Patent Application
20110092521
The invention relates to treating a disease or condition in which it is desirable to inhibit 5′-methylthioadenosine phosphorylase (MTAP) and/or 5′-methylthioadenosine nucleosidase (MTAN). The invention is further concerned with treating a disease or condition in which it is desirable to inhibit MTAP/MTAN by administering to a patient 5′-methylthioadenosine (MTA), or a prodrug of MTA, and one or more MTAP/MTAN inhibitors. The invention also relates to compositions containing MTA and one or more inhibitors of MTAP and/or MTAN. In particular, the invention relates to methods of treating prostate cancer or head and neck cancer by administering to a patient 5′-methylthioadenosine (MTA), or a prodrug of MTA, and one or more MTAP/MTAN inhibitors.
Certain nucleoside analogues have been identified as potent inhibitors of 5′-methylthioadenosine phosphorylase (MTAP) and 5′-methylthioadenosine nucleosidase (MTAN). These are the subject of U.S. Pat. No. 7,098,334.
Compounds where the location of the nitrogen atom in the sugar ring is varied or where two nitrogen atoms form part of the sugar ring, have also been identified as inhibitors of MTAP and MTAN. These compounds are described in U.S. Ser. No. 10/524,995.
MTAP and MTAN function in the polyamine biosynthesis pathway, in purine salvage in mammals, and in the quorum sensing pathways in bacteria. MTAP catalyses the reversible phosphorolysis of methylthioadenosine (MTA) to adenine and 5-methylthio-α-D-ribose-1-phosphate (MTR-1P). MTAN catalyses the reversible hydrolysis of MTA to adenine and 5-methylthio-α-D-ribose and of S-adenosyl-L-homocysteine (SAH) to adenine and S-ribosyl-homocysteine (SRH). The adenine formed is subsequently recycled and converted into nucleotides. Essentially, the only source of free adenine in the human cell is a result of the action of these enzymes. The MTR-1P is subsequently converted into methionine by successive enzymatic actions.
MTA is a by-product of the reaction involving the transfer of an aminopropyl group from decarboxylated S-adenosylmethionine to putrescine during the formation of spermidine. The reaction is catalyzed by spermidine synthase. Likewise, spermine synthase catalyses the conversion of spermidine to spermine, with concomitant production of MTA as a by-product. The spermidine synthase is very sensitive to product inhibition by accumulation of MTA. Therefore, inhibition of MTAP or MTAN severely limits the polyamine biosynthesis and the salvage pathway for adenine in the cells.
Although MTAP is abundantly expressed in normal cells and tissues, MTAP deficiency due to a genetic deletion has been reported with many malignancies. The loss of MTAP enzyme function in these cells is known to be due to homozygous deletions on chromosome 9 of the closely linked MTAP and p16/MTS1 tumour suppressor gene. As absence of p16/MTS1 is probably responsible for the tumour, the lack of MTAP activity is a consequence of the genetic deletion and is not causative for the cancer. However, the absence of MTAP alters the purine metabolism in these cells so that they are mainly dependent on the de novo pathway for their supply of purines.
MTA has been shown to induce apoptosis in dividing cancer cells, but to have the opposite, anti-apoptotic effect on dividing normal cells such as hepatocytes (E. Ansorena et al., Hepatology, 2002, 35: 274-280).
MTAP inhibitors may therefore be used in the treatment of cancer. Such treatments are described in U.S. Pat. No. 7,098,334 and U.S. Ser. No. 10/524,995.
The need for new cancer therapies remains ongoing. For some prevalent cancers the treatment options are still limited. Prostate cancer, for example, is the most commonly diagnosed non-skin cancer in the United States. Current treatment options include radical prostatectomy, radiation therapy, hormonal therapy, and watchful waiting. Although the therapies may offer successful treatment of an individual's condition, the pitfalls are quite unfavorable and lead to a decrease in a man's overall quality of life. Surgery may inevitably result in impotence, sterility, and urinary incontinence. Side effects associated with radiation therapy include damage to the bladder and rectum as well as slow-onset impotence. Hormonal therapy will not cure the cancer and eventually most cancers develop a resistant to this type of therapy. The major risk associated with watchful waiting is that it may result in tumour growth, cancer progression and metastasis. It is therefore desirable that alternative treatment options are made available to patients diagnosed with prostate cancer.
