases the survival of normal cells but also increases the death of cancer cells during treatments that inhibit cell growth (e.g. with oxaliplatin). The agent for inhibiting cell growth can be used for increasing intracellular H₂O₂And triggering apoptosis to cause cancer cell death. Laurent et al postulated that MnDPDP increases intracellular H due to SOD-like activity₂O₂And thus act synergistically with the drug inhibiting cell growth. Due to the normal cell ratio to H in cancer cell₂O₂The basal level was much lower, so the authors suggested that the H would be lower₂O₂The level is increased to cause cell survival in normal cells. They also suggest that much higher H in cancer cells is elevated simultaneously₂O₂At basal level, apoptotic signals are generated and thus cell death results. Thus, these authors suggested that both effects, i.e. an increase in cancer cell death and an increase in normal cell survival, are caused by the SOD-like activity of MnDPDP, which is completely dependent on manganese with redox activity. It has also been shown that intravenous injection of the parent compound MnDPDP and its metabolite MnPLED in mice results in protection against certain cytostatic drugs (EP0910360 and US 6147094).

When MnDPDP is injected intravenously into humans, approximately 70% of the administered manganese is released. The dissociation of manganese from MnDPDP does not represent a serious problem for diagnostic imaging applications and occasionally therapeutic applications. However, for more frequent applications, the toxicity of the accumulated manganese can represent a serious toxicological problem, especially when it reaches neurotoxicity (Crosgrove & Zheng; NMR biomed.2004; 17: 544-53). Therefore, for frequent therapeutic applications (such as in cancer treatment), compounds that dissociate manganese should be avoided.

Many antineoplastic agents have adverse side effects. For example, paclitaxel is a cytostatic drug that exhibits anti-tumor activity against a variety of malignant tissues, including breast malignant tissues. However, at doses required for antitumor effects, paclitaxel has a number of adverse side effects including cardiovascular disorders as well as hematologic and gastrointestinal toxicity. Oxaliplatin is another example of a cytostatic drug, especially when combined with 5-fluorouracil (5-FU), which is effective for the treatment of colorectal cancer but whose use is also limited by serious adverse side effects, especially hematologic and neurotoxicity. Serious side effects also limit the use of radiation therapy in cancer.

Thus, there is an unmet medical need to discover new chemotherapeutic drugs with fewer side effects and to discover methods to protect normal cells from damage caused by cancer treatment.

Detailed Description

The present invention provides compounds for treating cancer having the ability to kill cancer cells. The invention also provides the use of a compound of the invention in the manufacture of a medicament for the treatment of cancer. The invention further comprises a method of treating cancer in a human or non-human body, wherein said method comprises administering to said body a compound of the invention. Also disclosed are pharmaceutical compositions comprising the above compounds and a second compound having cytoprotective ability (i.e., the ability to protect normal cells from the side effects of chemotherapeutic drugs and radiation during cancer therapy). Also provided is the use of a pharmaceutical composition according to the invention in the manufacture of a medicament for the treatment of cancer. The invention also relates to a method of treating cancer in a human or non-human body, wherein said method comprises administering to said body a compound of the invention.

A first aspect of the invention relates to a compound of formula I, or a salt thereof, for use in the treatment of cancer:

formula I

Wherein

X represents a group of a CH or an N,

each R¹Independently represent hydrogen or-CH₂COR⁵；

R⁵Represents a hydroxyl group, an optionally hydroxylated alkoxy, amino or alkylamido group;

each R²Independently represent a group ZYR⁶(ii) a Z represents a bond or optionally a group R⁷Substituted C_1-3Alkylene or oxyalkylene;

y represents a bond, an oxygen atom or a group NR⁶；

R⁶Is a hydrogen atom, a group COOR⁸Alkyl, alkenyl, cycloalkyl, aryl or aralkyl optionally substituted with a substituent selected from COOR⁸、CONR⁸ ₂、NR⁸ ₂、OR⁸、＝NR⁸、＝O、OP(O)(OR⁸)R⁷And OSO₃One or more groups of M;

R⁷is hydroxy, optionally hydroxylated, optionally alkoxylated alkyl or aminoalkyl;

R⁸is a hydrogen atom or an optionally hydroxylated, optionally alkoxylated alkyl group;

m is a hydrogen atom or the equivalent of a physiologically tolerable cation; for example, alkali or alkaline earth cations, ammonium ions or organic amine cations, such as meglumine ions;

R³is represented by C_1-8Alkylene, preferably C_1-6Alkylene radicals such as C_2-4Alkylene, 1, 2-cycloalkylene, or 1, 2-arylene optionally substituted with R⁷Substitution; each R⁴Independently represent hydrogen or C_1-3Alkyl, and wherein the compound is optionally with one or two Na' s⁺Or K⁺The chelate formed, but may also be a Na⁺And a K⁺Combinations of (a) and (b).

