Patent Application
20120258944
The present invention relates to selenium-containing compounds for use as medicaments, and in particular to compounds comprising a phenol fused to a five-membered selenium-containing ring, for use in the treatment of disorders or conditions caused by or involving free radical-mediated or oxidative tissue damage.
Antioxidants are useful in many fields, including medical fields where problems caused by or involving free radical-mediated or oxidative tissue damage need treatment.
Numerous potential antioxidants have been studied in the hope of finding some which can be therapeutically useful. Amongst the large number of publications in this field, some relate to selenium-containing compounds. The use of selenium-containing compounds is associated with several disadvantages. One disadvantage is that their toxicology can be difficult to predict, and in many cases they exhibit toxicity by various mechanisms as summarised in for example Chem. Rev. 2004, 104, 6255-6285.
Several investigations and publications have indicated that certain selenium-containing compounds are not effective as medically useful antioxidants, and in some cases have taught that tellurium-containing compounds are highly effective whereas corresponding selenium-containing compounds are not.
For example, J. Am. Chem. Soc. 2001, 123, 3434-3440, a publication by the present inventor and others, discloses various studies on the following range of compounds:
This publication tries to assess the antioxidant character of these compounds by several tests and analyses.
Firstly, from a theoretical chemistry viewpoint, by considering redox properties of the compounds, the authors of J. Am. Chem. Soc. 2001, 123, 3434-3440 make postulations regarding bond dissociation enthalpies and oxidation potentials. However, because many other factors come into play, it is not reasonable to extrapolate such characteristics to predict how the compounds would behave as antioxidants in a real biological system.
Secondly, the extent to which the compounds inhibit the azo-initiated peroxidation of linoleic acid in a two-phase system is studied, and the publication states that this gives a reasonable indication of how the compounds inhibit lipid peroxidation. The publication indicates that, in the absence of thiol reducing agent, none of the compounds are as good as the natural antioxidant alpha-tocopherol at inhibiting peroxidation, and that the organoselenium compound (X═Se) is a poor inhibitor. In the presence of a reducing agent, according to this test method, the tellurium compound (X═Te) has the best antioxidant capacity.
Thirdly, the J. Am. Chem. Soc. 2001, 123, 3434-3440 publication teaches that hydroperoxide decomposition is perhaps the most important duty for preventive antioxidants, and that in biological systems this task is fulfilled by selenium-containing glutathione peroxidases and catalase. The publication refers to a coupled reductase method as providing a convenient test for assessing thiol peroxidase activity. It discloses that catalyst activity in the reaction between glutathione and hydroperoxide is insignificant for the organoselenium derivative (X═Se). In contrast, the organotellurium derivative (X═Te) is shown to be a highly active catalyst according this method.
Fourthly, the publication investigates the inhibition of lipid peroxidation in liver microsomes. Whereas the organotellurium derivative (X═Te) is stated to be a potent inhibitor, the other three analogues (X═O,S,Se) are not effective according to this method.
Another publication by the present inventor and others is J. Org. Chem. 2007, 72, 2583-2595. This uses similar test methods to those used in J. Am. Chem. Soc. 2001, 123, 3434-3440, including the two-phase method to assess the inhibition of peroxidation of linoleic acid. Although J. Org. Chem. 2007, 72, 2583-2595 studies variously substituted selenium-containing compounds wherein X is Se, in more detail, it reaches no clear conclusions. It finds that compounds often do not behave as predicted, and that there are several trends and aspects which are incompletely understood. Several selenium-containing compounds are disclosed as having poor regenerability.
Now, however, further work by the inventor has shown that organo-selenium compounds as defined below are surprisingly effective in biological systems, and in fact that some of the previous test results cannot be extrapolated to give a true indication of efficacy in vivo. Experiments as described below have compared compounds of the invention with various other compounds which fall outside the definition of the present invention (“comparative compounds”). The comparative compounds work better than compounds of the invention in the two-phase model. However, in biological systems, the compounds of the present invention work better than comparative compounds. This was completely unexpected to the present inventor.
It is surprising that the compounds of the present invention work better than comparative compounds, when in the two-phase model they perform less well than the comparative compounds. Furthermore, the inventor has shown that the compounds of the present invention possess the additional advantage of being pharmaceutically acceptable when so many other selenium-containing compounds are toxic.
From a first aspect, therefore, the present invention provides a compound comprising the following formula I, or a pharmaceutically acceptable salt thereof, for use in therapy:
wherein
each of R1, R2, R3 and R4 is independently selected from:
From a second aspect, the present invention provides a compound comprising the following formula II, or a pharmaceutically acceptable salt thereof, for use in therapy:
wherein
each of R1, R2, R3 and R4 is independently selected from:
It will be seen that formula II differs from formula I only in that R7 and OH are exchanged. This means that, in both formula I and formula II, the selenium atom, R5 and R6 are all ortho- or para- to OH. Therefore, the two structural types are closely related in terms of electronic effects.