MTAP and MTAN inhibitors may also be used in the treatment of diseases such as bacterial infections or protozoal parasitic infections, where it is desirable to inhibit MTAP/MTAN. Such treatments are described in U.S. Pat. No. 7,098,334 and U.S. Ser. No. 10/524,995. However, the search continues for more effective treatments using these inhibitors.
It has now been found that the treatment of diseases in which it is desirable to inhibit MTAP/MTAN may be enhanced by administering exogenous MTA and/or a prodrug of MTA together with an MTAP/MTAN inhibitor. Thus, the combination of MTA and/or a prodrug of MTA and the MTAP/MTAN inhibitors employed according to the present invention provides a potentially effective treatment against diseases or disorders such as cancer and bacterial infections.
It is therefore an object of the invention to provide an improved method of treating a disease or condition in which it is desirable to inhibit MTAP or MTAN, or at least to provide a useful choice.
In a first aspect, the invention provides a method of treating a disease or condition in which it is desirable to inhibit MTAP or MTAN comprising administering to a patient in need thereof MTA, or a prodrug of MTA, and one or more MTAP inhibitor(s) or one or more MTAN inhibitor(s).
Preferably the inhibitor of MTAP or MTAN is a compound a compound of the formula (I):
wherein:
Preferably the compound of formula (I) excludes (3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(methylthiomethyl)pyrrolidine.
In preferred embodiments of the invention Z is SQ. In some embodiments Z is not methylthio.
Preferably Q is an alkyl group, optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, and carboxy. It is further preferred that the alkyl group is a C1-C8 alkyl group, most preferably a methyl group.
It is also preferred that Q is an aryl group, optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, and carboxy. More preferably the aryl group is a phenyl or benzyl group.
Preferably G is CH2. It is also preferred that V is CH2 and W is NR1. It is further preferred that B is NH2. It is also preferred that D is H, and it is preferred that A is CH.
Preferably any halogen is chlorine or fluorine.
In preferred embodiments of the invention the compound of formula (I) is a compound of the formula (IV):
where J is aryl, aralkyl or alkyl, each of which is optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, and carboxy.
Preferably J is C1-C7 alkyl. More preferably J is methyl, ethyl, n-propyl, i-propyl, n-butyl, cyclobutyl, cyclopentyl, cydohexyl, cyclohexylmethyl, or cycloheptyl.
It is also preferred that J is phenyl, optionally substituted with one or more halogen substituents. More preferably J is phenyl, p-chlorophenyl, p-fluorophenyl, or m-chlorophenyl.
It is also preferred that J is heteroaryl, 4-pyridyl, aralkyl, benzylthio, or —CH2CH2(NH2)COOH.
In other preferred embodiments of the invention the compound of the formula (I) is a compound of the formula (V):
where T is aryl, aralkyl or alkyl, each of which is optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, carboxy, and straight- or branched-chain C1-C8 alkyl.
Preferably T is C1-C6 alkyl, optionally substituted with one or more substituents selected from halogen and hydroxy. More preferably T is methyl, ethyl, 2-fluoroethyl, or 2-hydroxyethyl. Most preferably T is methyl.
It is also preferred that T is aryl, optionally substituted with one or more substituents selected from halogen and straight-chain C1-C6 alkyl. More preferably T is phenyl, naphthyl, p-tolyl, m-tolyl, p-chlorophenyl, m-chlorophenyl, or p-fluorophenyl.
It is also preferred that T is aralkyl. More preferably T is benzyl.
Preferably the inhibitor of MTAP or MTAN is:
The inhibitor of MTAP or MTAN may be administered simultaneously with the MTA or prodrug of MTA. Alternatively, the inhibitor of MTAP or MTAN may be administered prior to administration of the MTA or prodrug of MTA or after administration of the MTA or prodrug of MTA.
In another aspect, the invention provides a composition comprising synergistically effective amounts of i) one or more MTAP inhibitors or one or more MTAN inhibitors; and ii) MTA, or a prodrug of MTA.