In one embodiment of the invention, R⁵Is hydroxy, C_1-8Alkoxy, ethylene glycol, glycerol, ammoniaRadical or C_1-8An alkylamide group;

z is a bond or selected from CH₂、(CH₂)₂、CO、CH₂CO、CH₂CH₂CO or CH₂COCH₂A group of (a); y is a bond;

R⁶is mono-OR poly (hydroxy OR alkoxylated) alkyl OR of the formula OP (O) (OR)⁸)R⁷A group of (a); and R is⁷Is hydroxyl or unsubstituted alkyl or aminoalkyl.

In another embodiment of the invention, R³Is ethylene and each radical R¹represents-CH₂COR⁵Wherein R is⁵Is a hydroxyl group.

In another embodiment of the invention, the compound of formula I is N, N '-dipyridoxyl ethylenediamine-N, N' -diacetic acid (PLED).

In yet another embodiment of the invention, the compound of formula I is N, N '-bis- (pyridoxal-5-phosphate) -ethylenediamine-N, N' -diacetic acid (DPDP).

Another embodiment of the invention describes the use of a compound of formula I as defined above for the preparation of a medicament for the treatment of cancer. The cancer may be any type of cancer, such as leukemia, breast cancer, colorectal cancer, liver cancer, and metastatic forms thereof. The medicament may comprise a pharmaceutically acceptable carrier or excipient.

A second aspect of the invention relates to a method of treating cancer in a human or non-human body, which method comprises administering to said body a compound of formula I according to the invention.

A third aspect of the invention relates to a pharmaceutical composition comprising a first compound of formula I as defined above and a second compound having cytoprotective ability.

In another embodiment of the present invention said second compound comprised in said pharmaceutical composition is a metal chelate comprising a compound of formula I as defined above.

In another embodiment of the present invention, the metal chelate compound contained in the pharmaceutical composition preferably has a Ka value of 10⁸To 10²⁴More preferably 10¹⁰To 10²²Most preferably 10¹²To 10²⁰。

In yet another embodiment of the present invention, said metal chelate is comprised in said pharmaceutical composition with a Ka value that is higher than the Ka value of iron (Fe) comprising a compound of formula I as defined above³⁺) The Ka value of the chelate is at least 10 lower³And (4) doubling.

In still another embodiment of the present invention, the metal in the metal chelate compound contained in the pharmaceutical composition is manganese (Mn)²⁺Or Mn³⁺) Or copper (Cu)⁺Or Cu²⁺)。

In another embodiment of the invention, the first compound in the pharmaceutical composition is N, N '-dipyridoxyl ethylenediamine-N, N' -diacetic acid and the second compound is a metal chelate comprising N, N '-dipyridoxyl ethylenediamine-N, N' -diacetic acid. The metal in the metal chelate is preferably manganese or copper.

In a preferred embodiment of the invention, the first compound in the pharmaceutical composition is N, N '-bis- (pyridoxal-5-phosphate) -ethylenediamine-N, N' -diacetic acid and the second compound is a metal chelate comprising N, N '-bipyridoylethylenediamine-N, N' -diacetic acid. The metal in the metal chelate is preferably manganese or copper.

In another embodiment of the present invention, said second compound in the pharmaceutical composition of the present invention may preferably represent 1/100 to 99/100 of said first compound on a molar concentration basis.

In another embodiment of the present invention, there is provided a pharmaceutical composition of the present invention for use in the treatment of cancer.

A fourth aspect of the invention provides a kit comprising a formulation of a first active ingredient which is a compound of formula I as defined above and a formulation of a second active ingredient which is a metal chelate comprising a compound of formula I as defined above, optionally together with instructions for simultaneous, sequential or separate administration of the formulations to a patient in need thereof.

A fifth aspect of the invention provides the use of a pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of cancer. The cancer may be any type of cancer, such as leukemia, breast cancer, colon cancer, liver cancer, and metastatic forms thereof.

In another embodiment of the present invention, there is provided the use of a pharmaceutical composition of the present invention, wherein the medicament further comprises a pharmaceutically acceptable carrier or excipient.