The compounds of formulae I and II are useful as medicaments in the treatment of disorders or conditions caused by or involving free radical-mediated or oxidative tissue damage.
The disorders or conditions include for example: ischemic or reperfusion injuries, thrombosis, embolism, neoplasms, cancer, Parkinson's disease, Alzheimer's disease, atherosclerosis, allergic/inflammatory conditions such as bronchitis, asthma, rheumatoid arthritis, ulcerative cholitis, Crohn's disease, cataract, respiratory distress syndrome, damage caused by chemicals, radiation, antineoplastic or immunosuppressive agents, ischemia/reperfusion injury in the heart, kidney and CSN and post-operative ischemia/reperfusion injury, organ preservation, burn injury, wound healing, and IBS (irritable bowel syndrome).
Preferred therapeutic applications are organ preservation, treatment of burn injury and treatment of rheumatoid arthritis.
Also covered by the present invention are pharmaceutical compositions comprising the compounds of the present invention and pharmaceutically acceptable diluents, excipients and/or carriers.
The core structures of formulae I and II are responsible for the enhanced activity of the compounds based on them. It is within the scope of the present invention to add substituents or moieties to the core structure, so long as the compounds still exhibit the therapeutic activity. For example, various groups are listed within the definitions of each of R1, R2, R3, R4, R5, R6 and R7 above, including some with optional substituents. The substituents may for example be: halogen (e.g. F, Cl, Br or I); OH, alkoxy, SH, NH2, N-alkylamino, N,N-dialkylamino, COOH, CHO, NO2, aryl or heteroaryl. The substituents may be further substituted by for example any of these, e.g. an alkoxy substituent may have a further substituent on its alkyl chain.
Alkyl as used herein preferably denotes C1-C15 alkyl. The alkyl may be saturated or unsaturated, unbranched or branched, or any combination thereof. The skilled person is well aware that different alkyl chain lengths affect the hydrophilicity/lipophilicity of compounds, and therefore in some cases short alkyl chain lengths are preferred whereas in other cases more “greasy” longer alkyl chains are preferred. This is an advantage of the present invention because it allows the tailoring of molecules.
The nature of the alkyl groups will now be discussed in more detail with reference to R1, R2, R3 and R4. Whilst all of these are shown in formulae I and II, in many of the preferred compounds some of R1, R2, R3 and R4 are hydrogen. In one preference, three of these are hydrogen and one of these is alkyl. In a further preference, two of these are hydrogen and two of these are alkyl (the same or different). It is within the scope of the present invention that all four may be hydrogen. It is also within the scope of the present invention that none, or one, may be hydrogen, and the remainder alkyl.
One or more of R1, R2, R3 and R4 may be alkyl groups such as C1-C15 alkyl. One preferred type of alkyl is lower alkyl (C1-C5 alkyl). A preferred alkyl group is methyl. In one preference R1 is methyl and R2, R3 and R4 are hydrogen. In another preference R3 is methyl and R1, R2, and R4 are hydrogen. In another preference each of R1, R2, R3 and R4 is hydrogen.
In a further preference, R1 is methyl and R2, R3, R4, R5, R6 and R7 are hydrogen. In another preference R3 is methyl and R1, R2, R4, R5, R6 and R7 are hydrogen. In another preference each of R1, R2, R3, R4, R5, R6 and R7 is hydrogen.
A further preferred alkyl group for R1, R2, R3 and/or R4 is ethyl.
Further preferences are the alteration of the above preferences by the addition of optional substituents on the alkyl groups; various possible substituents are listed above.
Thus it will be noted that the possibilities on the selenium-containing ring are defined in a focused way to cover those patterns which exhibit surprisingly effective results.
The possible substituents on the aromatic ring will now be discussed. Various possibilities for R5, R6 and R7 are defined above. It should be noted, firstly, that hydrogen may be present at one, two, or all of these positions, and that there is no need for other substituents in order for the compounds to be effective. However, some of the effective compounds do contain other moieties at one or more of these positions, and these moieties may optionally themselves carry further substituents.
With reference to R5, R6 and R7, the term “alkyl” has the same meaning as it does for R1, R2, R3 and R4. The term “alkoxy” is defined analogously, i.e. the same possible carbon chain lengths and other features and preferences apply as for “alkyl”. Similarly the same definitions, preferences and features apply, mutatis mutandis, to alkyl component(s) within alkylamino moieties. In particular, for example, a preferred alkyl part of these moieties is methyl.
It will also be noted that R5, R6 and R7 may be electron-donating substituents. Many of the moieties already listed for R5, R6 and R7 are of course inherently electron-donating substituents. This term also includes other substituents which are known to the skilled person as being electron-donating substituents, as well as those explicitly listed.
It is preferred for there to be electron-donating moieties at R5 and/or R6, in order to result in compounds which are particularly effective. Thus, R7 may be hydrogen whilst R5 and/or R6 is not hydrogen.