Preferably the MTAP inhibitor or the MTAN inhibitor is a compound of the formula (I) as defined above.
Most preferably the MTAP inhibitor or the MTAN inhibitor is:
Definitions
The term “alkyl” is intended to include straight- and branched-chain alkyl groups, as well as cycloalkyl groups. The same terminology applies to the non-aromatic moiety of an aralkyl radical. Examples of alkyl groups include: methyl group, ethyl group, n-propyl group, iso-propyl group, n-butyl group, iso-butyl group, sec-butyl group, t-butyl group, n-pentyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, 2-ethylpropyl group, n-hexyl group and 1-methyl-2-ethylpropyl group.
The term “aryl” means an aromatic radical having 6 to 18 carbon atoms and includes heteroaromatic radicals. Examples include monocyclic groups, as well as fused groups such as bicyclic groups and tricyclic groups. Some examples include phenyl group, indenyl group, 1-naphthyl group, 2-naphthyl group, azulenyl group, heptalenyl group, biphenyl group, indacenyl group, acenaphthyl group, fluorenyl group, phenalenyl group, phenanthrenyl group, anthracenyl group, cyclopentacyclooctenyl group, and benzocyclooctenyl group, pyridyl group, pyrrolyl group, pyridazinyl group, pyrimidinyl group, pyrazinyl group, triazolyl group, tetrazolyl group, benzotriazolyl group, pyrazolyl group, imidazolyl group, benzimidazolyl group, indolyl group, isoindolyl group, indolizinyl group, purinyl group, indazolyl group, furyl group, pyranyl group, benzofuryl group, isobenzofuryl group, thienyl group, thiazolyl group, isothiazolyl group, benzothiazolyl group, oxazolyl group, and isoxazolyl group.
The term “halogen” includes fluorine, chlorine, bromine and iodine.
The compounds are useful for the treatment of certain diseases and disorders in humans and other animals. Thus, the term “patient” as used herein includes both human and other animal patients.
The term “prodrug” as used herein means a pharmacologically acceptable derivative of the compound of formula (I), (IV) or (V), such that an in vivo biotransformation of the derivative gives the compound as defined in formula (I), (IV) or (V). Prodrugs of compounds of formulae (I), (IV) or (V) may be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved in vivo to give the parent compound.
Prodrugs include compounds of formulae (I), (IV) or (V), tautomers thereof and/or pharmaceutically acceptable salts thereof, which include an ester functionality, or an ether functionality. It will be clear to the skilled person that the compounds of formulae (I), (IV) or (V) may be converted to corresponding ester or ether prodrugs using known chemical transformations. Suitable prodrugs include those where the hydroxyl groups of the compounds of formula (I), (IV) or (V) are esterified to give, for example, a primary hydroxyl group ester of propanoic or butyric acid. Other suitable prodrugs are alkycarbonyoxymethyl ether derivatives on the hydroxyl groups of the compounds of formula (I), (IV) or (V) to give, for example, a primary hydroxyl group ether with a pivaloyloxymethyl or a propanoyloxymethyl group.
The term “pharmaceutically acceptable salts” is intended to apply to non-toxic salts derived from inorganic or organic acids, including, for example, the following acid salts: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, p-toluenesulfonate, salicylate, succinate, sulfate, tartrate, thiocyanate, and undecanoate.
Discussion of Cancer Treatment
The present invention relates to methods of treating diseases in which it is desirable to inhibit MTAP/MTAN by administering to a patient in need thereof one or more inhibitors of MTAP/MTAN together with MTA, or a prodrug of MTA. In particular, the invention relates to methods of treating certain cancers, such as prostate cancer or head and neck cancer by administering to a patient in need thereof one or more inhibitors of MTAP/MTAN together with MTA, or a prodrug of MTA.
Suitable MTAP/MTAN inhibitors which may be employed in the method of the present invention and the methods for preparing these inhibitors are described in U.S. Pat. No. 7,098,334 and U.S. Ser. No. 10/524,995.
Certain MTAP/MTAN inhibitor compounds are surprisingly effective for treating prostate and head and neck cancers. These are compounds of general formula (IV).