A sixth aspect of the invention provides a method of treating cancer in a patient in need of such treatment comprising the step of administering to said patient a cancer inhibiting amount of a pharmaceutical composition of the invention, optionally together with pharmaceutically acceptable carriers and excipients.

In another embodiment of the present invention, a method is provided wherein the pharmaceutical composition is administered with one or more other anti-cancer drugs. The anticancer drug may be any anticancer drug, such as doxorubicin, epirubicin, oxaliplatin, cisplatin, carboplatin, paclitaxel, 5-fluorouracil, cyclophosphamide, gemcitabine, irinotecan, and methotrexate.

In another embodiment of the present invention, there is provided a method wherein the pharmaceutical composition as described above and the one or more other anti-cancer drugs are administered to the patient simultaneously, separately or sequentially.

In another embodiment of the present invention, there is provided a method of treatment as described above, wherein said treatment is performed in combination with radiation therapy.

The invention is also to be understood as including the use of a pharmaceutical composition of the invention for the manufacture of a medicament for the treatment of cancer.

The compounds of formula I as described above for use in the present invention are to be understood as therapeutically active and physiologically preferred compounds.

The terms "alkyl" and "alkylene" as used herein include straight and branched chain, saturated and unsaturated hydrocarbons. The term "1, 2-cycloalkylene" includes cis-and trans-cycloalkylene and alkyl-substituted cycloalkylene having 5 to 8 carbon atoms. The term "1, 2-arylene" includes phenyl and naphthyl as well as alkyl-substituted derivatives thereof having 6 to 10 carbon atoms.

Unless otherwise indicated, any alkyl, alkylene or alkenyl moiety may conveniently contain from 1 to 20, preferably from 1 to 8, more preferably from 1 to 6 and particularly preferably from 1 to 4 carbon atoms.

Cycloalkyl, aryl and aralkyl moieties may conveniently contain 3 to 18, preferably 5 to 12 and particularly preferably 5 to 8 ring atoms. Aryl moieties comprising phenyl or naphthyl groups are preferred. For arylalkyl, phenyl C is preferred_1-8Alkyl, in particular benzyl.

When a group may be optionally substituted with hydroxy, it may be mono-or polysubstituted, in which case the alkoxy and/or hydroxy substituent may carry an alkoxy substituent.

In the formula I, R⁵Preferably hydroxy, C_1-8Alkoxy, glycol, glycerol, amino or C_1-8An alkylamide group. Preferably each radical R¹represents-CH₂COR⁵Wherein R is⁵Is a hydroxyl group.

In the compounds of formula I, Z is preferably a bond or selected from CH₂、(CH₂)₂、CO、CH₂CO、CH₂CH₂CO or CH₂COCH₂A group of (1). Preferably, Y represents a bond.

The compounds of formula I may have the same or different R on the two pyridine rings²Groups, and these groups may be attached at the same or different ring positions. However, substitution at the 5-and 6-positions is particularly preferred, with substitution at the 6-position, i.e., para to the hydroxyl group, being most preferred. R²Compounds with identical radicals and identical positions, for example 6, 6', are particularly preferred.

R⁶The radical is preferably a mono-OR poly (hydroxy OR alkoxylated) alkyl radical OR a radical of the formula OP (O) (OR)⁸)R⁷A group of (1).

R⁷Preferably a hydroxyl group or an unsubstituted alkyl or aminoalkyl group.

Particularly preferred R²Comprising a CHR⁷OCO(CH₂)_xPh and CHR⁷OCO(CH₂CO)_xPh (wherein x is 1-3), CHR⁷OCOBu^t、CH₂N(H)R⁶’、CH₂N(H)R⁶’、N(H)R⁶’、N(R⁶’)₂、CH₂OH、CH₂OR⁶’、COOR⁶’、CON(H)R⁶’、CON(R⁶’)₂OR OR⁶' (wherein R is⁶' is mono-or polyhydroxylated, preferably C_1-4Alkyl, particularly preferably C_1-3Alkyl), (CH), (c h)₂)_nCOOR⁷' (wherein n is 1 to 6), COOR⁷' (wherein R is⁷Is' a C_1-4Alkyl, preferably C_1-3Alkyl, particularly preferably methyl), CH₂OSO₃ ^-M、CH₂CH₂COOH、CH₂OP(O)(OH)(CH₂)₃NH₂、CH₂OP(O)(OH)CH₃Or CH₂OP(O)(OH)₂A group. More preferably, R²Is represented by formula CH₂OP(O)(OH)₂A group of (1).

Particular preference is given to R³Is alkylene and R²A compound of formula I which is any of the groups listed above.