Thus the possibilities on the aromatic ring are defined in a focused way to cover those patterns which exhibit surprisingly effective results.
Some preferred compounds, and groups of compounds, of the invention are shown below. In these,
Compounds of formulae I and II may be made according to procedures known in the art, adapted where necessary within the ability of the skilled person. For example, preparative procedures for some of the compounds are given in J. Org. Chem. 2007, 72, 2583-2595 and J. Am. Chem. Soc. 2001, 123, 3434-3440.
The compound according to the present invention may optionally be used as a catalytic antioxidant. For example, it may be used under conditions which allow its regeneration, either because the system in which it is used inherently regenerates the compound and cycles the catalyst, or because an additional component is used to regenerate the catalyst.
Thus, the compound according to the present invention may also be combined with, or used in combination with, a reducing agent. The reducing agent may for example be a mild reducing agent. For example, suitable reducing agents include those which are suitable for regenerating the catalyst without causing undesirable side-effects. One suitable class of reducing agents is the class of thiols. Specific examples include N-acetylcysteine, cysteine, dithiothreitol, glutathione, ascorbic acid and sodium ascorbate.
In drug development, catalytic compounds are particularly advantageous since only small amounts are needed to achieve the pharmacological effects. The relatively low amounts needed significantly reduce the risks of toxic effects or other side effects, including those which may be associated with impurities in a final drug substance.
Additionally, catalytic use is economically and environmentally advantageous and minimizes the preparation and use of potentially hazardous materials. It allows the use of reducing agents which are low-cost, readily available and safe.
The compounds of the present invention can be used in a non-catalytic sense, but in that case they only have a finite effect, and for example can only destroy a limited number of peroxyl radicals before they are themselves converted to inactive compounds. It is therefore preferable for them to be regenerated by cheap, nontoxic reducing agents. It is advantageous that the compounds can act as chain-breaking antioxidants by catalyzing the decomposition of peroxyl radicals in the presence of mild reducing agents.
The present invention describes novel, regenerable compounds, and any acid or base addition salt or prodrug thereof, which are capable of acting as chain-breaking antioxidants by catalyzing the decomposition of peroxyl radicals in the presence of mild reducing agents. Compounds according to the present invention interfere with pathophysiologically important reactions in man and animals and thus effectively hamper the degradation of tissue constituent molecules as well as act to remove harmful products from such degradation. The compounds possess an ability to protect tissues against oxidative damage induced by overreacting host defence systems. Compounds according to the present invention are therefore useful for the pharmacological treatment of diseases in which free radical-mediated or oxidative tissue degradation occurs or where oxidants trigger pro-inflammatory receptors on cell surfaces. The reducing agents required for a catalytic mode of action of compounds according to formulae I and II could either be exogenous and present in the environment where the antioxidant effect is desired (for example glutathione or ascorbate) or they have to be supplemented together with compounds according to the present invention. Diseases such as inflammatory (including autoimmune inflammatory) conditions like asthma, bronchitis, various allergic skin and systemic disorders, Crohn's disease, ulcerative colitis, rheumatoid arthritis and other kinds of arthritis respond to such treatment. Compounds according to the present invention together with suitable reducing agents may also be used for intervention of cataract and the respiratory distress syndrome. Further, the involvement of oxidative damage in atherosclerosis and in ischemia/reperfusion injury in the heart, kidney, CSN or post-operative ischemia/reperfusion injury as well as in thrombosis and embolism makes these disorders liable to intervention by the compounds according to the present invention together with suitable reducing agents. The free radical dependent pathology of ageing and neoplasm development as well as disorders such as Parkinson's and Alzheimer's diseases may also be influenced in a favorable manner by the compounds according to the present invention together with suitable reducing agents. The oxidative damage to tissues caused by radiation, but also by antineoplastic or immunosuppressive agents and other xenobiotics can be prevented or limited by the use of compounds according to the present invention together with suitable reducing agents.
The present invention will now be described in further non-limiting detail and with reference to various Figures and Examples which are described and discussed below.
In the following experiments, organoselenium compound 1 is a compound of the present invention having the following formula:
Comparative compounds 2 and 3 are not compounds of the present invention. They have the following formulae:
Previously, results according to the two-phase model (discussed above and in J. Org. Chem. 2007, 72, 2583-2595 and Org. Lett. 2008, 10, 21, 4895-4898) indicated that comparative compounds 2 and 3 (which fall outside the definition of the present invention), in comparison to compound 1, are much better chain-breaking antioxidants as determined by the inhibited rate of peroxidation, Rinh, in the presence of N-acetylcysteine in the aqueous phase. The table below shows this data. In addition, both of the comparative compounds have a capacity to act as catalytic preventive antioxidants by decomposing hydroperoxides via redox cycling of tellurium between the oxidation states II (telluride) and IV (telluroxide).