This sub-class of MTAP/MTAN inhibitors incorporates an adenine-like base moiety and a pyrrolidine moiety having an alkyl- aryl- or aralkylthiomethyl group at the 4-position.
Other MTAP/MTAN inhibitor compounds are also surprisingly effective for treating prostate and head and neck cancers. These are compounds of general formula (V).
This sub-class of MTAP/MTAN inhibitors also incorporates the adenine-like base moiety but has an iminoribitol moiety with an alkyl- aryl- or aralkylthiomethyl group at the 5′-position.
Examples of the first sub-class of inhibitors include compounds (1) and (2).
The Examples below show that compounds (1) and (2) are effective both in vitro and in vivo against a variety of cell lines (PC3, RM1, SCC25 and FaDu). These compounds are therefore particularly useful in the treatment of prostate and head and neck cancers.
The MTAP/MTAN inhibitor compounds inhibit cell growth in vitro of the prostate cancer cell lines PC3 and RM1 and the head and neck cancer cell lines SCC25 and FaDu. A surprising enhancement in the cell-killing effect is seen in vitro with combined administration of the MTAP/MTAN inhibitor compound plus MTA. Examples of this effect are shown in
Furthermore, the inhibitor compounds, when co-administered with MTA, exhibit a cytostatic effect on PC3 cells in vitro.
In order to determine whether the inhibition is selective for malignant cells, normal human fibroblast cells (GM02037) were also treated with compound (2) and MTA for 3 weeks. No cytotoxicity was observed. Compound (2) is therefore cytotoxic for human HNSCC (human head and neck squamous cell carcinoma) cells at doses that exhibit minimal toxicity for normal cells. This selectivity is a further indication that the MTAP/MTAN inhibitors described above are useful agents for the treatment of head and neck cancer.
The present in vivo studies further demonstrate the efficacy of the compounds. In a NOD-SCID mouse model, compound (2) significantly delays the growth of established FaDu xenografts. The in vivo effect is seen either with or without co-administration of the inhibitor compound with MTA.
In addition, prostate cancer progression in the TRAMP mouse model is inhibited in mice treated with compound (2), either alone or in combination with MTA.
An example of the second sub-class of inhibitors is compound (3).
This compound also inhibits prostate cancer progression in the TRAMP mouse model, when administered either alone or in combination with MTA.
For the above in vivo models, the inhibitor compounds exhibit activity when administered with exogenous MTA and when administered alone. There is not a significant enhancement observed when the inhibitors are administered together with MTA. However, the in vitro results clearly demonstrate a surprising enhancement in activity when the inhibitors are administered in conjunction with MTA. Thus, the combined administration method provides a potential alternative treatment method for patients suffering from diseases where the administration of MTAP/MTAN inhibitors is indicated.
It will be clear to the skilled person that the above-mentioned compounds, which are also inhibitors of MTAN, will be useful in treating diseases where it is desirable to inhibit MTAN. Such diseases include bacterial infections and protozoal parasitic infections. Thus, the invention further relates to a method of treating such diseases, comprising administering to a patient one or more MTAN inhibitor compounds, together with MTA.
The MTAP/MTAN inhibitor compounds of formulae (I), (IV) and (V) (in particular the compounds of formulae (IV) and (V)), when co-administered with MTA, provide an effective alternative treatment option for sufferers of diseases such as cancer, bacterial infections or protozoal parasitic infections.
General Aspects
The MTAP/MTAN inhibitor compounds are useful in both free base form and in the form of salts.
It will be appreciated that the representation of a compound of formula (I) where B and/or D is a hydroxy group, is of the enol-type tautomeric form of a corresponding amide, and this will largely exist in the amide form. The use of the enol-type tautomeric representation is simply to allow fewer structural formulae to represent the compounds of the invention.
Similarly, it will be appreciated that the representation of a compound of formula (I), where B and/or D is a thiol group, is of the thioenol-type tautomeric form of a corresponding thioamide, and this will largely exist in the thioamide form. The use of the thioenol-type tautomeric representation is simply to allow fewer structural formulae to represent the compounds of the invention.
It will also be appreciated that the compounds depicted with bold solid lines are representations of the D-ribo or 2′-deoxy-D-erythro-stereochemical arrangement of substituents on the pyrrolidine ring, such as shown here.