The pharmaceutical compositions of the invention and the formulations contained in the kits of the invention may be formulated with conventional pharmaceutical or veterinary formulation aids, such as stabilizers, antioxidants, tonicity modifiers, buffers, pH modifiers and the like, and may be in a form suitable for parenteral or enteral administration, such as injection or infusion. Thus, the pharmaceutical compositions of the present invention are in a form for conventional administration, such as tablets, capsules, powders, solutions, suspensions, dispersions, syrups, suppositories, and the like.

Thus, the compounds of formula I and metal chelates comprising compounds of formula I can be formulated for administration using physiologically acceptable carriers and/or excipients in a manner well known to those skilled in the art. For example, the compound of formula I and the metal chelate comprising the compound of formula I can be suspended or dissolved in an aqueous medium, optionally with the addition of a pharmaceutically acceptable excipient.

The medicaments and pharmaceutical compositions of the invention can be administered by different routes, for example orally, transdermally, rectally, intrathecally, topically or by inhalation or injection, especially subcutaneous, intramuscular, intraperitoneal or intravascular injection. Other routes of administration may also be included if the efficacy, bioavailability or tolerability of the product is improved. The most suitable route can be selected by those skilled in the art depending on the formulation used.

The amount of cancer inhibition of the drug administered to the patient will depend on several different factors, such as the type of cancer, the age and weight of the patient, etc., and the attending physician will perform the treatment according to laboratory tests to adjust the dosage, if necessary.

Typically, the dose of the active compounds (i.e. the first and second compounds) in the pharmaceutical composition of the invention will comprise from 0.01 μmol to 100 μmol of compound per kilogram of body weight of the patient.

Thus, the pharmaceutical compositions of the present invention may comprise compounds of formula I, in particular DPDP or its dephosphorylated compounds, DPMP and PLED, representing methods for treating various cancer diseases, either alone or in combination with other cytostatic drugs or radiation therapy.

If the labile hydrogen atoms of the chelating agent of the present invention are not all replaced by complexed metal ions,the biological tolerance and/or solubility of the chelating agent can be enhanced by replacing the remaining labile hydrogen atoms with physiologically biocompatible cations of inorganic and/or organic bases or amino acids. Examples of suitable inorganic cations include Li⁺、K⁺、Na⁺And especially Ca²⁺. Suitable organic cations include ammonium, substituted ammonium, ethanolamine, diethanolamine, morpholine, glucosamine, N, -dimethylglucamine, lysine, arginine, or ornithine.

Where the first or second compound of the invention is generally charged, it may conveniently be used in the form of a salt with a physiologically acceptable counter ion, for example an ammonium ion, a substituted ammonium ion, an alkali metal or alkaline earth metal (e.g. calcium) cation or a cation from an inorganic or organic acid. In this respect, meglumine salt is particularly preferred.

The therapeutic agents of the present invention may be formulated with conventional pharmaceutical or veterinary formulation aids such as stabilizers, antioxidants, tonicity adjusting agents, sweeteners, and the like.

As mentioned previously, the present invention provides a compound of formula I as defined above for use in the treatment of cancer. When the present inventors compared MnDPDP and DPDP, they unexpectedly found that DPDP was more effective in killing cancer cells than MnDPDP, and they concluded that the previously described ability of MnDPDP to kill cancer cells was an intrinsic property of DPDP. Thus, the present invention provides novel compounds for the treatment of cancer while avoiding toxicity problems due to manganese release.

The compounds described above may also be used in combination with a second compound having cytoprotective properties. In one embodiment of the present invention, the use of metal chelates comprising compounds of formula I as the compound having cytoprotective ability is described. It was surprisingly found that the metal chelate is much more stable than MnDPDP and thus avoids the problem of metal release. Thus, suitable pharmaceutical combinations for the treatment of cancer are provided.

According to the prior art, the stability of MnDPDP after administration to humans depends mainly on DPDP and Mn²⁺And other competing metals (mainly non-redox active Zn)²⁺Having a specific Mn of²⁺Higher DPDP affinity) between the two (rockage et al, Inorg Chem 1989; 28: 477-485 and Toft et al, Acta radio 1997; 38: 677-689). After intravenous injection into humans, except for Mn²⁺And both phosphates are hydrolyzed from DPDP to give PLED. Shortly after intravenous injection, approximately 30% of the injected MnDPDP is converted to MnPLED, and according to the prior art (Toft et al, 1997), Mn²⁺Will also dissociate from PLED, substantially more readily than from DPDP. The said expression of MnPLED is highly supported by the stability constants reported in the literature (Rocklage et al, 1989).