Compound 1
Comparative Compound 2
Comparative Compound 3
Based on the results from the two-phase model, it would be difficult even for a person skilled in the art to predict how these compounds would perform as antioxidants in complex biological systems where so many other factors come into play. If anything, the above data rather teaches away from the use of compound 1 as an effective antioxidant.
Surprisingly, as shown in the experiments below, compound 1 is not only highly effective in cellular systems, but is actually significantly more effective than comparative compounds 2 and 3. Thus, the results from the two-phase model cannot always be extrapolated to give a true indication of efficacy in vivo. Furthermore, compound 1 displays excellent results in terms of non-toxicity that could also not be predicted.
It is surprising that the compounds of the present invention work better than comparative compounds, when in the two-phase model they perform less well than the comparative compounds. Furthermore, the inventor has shown that the compounds of the present invention possess the additional advantage of being pharmaceutically acceptable when so many other selenium-containing compounds are toxic.
Furthermore, compounds of the present invention display unexpectedly good results with respect to concentrations needed to induce cell toxicity in human endothelial cells. In experiments described below, cell toxicity was monitored for up to 72 hours in presence of different concentrations of test compounds and it was found that compound 1 was far less toxic than compounds 2 and 3 and also that the concentration needed for toxicity was about two orders of magnitude higher than the concentrations needed for ROS-inhibition. This fact strongly indicates the presence of a therapeutic window well separated from concentrations where the compounds become toxic.
Previously, the effectiveness and validity in biological systems of compounds of the present invention had not been established. Therefore, the following experiments were carried out to investigate their effect in cell systems with induced ROS (reactive oxygen species) production. These experiments were carried out on the following potential antioxidants: Compound 1 and Comparative Compounds 2 and 3.
The purpose of the experiments was to validate the antioxidative effect of these substances on human neutrophil cell line PLB985, human neutrophils and PBMC (peripheral blood mononuclear cells) and rat neutrophils and PBMC.
Validation was performed by stimulating cells with PMA and fMLF. When treated with the antioxidants, altered level of ROS production from the cells was measured using luminometry. Both intracellular and extracellular effect on ROS on the cells was analyzed using isoluminol (extracellular) and luminol (intracellular when co-administered with SOD and catalase) (Dahlgren et al. “Respiratory burst in human neutrophils”, Journal of Immunological Methods 232 1999; 3-14).
The viability of cells treated with the compounds was also analyzed using a Resazurin based in vitro Toxicology Assay Kit (SigmaAldrich TOX8). Resazurin systems measure the metabolic activity of living cell. Resazurin in its oxidized form (blue) is reduced (red) by living cells and the amount of dye conversion is measured fluorometrically or spectrophotometrically indicating the degree of cytotoxicity.
Thus, the experiments investigate the following in detail:
The following reagents were used:
PMA is a potent tumor promoter; activates protein kinase C in vivo and in vitro and is a very potent NADPH activator.
fMLF is a potent inducer of leucocyte chemotaxis and macrophage activator. It induces a metabolic burst in macrophages accompanied by an increase in respiratory rate, secretion of lysosomal enzymes, and production of superoxide anion. Receptors that bind formylpeptides are found on phagocytic neutrophils and have recently been identified on cells of the intestinal mucosa.
Compound 1 and Comparative Compounds 2 and 3 were tested. Fresh solutions in DMSO were prepared on the day of experiment.
The compounds were tested in all experiments in dilution series of 120 microM to 0 microM of final concentration.
The NAC concentration was tested in dilution series 200, 100 and 0 microM final concentration.
PMA and fMLF were used in final reaction concentration of 30 ng/ml.
White 96 well plates (VWR 732-2698 microplate 96 f fluoronunc white) and detection in luminescence detector (FluoStar).
Transparent 96 well culture plate, detection in Spectra MAX 250 using Soft MAX PRO software.
Human blood was purchased in day-old buffy coats from Blodcentralen, Sahlgrenska Univeristy hospital (Blodcentralen, 031-342 36 54). Rat blood was taken from rats at the central animal facility EBM in Göteborg (Sofia Berntsson 031-7865874, Rosita Olden 031-7865845). Various strains of rats can be used, like Sprague-Dawley, Wistar or Fischer rats. The rats were sacrificed using carbon dioxide and blood was taken by heart puncture. Isolated blood was heparinised to prevent coagulation. Whole blood was mixed with equal volume of room temp. 0.9% NaCl and Dextran 2%, diluted in 0.9% NaCl to a final concentration of 1%. Cells were allowed to sediment for 20-35 min before the upper layer was isolated and transferred to centrifugation tubes, ca 15 ml/tube. Using a syringe with injection needle an equal volume of Ficoll-Paque Plus (GE Healthcare) was added to the bottom of each tube. These were centrifuged at 2000 rpm, 4° C. for 15 min.