Formulations and Modes of Administration
For oral administration the active compounds can be formulated into solid or liquid preparations, for example tablets, capsules, powders, solutions, suspensions and dispersions. Such preparations are well known in the art as are other oral dosage regimes not listed here. In the tablet form the compounds may be tableted with conventional tablet bases such as lactose, sucrose and corn starch, together with a binder, a disintegration agent and a lubricant. The binder may be, for example, corn starch or gelatin, the disintegrating agent may be potato starch or alginic acid, and the lubricant may be magnesium stearate. For oral administration in the form of capsules, diluents such as lactose and dried cornstarch may be employed. Other components such as colourings, sweeteners or flavourings may be added.
When aqueous suspensions are required for oral use, the active ingredient may be combined with carriers such as water and ethanol, and emulsifying agents, suspending agents and/or surfactants may be used. Colourings, sweeteners or flavourings may also be added.
The compounds may also be administered by injection in a physiologically acceptable diluent such as water or saline. The diluent may comprise one or more other ingredients such as ethanol, propylene glycol, an oil, or a pharmaceutically acceptable surfactant.
The compounds may also be administered topically. Carriers for topical administration of the compounds include mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. The compounds may be present as ingredients in lotions or creams, for topical administration to skin or mucous membranes. Such creams may contain the active compounds suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
The compounds may further be administered by means of sustained release systems. For example, they may be incorporated into a slowly dissolving tablet or capsule.
Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences (Mack Publishing Company).
Inhibitor Compounds Inhibitors of MTAP/MTAN were synthesized as described earlier (Singh, V., Shi, W., Evans, G. B., Tyler, P. C., Fumeaux, R H, Almo, S C, and Schramm, V L (2004) Biochemistry 43, 9-18; Evans G B, Fumeaux R H, Lenz D H, et al., J Med Chem 2005: 48, 4679-89). Solutions were standardized by the UV absorbance of the 9-deazaadenine ring. Sterile solutions of inhibitors were prepared by filtration.
Protocol for Clonogenic Survival Assay of Cancer Cells
1. 60% confluent plates of experimental cell line was taken and subjected to trypsinization
2. Single cell suspension of the experimental cell line was made in the regular growth medium and number of cells per mililitre of suspension counted
3. A fixed low number of cells were plated out in a volume of 3ml of growth medium in each well of 6 well culture dishes and incubated overnight at 37° C. in a CO2 incubator
4. Measured volumes of the inhibitor and substrate solutions in sterile deionised cell culture water was added to each well of the 6 well plates. Typically each concentration of inhibitor and/or substrate was added in triplicate wells to calculate error bars. Final concentrations were calculated based on a total volume of 3ml of culture medium such that dilution factor did not exceed 1% of final volume.
5. Treated cell culture plates were incubated at 37° C. in a CO2 incubator for a period of 7 days
6. At the end of the period of incubation growth medium was removed from each well, attached cells were washed once with PBS and fixed by addition of 100% Formalin solution to each well and keeping at room temperature for ˜1 hour.
7. At the end of 1 hour, formalin was removed from the wells and ˜150 μL of Crystal Violet staining solution was added to each well and let stand at room temperature for 30 min.
8. After staining is complete, wells were flushed with running tap water to remove traces of residual stain and dried by inverting over paper towels.
9. Number of crystal violet stained colonies in each well containing more than 60 cells per colony was counted.
10. Assuming each colony originated from a single surviving cell post-treatment and taking the number of colonies in the untreated control well as 1, the fraction of surviving cells in each well was calculated and plotted in a graph.
PC3 cells were grown in equal (1:1) portions of Dulbecco's modified Eagle's medium and F12 containing 10% fetal bovine serum, 10 U/mL penicillin-G and 10 μg/mL streptomycin in monolayers to near confluency at 37° C. Cells were lysed in 50 mM sodium phosphate pH 7.5, 10 mM KCl and 0.5% Triton X-100.