However, a different explanation of the previously published results may in fact indicate that MnPLED is much more stable (in terms of metal stability) than MnDPDP under in vivo conditions. If the human plasma concentration data studied by Toft et al in 1997 were recalculated, it would be seen that MnDPDP and its five metabolites disappeared from plasma between 30 and 60 minutes, approximately simultaneously MnPLED disappeared (after the initial distribution phase). All these compounds are eliminated from the body by renal excretion and if manganese is dissociated from MnPLED, it is expected that these two processes will be separated during that time. This finding may indicate that MnPLED is stable under in vivo conditions.

Taking into account the reported Mn²⁺And Fe³⁺When Mn occurs²⁺The results of example 3 quite clearly support the opposite observation further that MnPLED is much more stable than MnDPDP. From pharmacokinetic data it is further expected that target cells and tissues will not be exposed to MnPLED concentrations above 5 μ M, i.e. concentrations where MnPLED is expected to be stable.

The present invention shows that MnPLED is much more stable than MnDPDP and most importantly, by replacing its parent substance, MnDPDP, with MnPLED, the serious manganese poisoning problem during frequent therapeutic use in humans can be solved.

It should be further emphasized that pretreatment with MnPLED in mice has been shown to be about 100 times more effective than MnDPDP (EP0910360 and US 6147094). This suggests that MnPLED doses can be considerably reduced compared to MnDPDP, which can further reduce the toxic potential of the pharmaceutical composition and thus further improve the therapeutic index. Furthermore, lower doses of MnPLED (3. mu. mol/kg) compared to the dose administered for enhanced diagnostic imaging of MnPDPD (5-10. mu. mol/kg) have been shown to reduce infarct size in pigs (Karlsson et al, actaRadiol 2001; 42: 540-.

Interestingly, MnDPDP did not reduce the infarct size in pigs. This is presumably because substitution of manganese for zinc is more rapid in pigs than in humans. Ten minutes after MnDPDP injection all manganese has been replaced by zinc (Kalsson et al, 2001), which is different from about 30% of the injected manganese in the human body (and some other species studied) binding to the chelator for a considerable time. As previously mentioned, protection of normal cells (in this case cardiomyocytes) is dependent on redox active manganese. According to the prior art (Rockcage et al, 1989), Mn²⁺The stability constant with DPDP is 15.10(logK), and Zn²⁺The stability constant with DPDP is 18.95, Mn²⁺Dissociation ratio Zn from DPDP²⁺About 1000 times easier. Mn²⁺With PLED and Zn²⁺The corresponding stability constants with PLED are 12.56 and 16.68, respectively, Mn²⁺Dissociation repetition ratio of Zn²⁺About 1000 times easier. With this result and the published metabolic protocol (Toft et al, 1997), a large difference in the stability of manganese to zinc between MnDPDP and MnPLED after administration to pigs would not be expected. Thus, the above-described reduction in infarct after administration of MnPLED, but not MnDPDP, is an unreasonable consequence. However, as illustrated in embodiment 3 of the present invention, the present invention gives a proper explanation to the unreasonable that MnPLED is more preferable than MnDPDPStable complexes and most importantly it solves the problem of manganese instability toxicity.

The advantage of combining the anticancer activity of DPDP with the cytoprotective ability of MnPLED for normal cells and tissues can be exemplified by the problem of anthracycline-induced cardiotoxicity with dexrazoxane as a cardioprotective agent. Because of its potential (although far less pronounced) to reduce the anticancer effects of anthracyclines, dexrazoxane is not recommended for use at the start of anthracycline therapy in patients with metastatic breast cancer (Yeh et al, Circulation 2004; 109: 3122-3132). However, as the present inventors and others have demonstrated for MnDPDP, preclinical data relatively clearly show that this is not a problem when our method is employed. One possible explanation for this phenomenon is the two distinct and intrinsic activities of MnDPDP, namely its anticancer activity and its cytoprotective activity, and in our invention it has been further separated into two distinct chemical entities, namely DPDP with anticancer activity, and MnPLED with cytoprotective ability for normal cells and tissues.

Drawings

Figure 1. MTT assay on human colon cancer SW480 cells in the absence (control) and presence of MnDPDP, DPDP, oxaliplatin or DPDP + oxaliplatin (mean ± SD; n ═ 3).

Figure 2 MTT assay of mouse lymphoma J774 cells in the absence (control) and presence of MnDPDP, DPDP, oxaliplatin or DPDP + oxaliplatin (mean ± SD; n ═ 3).