PBMCs were isolated from the middle layer using a Pasteur pipette and were transferred to new, pre-chilled tubes. These were diluted in cold HBSS, counted and pelleted. Dilution was carried in proper volume of cold HBSS to get 2 million cells/ml, and storage was on ice.
To isolate the neutrophils, all liquid was removed and the bottom pellet was saved, containing neutrophils and remaining erytrocytes. These cells were washed in 20 ml KRG without Ca2+, 900 rpm, 10 min. Erythrocytes were lysed by adding 6 ml dH2O for 30 seconds. 2 ml 2.4% NaCl in PBS was added, and washing was in KRG without Ca2+, ca 10 ml. Centrifuging was at 900 rpm, 4° C., 10 min. The lysation was repeated 2-4 times. Isolated neutrophils were resuspended in proper volume KRG with Ca2+ to get 2 million cells/ml.
Human neutrophil cell line PLB985 was grown in RPMI 1640 (Gibco) supplemented with 10% FCS, Penicillin/Streptomycin. The cells were grown at 37° C. at 5% CO2. The cell are in suspension and when the growth is close to “saturation”. The cells are pelleted and resuspended in new media with the addition of 1.25% DMSO (625 micro- 1/50 ml media). This treatment induces differentiation to neutrophils after 5-8 days (Then, L.; King, A. A. J.; Xiao, Y.; Chanock, S. J.; Orkin, S. H.; Dinauer, M. C. Proc. Natl. Acad. Sci. 1993, 90, 9832-9836) (Tucker, K. A.; Lilly, M. B.; Heck, L.; Rado, T. A. Blood 1987, 70, 372-378). These cells duplicate once every 24 hours. So 1 million cells in 50 mL media will reach saturation after 7 days of culture and be about 1-1.5 million cells/ml. Cells were washed in 20 mL HBSS and resuspended at 2 million cells/ml.
PMA and fMLF were used at a working concentration of 120 ng/ml and final reaction concentration of 30 ng/ml. Dilutions to final concentrations were done in HBSS.
The test compounds were diluted in DMSO to 10 mM concentrations. Further dilutions were made in HBSS. The activity of the test compounds was tested in a dose titration of 120 micro-M to 0 micro-M range of final reaction concentration.
NAC was diluted in HBSS to 10 mM, final reaction concentrations were 200, 100 and 0 micro-M. Dilution to final reaction concentrations was done in 2× Assay (Isoluminol/Luminol) buffer.
The isoluminol reagent buffer was made of HBSS buffer (with Ca2+), Isoluminol and HRP.
Isoluminol (10 mg/ml) should be kept dark at RT. HRP should be kept in the fridge, or can be frozen if stored for longer periods. The Isoluminol Assay buffer is quite stable but should be prepared fresh every day or twice daily.
Luminol Assay buffer
The luminol reagent buffer is made of HBSS buffer (with Ca2+), luminol and SOD/Catalase.
Luminol (10 mg/ml) should be kept dark at RT. The Luminol Assay buffer is quite stable but should be prepared fresh every day or twice daily.
The antioxidants and activators were diluted in Isoluminol/Luminol Assay buffers
1) Add 25 microL of antioxidants to assay plate to get proper concentrations in a total reaction volume of 100 micro-L.
2) Add 25 microL of PMA (120 ng/ml) and fMLF (120 ng/ml) to assay plate to get concentration of 30 ng/ml in a total reaction volume of 100 microL.
3) Add 50 microL of cell suspension (2 million cells/ml) to each well. Immediately after addition of cell suspension initiate luminescence measurement; the reaction starts at once when the cells are added to the assay plate. Measurement points are taken at 1-minute intervals for 25 minutes. Data is presented as AUC (Area Under Curve) by a summary of all collected measurements for each well.
1) 75 micro-l of PLB985 cells (1 million cells/ml) is mixed with 25 micro-l of Compounds 1, 2, 3 and D (480, 120, 15 and 0.48 micro-M) in a 96 well plate in duplicates.
2) 10 micro-L of Resazurin solution is added to each well and the plate is incubated at 37° C. for 3 hours.
3) Spectrophotometrical analysis is performed in plate reader at 600 and 690 nm.
Results are shown in
The annotations “1”, “2” and “3” under the bar charts indicate the results in respect of compound 1 and comparative compounds 2 and 3 respectively.
The graphs shown in
Several conclusions can be drawn from the above experiments and the graphs in
Compound 1 has a strong antioxidative effect. Without wishing to be bound by theory, the lesser effect of Compounds 2 and 3 could be due to lower potency or due to problems in passing the cell membrane to the intracellular compartment (
Compound 1 has an antioxidative effect on extra cellular ROS. Compounds 2 and 3 are less potent (
We see no species differences between rat and human cell produced ROS (
We see no cell type specific effect between monocytes and Neutrophils (
We see no clear effect of inclusion of NAC (100 or 200 micro-M) or that NAC is critical for the antioxidative effect (differs between all experiments, but clearly no general effect that NAC should be required for the effect of the compounds). It is possible that, as observed in
Compound 1 shows no signs of cytotoxicity. In their highest concentrations (120 μM) Compounds 2 and 3 cause a decrease in cell viability.