PC3 cells were maintained in MEM Eagle's media supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/mL streptomycin, 0.1 mM non essential amino acids and 1 mM sodium pyruvate. Cell survival was evaluated using the WST-1 assay (Kicska G A, Iong Li, Horig H, et al. Proc Natl Acad Sci USA 2001; 98:4593-98). Cells were seeded onto 96 well plates at a density of 104 cells per well, with either no additions, 1 μM compound (2), 20 μM MTA or 1 μM compound (2)+20 μM MTA. IC50 was determined following the manufacturer's protocol (Roche Applied Science, IN). Cells were grown and measured in triplicate or quadruplicate and the error bars show the mean±SD of the multiple samples.
SCC25 cells were maintained in MEM Eagle's media supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/mL streptomycin, 0.1 mM non essential amino acids and 1 mM sodium pyruvate. Cell survival was evaluated using the WST-1 assay (Kicska G A, Iong Li, Horig H, et al. Proc Natl Acad Sci USA 2001; 98:4593-98). Cells were seeded onto 96 well plates at a density of 104 cells per well, with either no additions, 1 μM MT-compound (2), 20 μM MTA or 1 μM compound (2)+20 μM MTA. IC50 was determined following the manufacturer's protocol (Roche Applied Science, IN). Cells were grown and measured in triplicate or quadruplicate and the error bars show the mean±SD of the multiple samples.
FaDu cells were maintained in MEM Eagle's media supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/mL streptomycin, 0.1 mM non essential amino acids and 1 mM sodium pyruvate. Cell survival was evaluated using the WST-1 assay (Kicska G A, Iong Li, Horig H, et al. Proc Natl Acad Sci USA 2001; 98:4593-98). Cells were seeded onto 96 well plates at a density of 104 cells per well, with either no additions, 1 μM compound (2), 20 μM MTA or 1 μM compound (2)+20 μM MTA. IC50 was determined following the manufacturer's protocol (Roche Applied Science, IN). Cells were grown and measured in triplicate or quadruplicate and the error bars show the mean±SD of the multiple samples.
FaDu cells were subjected to six days in culture using the same conditions described as for Example 4.
FaDu cells were subjected to six days in culture using the same conditions described as for Example 4, before staining with propidium bromide and FACS cell sorting analysis.
Two groups of 3 C57BL6 mice received a single oral dose of compound (2) dissolved in sterile, deionized water, pippeted onto a crumb of food. Treated food was fed to each mouse individually under close observation at time zero. Two different single doses of inhibitor were administered: 50 μg and 100 μg. Mice were individually fed and closely observed for consumption of food. At specific time points, 4 μL blood samples were collected from the tail vein. The blood was mixed with 4 μL 0.6% Triton X-100 in PBS and stored at −80° C. until time of analysis. The amount of adenine produced was measured by the following MTAP activity assay: Cells were harvested, washed three times with PBS and lysed with RIPA buffer. The reaction mixture for MTAP activity assays contained the following: ˜75 μg protein from cell lysates, 50 mM HEPES pH 7.4, 50 μM MTA, and 20,000 dpm [2,8-3H]MTA. Labeled MTA was synthesized from [2,8-3H]S-adenosylmethionine by a known method. Products of the MTAP reaction were resolved using TLC silica plates with 1 M ammonium acetate, pH 7.55, and 5% isopropanol. Adenine spots were excised and counted for label incorporation.
Oral dosing was performed in essentially the same manner as for Example 7. For IP availability, 100 μg of the inhibitor was dissolved in around 200 μl of sterile deionised water and taken up in a 1 ml syringe attached to a 26 G needle and injected intraperitonially in the mouse at 0 min time point. Blood (4 μl) was collected from the tail of the mouse at specific time points, mixed with 4 μl of 0.6% TritonX-100 solution in PBS and stored at −80° C. until ready for enzyme assay. Blood (4 μl) was collected from each mouse prior to injection which served as 0 min control time point. Each experiment was repeated three times with three different mice to get standard error bars.