FIG. 3 cytostatic effect achieved by increasing oxaliplatin concentration in SW620, HCT-8 and hTERT-RPE1 cells (A). Cell growth inhibitory effects of oxaliplatin alone or in combination with MnDPDP or DPDP (mean. + -. SD; n ═ 3) at low concentrations in SW620(B), HCT-8(C) and hTERT-RPE1(D) cells.

Figure 4. Fenton assay in the presence of different concentrations of DPDP, MnDPDP and MnPLED (mean ± SD; n ═ 3).

(A) Control determinations (mean ± SD; n ═ 3) were performed in parallel and at the end of the experiment.

(B) The controls also included the absence of iron (-Fe), the presence of the iron chelator desferoxamine (15 μ M DFO) or the hydroxyl radical scavenger DMSO (10% DMSO) (mean ± SD; n ═ 3).

Examples

The invention will now be further demonstrated and described by the following non-limiting examples. The examples are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

Example 1

The cell growth inhibitory activity of DPDP and MnDPDP was compared by co-incubation of human colon cancer cells (SW480) with mouse lymphoma cells (J774) with MnDPDP, DPDP and/or oxaliplatin.

Method of producing a composite material

Cell viability was measured using the MTT method. Briefly, 20,000 SW480 or J774 cells were seeded in each well of a 96-well plate and cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum, 2mML glutamine, 100Ul/ml penicillin, and 100 μ g/ml streptomycin at 37 ℃ in 5% CO₂Overnight in a humidified atmosphere. Cells were then exposed to 50 μmmnpdp, 50 μ M DPDP or 10 μ M oxaliplatin for 24 hours at 37 ℃. The effect of the combination of 10 μ M oxaliplatin with 50 μ M DPDP on viability was also tested. Cell viability was then assessed by adding 5mg/ml thiazolyl blue (MTT) to a final concentration of 0.5mg/ml and incubating the cells for a further 4 hours at 37 ℃. The blue color formed by the mitochondrial dehydrogenase of living cells was then caused by the addition of 10% SDS and 10mM HCl to reach a final concentration of 5% SDS and 5mM HClFormazan is dissolved overnight at 37 ℃. Finally, the absorbance of the solution at 570nm was read at a reference wavelength of 670nm on a microplate reader Spectramax 340(Molecular Devices, Sunnyvale, Calif., USA) connected to the Apple Macintosh computer running the program Softmax Pro V1.2.0(Molecular Devices, Sunnyvale, Calif., USA).

Results

In human rectal SW480 cells, the cell growth inhibitory activity of 50 μ M DPDP was statistically significantly more potent than that of 50 μmmnpdp (unpaired t-test) (fig. 1). Although statistically not significant (p < 0.07), there is a trend that DPDP in combination with oxaliplatin will be more effective than oxaliplatin alone on SW480 cells. Figure 2 shows the corresponding cell growth inhibitory activity of 50 μ M MnDPDP and 50 μ M DPDP in mouse lymphoma J774 cells, from which it is evident that DPDP is significantly more effective in killing lymphoma cells than MnDPDP. Moreover, DPDP in combination with oxaliplatin was significantly more effective than oxaliplatin alone.

Conclusion

When comparing MnDPDP and DPDP, it was unexpectedly found that DPDP is more effective than MnDPDP in killing cancer cells, it can be concluded that: the aforementioned cancer cell killing ability of MnDPDP is an inherent property of DPDP.

Example 2

The cell growth inhibitory activity of MnDPDP or DPDP in human adenocarcinoma cells (SW620), human ileocecal colorectal carcinoma cells and normal retinal epithelial telomerase immortalized cells (hTERT-RPE1) was tested in the absence and presence of oxaliplatin.

Method of producing a composite material

HCT-8 cells were cultured in RPMI 1640 medium containing sodium pyruvate (1mM) and 10% horse serum. SW620 was cultured in a Leibovitz's L-15 medium with ATCC containing 10% fetal bovine serum. The cells were kept in a humidified atmosphere containing 5% carbon dioxide at 37 ℃. Cells need to be harvested at log phase for the experiments. Microculture cytotoxic fluorescence assay (FMCA) was used to study the cell growth inhibitory activity of oxaliplatin, MnDPDP or DPDP and combinations thereof. FMCA is measured based on the fluorescence produced by cells with intact plasma membrane hydrolyzing fluorescein diacetate (FAD) to fluorescein. Briefly, 96-well titer plates were freshly prepared in triplicate with drug solutions at 10-fold the desired drug concentration. The cell suspension was seeded at 20,000 cells per well on a plate for drug preparation, and then the template was incubated for 72 hours. After incubation, the plates were washed, the FDA added, and the fluorescence generated (excitation wavelength 480nm) was measured in a fluorimeter (fluorostar optima, BMG Technologies) at 538nm after 50 minutes of incubation, proportional to the number of cells with intact plasma membranes present in the wells.