In summary, compounds of the present invention are highly and consistently effective in cellular systems.
Previously, the antioxidant capacity of compound 1 was tested in lipid (chlorobenzene) phase (J. Org. Chem. 2007, 72, 2583-2595).
In order to investigate its aqueous phase performance, we thought that it would be interesting to determine its Trolox Equivalent Antioxidant Capacity (TEAC) (Free Rad. Biol. Med. 1999, 26, 1231-1237) which has been used to quantify the antioxidant activity of biological fluids, extracts and pure compounds. The TEAC value is defined as the concentration of Trolox which has an equivalent antioxidant potential as a 1 mM solution of the compound under investigation (Free Rad. Biol. Med. 1996, 20, 933-956).
A 2,2′-azinobis(3-ethylbenzothiazoline-6 sulphonic acid radical cation (ABTS•+) assay was used to determine relative TEAC values. To a preformed solution of ABTS•+ in PBS buffer, pH 7.4, with an absorbance slightly above 0.7 were added various amounts (3-30 microM) of organoselenium compound 1, Trolox, sodium ascorbate and N-acetylcysteine. The absorbance at 734 inn was recorded immediately after mixing and then every minute for the next 8 minutes.
Experiments carried out at 6.08 micro-M concentrations could clearly distinguish the quenching capacity of compound 1 and the three water-soluble reference antioxidants (
It is clear from this part of the study that organoselenium compound 1 has a high capacity to quench radicals in an aqueous environment.
Antioxidant activity as determined by the capacity of the antioxidant to decolorize the ABTS+ radical cation was determined essentially as described in the literature (Acc. Chem. Res. 1986, 19, 194-201). ABTS, (2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt; 5.76 mg, 0.0105 mmol), was allowed to react with less than the stoichiometric amount of potassium persulfate (1.00 mg, 0.0037 mmol) in water (1.5 mL) to produce, after 15 h, a stock solution of the corresponding radical cation with a stable absorbance. This stock solution (0.0200 mL), diluted with PBS buffer-pH 7.4 (1.48 mL), showed an absorbance of 0.711 at 734 nm. Using an extinction coefficient of 1.5×104 for the radical cation ABTS+ in water, this value corresponds to a 47.4 microM concentration of the ABTS+ radical cation. In a typical experiment, the antioxidant (1.52 micro-L of a 3.00 mM solution in MeOH or water; final concentration=6.08 micro-M) was syringed into 0.750 mL of the ABTS+ radical cation solution. After initial mixing, the absorbance was recorded every minute for the next eight minutes.
Alamar Blue is a non-toxic metabolic indicator for viable cells. Following uptake, the dye becomes reduced and causes a change in the colour of the cell. The colour change correlates reasonably well with the number of living cells in the sample.
As can be seen in
Cell death and plasma membrane damage was also assayed by measuring the release of lactate dehydrogenase (LDH), a stable cytoplasmic enzyme present in most cells. In the kit provided by Sigma, LDH participates in a reaction which converts a yellow tetrazolium salt into a red formazan-class dye. The amount of formazan is directly proportional to the amount of LDH in the culture, which is in turn proportional to the number of dead or damaged cells.
The LDH assay correlated well with the proliferation data, that is, no significant signs of toxicity for any of the cell types could be seen as a consequence of addition of selenide 1 or Trolox at 60 micro-M (
MG-63, SHSY-5Y, MRC-5, Caco-2 and HEK 293 were cultured in DMEM:F12 (Sigma-Aldrich) supplemented with 10% FCS, 100 IU penicillin/ml, 100 μg streptomycin/ml, 2 nM L-glutamine and 1% non essential amino acids at 37° C., 5% CO2 in a humidified atmosphere.
The THP-1 cells were grown in RPMI-1640 (GIBCO®) supplemented with 10% FCS, 100 IU penicillin/ml, 100 μg streptomycin/ml, 2 nM L-glutamin and 1% non essential amino acids at 37° C., 5% CO2 in a humidified atmosphere.
MG-63, SHSY-5Y, MRC-5, Caco-2 and HEK 293 were harvested using trypsin-EDTA treatment. The cells were centrifuged at 400 g for 5 min after which they were resuspended in phenol red free Alpha medium (GIBCO®) containing 10% FCS, 100 IU penicillin/ml, 100 μg streptomycin/ml, 2 nM L-glutamin and 1% non essential amino acids. 30 000 cells/well (of each cell type) and 60 micro-M of the antioxidants Trolox or SeOH were added to a 24 well plate in triplicate samples. Cells w/o antioxidant were used as controls.