NOD-SCID mice (6-8 weeks old) were obtained from Jackson Laboratory (Bar Harbor, Me.). FaDu cells (106) were inoculated into the dorsum of the hind foot. After 5 days, mice were treated with 9 mg/kg or 21 mg/kg body weight of compound (2) in drinking water or by daily i.p. injections of 5 mg/kg body weight of compound (2). After inoculation mice were assigned to treatment or control groups (n=5). Tumor volume (V) was determined from: V=(4/3)*(22/7)*1/8*(length*width*height). Differences between treatment cohorts were determined using the Student's t test Mice were weighed every 4-5 days, monitored for hair loss, loss of appetite, vomiting and diarrhoea. Total and differential blood and bone marrow analyses were performed after treatment with compound (2).
MRI experiments were performed using a 9.4 T 21 cm bore horizontal bore magnet (Magnex Scientific) Varian INOVA MRI system (Fremont, Calif.) equipped with a 28 mm inner diameter quadrature birdcage coil. Mice were anesthetized with isoflurane inhalation anesthesia (1-1.5% in 100% O2 administered via a nose cone) and positioned in the MRI coil. Body temperature was maintained (37-38° C.) using a homeothermic warming system. After acquiring scout images, multi-slice spin-echo imaging with an echo time of 18 ms and a repetition time of 400 ms ms was performed. A 40 mm field of view with a 256×256 matrix size was used. Nine to 15 slices along the transverse, sagittal, and coronal planes were acquired in each multi-slice experiment with a slice thickness of 1 mm and the gap between slices of 0.5 mm. MRI data were processed off-line with MATLAB-based MRI analysis software.
Spent media and perchloric acid extracts of both PC3 cells and tissue samples were subjected to purification via cation exchange chromatography and dansyl-derivatized with minor changes. Disposable 10 ml BIO-RAD columns were centrifuged at 4,000 rpm for 3 minutes. Sodium carbonate used for derivatization was adjusted to pH 9.3 and the concentration of dansyl-chloride was adjusted to 100 mM. Dansyl-polyamines were quantitated by a Waters HPLC/Fluorescence system. A Phenomenex Luna 5 μ C18 column was used with a mobile phase of 30% acetonitrile in a 50 mM ammonium acetate buffer at pH 6.8 (eluent A) and 100% acetonitrile (eluent B). Fluorescence detection was monitored by excitation at 338 nm and emission at 500 nm.
Short-Term: Mice were treated with sterile solutions of 100 μM compound (2) (pH ˜6.4). Water bottles were autoclaved prior to filling with sterile inhibitor solutions. Mice were sacrificed at 1, 2, and 7 days, with three mice in each group, with the control group sacrificed after 7 days. Livers were immediately removed upon sacrifice for polyamine analysis, conducted as described above.
Long-Term: Sterile solutions of 100 μM compound (2) (pH ˜6.4). Water bottles were autoclaved prior to filling with sterile inhibitor solutions. Water consumption was monitored every other day, with fresh inhibitor solution being administered to prevent bacterial growth. Mice were control-sacrificed and tissues (genitourinary system, liver, lungs) were collected for histology and polyamine analysis. Mass and dimensions of excised genitourinary system tumours were recorded. Sections of small intestine were also removed for toxicity analysis via H&E staining.
Growth of 3LL and RM1 cells was in Dulbecco's modified Eagle's medium containing serum and antibiotics with 5 mM sodium pyruvate and 0.25 mM non essential amino acid mixture (Gibco). Compound (1) was added as a sterile solution and MTA was absent or present at 20 μM.
Discussion of the Examples
Thus, administration of MTA in circumstances where its degradation by MTAP is inhibited by an MTAP inhibitor leads to greater circulatory and tissue levels of MTA and consequently an enhanced effect in the treatment of cancer.
Another head and neck cancer cell line, Cal27 was also found to be susceptible to compound (2) and MTA. After 8 days of treatment, the number of viable Cal27 cells decreased as a result of G2/M arrest and apoptosis when compared to controls (
Longitudinal MRI provides a noninvasive means of monitoring prostate tumour growth in mice (Gupta S, Hastak K, Ahmad N, Lewin J S, Mukhtar H Proc Natl Acad Sci USA 2001 Aug. 28; 98(18):10350-5; Eng M H, Charles L G, Ross B D, Chrisp C E, Pienta K J, Greenberg N M, Hsu C X, Sanda M G Urology 1999 December:54(6):1112-9; Song S K, Qu Z, Garabedian E M, Gordon J I, Milbrandt J, Ackerman J J Cancer Res. 2002 Mar. 1:62(5):1555-8.).