Results

The cell growth inhibitory activity of oxaliplatin at elevated concentrations in cancer cells (SW620 and HCT-8) and normal cells (hTERT-RPE1) is shown in FIG. 3A. Oxaliplatin demonstrated a concentration-dependent inhibition of cell growth in both cancer cell lines, but only a slight effect was present at its highest concentration in normal cells. Neither MnDPDP nor DPDP showed any cell growth inhibitory effect in any of these cells (not shown). However, 100 μ M DPDP, but not MnDPDP significantly (unpaired t-test) enhanced the cell growth inhibitory effect of low concentrations (8 μ M) of oxaliplatin, not only in both cancer cell lines, but also in normal cells (fig. 3B-D).

Conclusion

When comparing MnDPDP and DPDP, it was unexpectedly found that DPDP is much more effective than MnDPDP in enhancing the cancer cell killing ability of oxaliplatin, it can be concluded that: the cancer cell killing ability of MnDPDP is an inherent property of DPDP.

Example 3

The stability of MnDPDP and MnPLED was compared by allowing the chelator DPDP, the metal complexes MnDPDP and MnPLED to compete with iron in the Fenton assay.

Method of producing a composite material

Ferric iron (10. mu.M) was partially reduced to ferrous iron (100. mu.M) in 150mM acetate buffer. Addition of H₂O₂(100. mu.M) causes the generation of hydroxyl radicals (HO.). The latter will be H₂DCF (non-fluorescent 2 ', 7' -dichlorodihydrofluorescein; 5. mu.M) was oxidized to fluorescent DCF (2 ', 7' -dichlorofluorescein). By hydrolysis of H₂Acetate of DCF (H)₂DCF-DA) to obtain H₂DCF. DMSO (10%) and DFO (10 μ M) were used to confirm the formation of HO · and the involvement of iron, respectively. The iron chelating capacity of DPDP, MnDPDP and MnPLED at different concentrations was determined. Fluorescence was detected at an excitation wavelength of 485nm and an emission wavelength of 530nm in a FL600 fluorescence microplate reader (Bio-Tex, Winooski, VT, u.s.a.).

Results

Figure 4 demonstrates that DPDP inhibits the Fenton response in a dose-dependent manner; inhibition started at 0.1. mu.M and completed at 10. mu.M. The inhibition pattern is consistent with the reported high affinity of DPDP for ferrous iron (logK. 33.52). However, in the case of MnPLED, there was no significant inhibition at concentrations up to and including 5 μ M, but the inhibition was complete when the concentration reached 10 μ M. The iron chelating capacity of MnDPDP is significantly higher than that of MnPLED.

Conclusion

The present results are contrary to and highly unexpected from the previously published expectations for both iron and manganese chelating capabilities of MnDPDP and MnPLED. Reported Fe³⁺And stability constant between DPDP and Fe³⁺And PLED are 33.52 and 36.88(logK), respectively, and the reported Mn²⁺And stability constant between DPDP and Mn²⁺And PLED are 15.10 and 12.56, respectively (Rocklage et al, 1989). MnPLED can therefore be expected to be a much better inhibitor of the Fenton reaction than MnDPDP.

Claims (22)

1. A compound of formula I or a salt thereof for use as a carcinostatic agent:

formula I

Wherein

X represents a group of a CH or an N,

each R¹Independently represents hydrogen or-CH₂COR⁵；

R⁵Representing hydroxy, optionally hydroxylated alkanesOxy, amino or alkylamido;

each R²Independently represent a group ZYR⁶(ii) a Z represents a bond or optionally a group R⁷Substituted C_1-3Alkylene or oxyalkylene;

y represents a bond, an oxygen atom or a group NR⁶；

R⁶Is a hydrogen atom, a group COOR⁸Alkyl, alkenyl, cycloalkyl, phenyl, naphthyl or phenyl C_1-8Alkyl optionally substituted by a group selected from COOR⁸、CONR⁸ ₂、NR⁸ ₂、OR⁸、＝NR⁸、＝O、OP(O)(OR⁸)R⁷And OSO₃One or more groups of M;

R⁷is hydroxy, optionally hydroxylated, optionally alkoxylated alkyl or aminoalkyl;

R⁸is a hydrogen atom or an optionally hydroxylated, optionally alkoxylated alkyl group;

m is a hydrogen atom or the equivalent of a physiologically tolerable cation;

R³is represented by C_1-8Alkylene, 1, 2-cycloalkylene or 1, 2-arylene optionally substituted by R⁷Substitution; each R⁴Independently represent hydrogen or C_1-3An alkyl group.