Culture medium was removed from the wells after 1, 3 and 7 days and replaced by 0.5 ml Alamar Blue stock solution (Serotec) diluted 1:10 in Hanks balanced salt solution (HBSS) and incubated at 37° C., 5% CO2 in a humidified atmosphere for 2.5 h. Alamar Blue is a non-toxic metabolic indicator for viable cells. Upon uptake into the cell the dye becomes reduced and changes colour. The colour change correlates approximately with the number of living cells in the sample. Aliquotes of 100 micro-1 from each well were transferred to a 96-well plate and the absorbance was read at 570 nm using a multiscan MS spectrophotometer (Labsystems).
Culture medium was removed from the wells after 1, 3 and 7 days and used for cytotoxicity measurements using an LDH in vitro toxicology assay kit (Sigma®) according to the manufacturer's protocol. Cell death and plasma membrane damage can be assayed measuring the release of lactate dehydrogenase (LDH), a stable cytoplasmic enzyme present in most cells. LDH participates in a coupled reaction, which converts a yellow tetrazolium salt into a red, formazan-class dye. The amount of formazan is directly proportional to the amount of LDH in the culture, which is in turn proportional to the number of dead or damaged cells. Absorbance was read at 570 nm using a multiscan MS spectrophotometer (Labsystems).
We investigated the capacity of the material to quench ROS produced by freshly isolated human neutrophils or THP-1 cells (human acute monocytic leukemia cell line) stimulated with PMA (Phorbol Myristate Acetate). The antioxidant capacity of selenide 1 was again compared to those of Trolox, a water soluble derivative of vitamin E, which is commonly used in biological and biochemical applications to reduce oxidative stress or damage.
In a first attempt total ROS (i.e., both extra- and intracellular) production was measured using modified luminol enhanced chemiluminescence (CL). As can be seen in
HBSS, selenide 1 at 1 micro-M, selenide 1 at 10 micro-M, and selenide 1 at 20 micro-M, respectively.
Neutrophils were isolated from heparinized blood of apparently healthy blood donors (Academic Hospital, Uppsala, Sweden) following a routine dextran sedimentation method essentially as described by H{dot over (a)}lansson and Venge (Scandinavian Journal of Immunology 1980, 11, 271-282). To each of three test tubes containing 5 ml heparinized blood, dextran (T500, Pharmacia, Uppsala, Sweden) was added (final concentration 1%) and the sedimentation was allowed to proceed for 30 min at room temperature. The neutrophil rich supernatant was harvested, pooled and centrifuged at 160 g for 5 min. The pellet was washed twice with 0.9% NaCl. Contaminating erythrocytes were lysed by a 30 s exposure to water (Milli Q) after which 3.6% NaCl was added to reach a final concentration of 0.9%. The obtained suspension was then centrifuged for 5 min at 160 g. Finally, the pellet was resuspended in Gey's buffer and the average content of neutrophils (approximately 90%) was calculated using Türks staining and hemocytometer method. The experiments were performed within 1-2 h after neutrophil isolation.
The generation of oxygen free radicals over time from neutrophils was monitored for 40 minutes in 24 well white optiplates (Greiner) using a Wallac Victor (Zweifel, H; “Stabilization of Polymeric Materials”, Springer Berlin, Germany, 1997) in the luminescence mode.
Approximately 300 000 neutrophils diluted in HBSS containing different concentrations of the antioxidants 1 and Trolox were added per well to a 24 well plate together with 100 micro-M luminol (5-amino-2,3-dihydro-1,4 phtallazinedione), 0.17 M NaOH and 6.4 U/ml horseradish peroxidase (HRP) (Research Trends 1991, 427-443). Luminescence intensity was read every 5 min. The measurements were commonly performed on quadruplicate samples.
The generation of oxygen free radicals over time from macrophages (THP-1) was monitored for 180 minutes in 96-well white optiplates (Greiner) using a using a TECAN reader and Diogenes chemiluminescence kit (National Diagnostics). Approximately 200 000 THP-1 cells diluted in HBSS containing different concentrations of the antioxidants 1 and Trolox were added per well to a 96 well plate together with the Diogenes reagent 20 v/% (following the manufacturers recommendations) and 40 micro-M Phorbol Myristate Acetate (PMA). Luminescence intensity was read every 6 min. The measurements were commonly performed on quadruplicate samples
Further experiments were carried out in order to investigate the toxic potential of Compound 1 and Comparative Compounds 2 and 3 on primary endothelial cells derived from human umbilical veins.
The experiments on human umbilical vein endothelial cells described below can be summarised as follows. Compound 1 was toxic at 500 μM after 24 hrs of incubation and at 200 μM after 72 hrs. Comparative Compound 2 was toxic to the cells at 200 after 48 hrs and at 500 μM after 24 hrs. Comparative Compound 3 demonstrated toxicity after only 24 hrs of incubation at 100 μM.