MRI was used to evaluate prostate tumour growth and progression longitudinally in TRAMP mice (either untreated or treated with a compound that may be, used according the methods of the invention). Mice were imaged approximately monthly from 12-33 weeks of age. Representative MRI images comparing untreated control TRAMP and treated TRAMP mice at approximately 30 weeks of age are shown in
Panels A and B show results from control mice. Panel A shows a coronal section through of a 30 week old TRAMP mouse with a large tumour (bright tissue) that weighed 8.76 g upon dissection at 34 weeks of age. The inset shows a more posterior coronal section. The bright tumour is smaller in this section but metastasis to the liver is observed (white arrow). Panel B shows a coronal section through the prostate region of a 30 week old TRAMP mouse. The seminal vesicles (SV) are enlarged. A large tumour (weighing 4.89 g upon dissection at 36 weeks of age) that spanned from the kidney to bladder (BL) is visible in the transverse section shown in the inset (white arrow).
Panels E and F show results for mice treated with 1 mM compound (2). Panel E shows a coronal section through the prostate region of a 30 week old treated TRAMP mouse. The tumour, weighing 0.41 g upon dissection at 34 weeks of age, was not observed during the imaging session. Panel F shows a similar section through a 30 week old treated TRAMP mouse that exhibited a 0.64 g tumour upon dissection at 39 weeks of age. The tumour is indicated by the white arrow in the MRI image shown in this panel.
Untreated TRAMP mice therefore demonstrate primary prostate tumour growth. However, prostate cancer progression in the TRAMP mouse is inhibited in mice treated with compound (2), either alone or in combination with MTA.
Treatment of PC3 cells with compound (2) resulted in numerous significant alterations in both intracellular and spent media polyamine levels. After 24 hours of treatment, the increase observed in cellular SPD levels as well as putrescine (PUT) cellular and spent media polyamine levels correlated with the effects expected with MTAP inhibition. MTA accumulated in the cells, began feedback inhibition of SPN synthase, resulting in accumulations of SPD and RUT, with PUT being significantly excreted into the media, and a slight decrease of SPN in the media. By day 6, cellular SPN levels were significantly reduced in combination treated cells, while maintaining the characteristic elevations in levels of PUT and SPD. Treatment of cells for 12 days showed a significant increase in cellular SPD levels and a slight decrease in spent media PUT levels, pointing to the fact that a compensatory pathway had been activated to make up for the block in MTAP. PUT may have been being taken up from the media for SPD synthesis. After combination treatment for approximately 2 weeks, PC3 cells displayed a cytostatic effect, as determined by the clonogenic assay. Initially, it was believed that MTAP inhibition would lead to MTA accumulation, causing feedback inhibition of polyamine biosynthesis, resulting in decreases in cellular proliferation. Although a halt in cellular proliferation was observed, this is clearly not due simply to polyamine depletion.
Mass (Table 1) and dimensions of excised genitourinary system tumors were recorded for all members of the treatment groups. Sections of small intestine were also removed for toxicity analysis via H&E staining. Histology of TRAMP mice revealed all animals showed extensive prostate intraepithelial neoplasia involving most prostate acini. However, the size and incidence of preinvasive tumors, as well as the incidence of invasive cancer and metastasis were all decreased in treated TRAMP mice (Table 1). No alterations, inflammations, or irregularities were observed in the intestinal crypts, neither were any hair loss or general GI problems noted, indicating a lack of drug toxicity.
Although the invention has been described by way of example, it should be appreciated the variations or modifications may be made without departing from the scope of the invention. Furthermore, when known equivalents exist to specific features, such equivalents are incorporated as if specifically referred to in the specification.
The co-administration of 5′-methylthioadenosine (MTA), or a prodrug of MTA, with one or more MTAP/MTAN inhibitors is effective for treating diseases or conditions in which it is desirable to inhibit MTAP or MTAN. Such diseases include prostate cancer and head and neck cancer.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/NZ2007/000038 | 2/23/2007 | WO | 00 | 1/4/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 60776471 | Feb 2006 | US |