2. A compound according to claim 1, wherein

R⁵Is hydroxy, C_1-8Alkoxy, glycol, glycerol, amino or C_1-8An alkylamide group;

z is a bond or selected from CH₂、(CH₂)₂、CO、CH₂CO、CH₂CH₂CO and CH₂COCH₂A group of (a);

y is a bond;

R⁶is mono-OR poly (hydroxy OR alkoxylated) alkyl OR of the formula OP (O) (OR)⁸)R⁷A group of (a); and

R⁷is hydroxyl or unsubstituted alkyl or aminoalkyl.

3. A compound according to claim 1 or 2, wherein R³Is ethylene and each radical R¹represents-CH₂COR⁵Wherein R is⁵Is a hydroxyl group.

4. The compound according to any one of claims 1 to 3, wherein the compound is N, N '-dipyridoxyl ethylenediamine-N, N' -diacetic acid.

5. The compound according to any one of claims 1 to 3, wherein the compound is N, N '-bis- (pyridoxal-5-phosphate) -ethylenediamine-N, N' -diacetic acid.

6. Use of a compound of formula I as defined in any one of claims 1 to 5 in the manufacture of a medicament for the treatment of cancer.

7. A pharmaceutical composition comprising a first compound of formula I as defined in any one of claims 1 to 5, and a second compound having cytoprotective properties.

8. The pharmaceutical composition according to claim 7, wherein the second compound is a metal chelate comprising a compound of formula I as defined in any one of claims 1 to 5.

9. The pharmaceutical composition according to any one of claims 7-8, wherein the metal chelate has 10⁸To 10²⁴K of the range_aThe value is obtained.

10. Pharmaceutical composition according to any of claims 7 to 9, wherein the metal chelate has a Ka value ratio of Fe comprising a compound of formula I as defined in any of claims 1 to 5³⁺The Ka value of the chelate is at least 10 lower³And (4) doubling.

11. The pharmaceutical composition according to any one of claims 8-10, wherein the metal is Mn²⁺Or Mn³⁺Or Cu⁺Or Cu²⁺。

12. The pharmaceutical composition according to any one of claims 7 to 11, wherein the first compound is N, N '-dipyridoxyl ethylenediamine-N, N' -diacetic acid and the second compound is a metal chelate comprising N, N '-dipyridoxyl ethylenediamine-N, N' -diacetic acid.

13. The pharmaceutical composition according to any one of claims 7 to 11, wherein the first compound is N, N '-bis- (pyridoxal-5-phosphate) -ethylenediamine-N, N' -diacetic acid and the second compound is a metal chelate comprising N, N '-dipyridoxyl ethylenediamine-N, N' -diacetic acid.

14. The pharmaceutical composition according to any one of claims 7-13, wherein the second compound comprises 1/100 to 99/100 of the first compound on a molar basis.

15. A pharmaceutical composition according to any one of claims 7 to 14 for use in the treatment of cancer.

16. A kit comprising a formulation of a first active ingredient which is a compound as defined in any one of claims 1 to 5 and a formulation of a second active ingredient which is a metal chelate comprising a compound as defined in any one of claims 1 to 5, optionally together with instructions for the simultaneous, sequential or separate administration of the formulations to a patient in need thereof.

17. Use of a pharmaceutical composition according to any one of claims 7 to 15 in the manufacture of a medicament for the treatment of cancer.

18. The use of a pharmaceutical composition according to claim 17, wherein the medicament further comprises a pharmaceutically acceptable excipient.

19. The use of a pharmaceutical composition according to claim 17, wherein the medicament is for the treatment of cancer together with one or more other anti-cancer drugs.

20. The use of a pharmaceutical composition according to claim 17, wherein the medicament is for simultaneous, separate or sequential administration to a patient with the one or more other anti-cancer drugs.

21. Use of a pharmaceutical composition according to claim 17, wherein the medicament is for use in combination with radiation therapy.

22. The use of a pharmaceutical composition according to claim 17, wherein the medicament further comprises a pharmaceutically acceptable carrier.