HUVECp were seeded according to the manufacturer's instructions and subcultured when reaching at least 80% confluence. Medium 200 supplemented with fetal bovine serum, fibroblast growth factor, hydrocortisone, epidermal growth factor, penicillin, streptavidine and gentamicin was utilized.
Cells were harvested and resuspended in supplemented medium 200 without phenol red. 50 000 cells were added in duplicates to 96-well plates. The compounds were tested on HUVECs according to the following concentrations.
The compounds were diluted in Medium 200 from the 0.1M stock solution. The toxic effects of the compounds were tested after 24 hrs, 48 hrs and 72 hrs.
As a negative control, cells incubated with medium containing 0.5% DMSO (corresponding to the highest DMSO concentration above) was used. As a positive control, cells were incubated in the presence of 20% EtOH.
After the incubation with the compounds, the wells were washed with PBS to remove substances which could interfere with the assay. 40 μl of fresh medium 200 without phenol red was added together with 10 μl XTT substance (according to the manufacturer's instructions). The plates were incubated for 8 hrs and the colour development was monitored by measuring the absorbance at 450 nm.
By visually looking at the cells under the microscope and estimating the dislodging from the surface, the state of the cells was evaluated. Cells incubated with the highest concentration of Compound 1 (500 μM) were affected after 24 hrs. Comparative Compound 2 affected cells at 200 μM after 48 hrs, but only at 500 μM after 24 hrs. For Comparative Compound 3, cells were affected already at 100 μM after 24 hrs.
When measuring the absorbance at 450 nm after incubation with the XTT compound, Compound 1 demonstrated toxic properties at 200 μM after 72 hrs, but only at 500 μM after 24-48 hrs (
The results of the viability measurements coincide with the visual observations made. Comparative Compound 3 was the most toxic compound demonstrating toxic properties at 100 μM concentrations already after 24 hrs of incubation. Comparative Compound 2 seems to be slightly more toxic than Compounds 1; cells were affected at 200 μM after 48 hrs of incubation. Compound 1 was toxic at 200 μM only after 72 hrs of incubation.
Some further compounds were tested as follows:
Compounds 4 to 9 were tested in a similar manner to that described under “example series 1”. In series 6, DTT was used instead of NAC. Results are also shown below for compound 1 using DTT in place of NAC.
The antioxidative effect of these substances on human neutrophil cell line PLB985 stimulated with PMA with and without addition of DDT was validated. When treated with the antioxidants, altered level of extracellular ROS production from the cells was measured using luminometry. (Dahlgren et al., Journal of Immunological methods, 1999, cited previously)
This analysis determined the effect of the test compounds:
1) Antioxidative effect of extracellular ROS in viable cells
2) Dependence of DTT for efficacy
Reagents used were as follows:
The compounds were coded with unique numbers and the identity of the structures of the compounds was not disclosed to the researchers during the experiment. The compounds were supplied as dry powder of 1-2 mg each. Two (2) vials of each test compound were supplied so that the test compound could be diluted fresh in DMSO for each days experiments.
The compounds were tested in dilution series of 120 μM to 0 μM as final concentration. DTT were tested at 100 μM as final concentration. Of PMA a final reaction concentration of 30 ng/ml were used.
All samples were analyzed in duplicates and presented as mean values of ROS production. Control samples (0) were wells analysed without addition of compound, while control (blank) wells were analysed without NADPH oxidase stimulation (PMA) or Catallox compound added.
Plates and Detection: White 96 well plates (VWR 732-2698 microplate 96 f fluoronunc white) and detection in luminescence detector (FluoStar).
Human neutrophil cell line PLB985 were grown in RPMI 1640 (Lonza) supplemented with 10% FCS, Penicillin/Streptomycin. The cells were grown at 37° C. at 5% CO2. The cell were in suspension and when the growth was close to “saturation” the cells were pelleted and resuspended in new media with the addition of 1.25% DMSO (625 ul/50 ml media). This treatment induced differentiation to neutrophils after 5-8 days (Zhen PNAS, 1993) (Tucker Blood 1987). These cells duplicate once every 24 hours. So 1 million cells in 50 mL media will reach saturation after 7 days of culture and be about 1-1.5 million cells/ml. Cells are washed in 20 mL HBSS and resuspended at 2 million/ml.
Dead Cells No dead cells counted
Diluted to: 4 million/ml in 13.5 ml HBSS
PMA was used at a working concentration of 120 ng/ml and final reaction concentration of 30 ng/ml. Dilutions to final concentrations were done in 2× Isoluminol Assay Buffer.
The test compounds were diluted in DMSO to 10 mM concentrations. Further dilutions were made in HBSS. The activity of the test compounds were tested in a dose titration of 120 μM to 0 μM range of final reaction concentration.
DTT were diluted in DMSO to 10 mM, final reaction concentration were 100 μM. Dilution to final reaction concentrations were done in HBSS.
The isoluminol Assay buffer was made of HBSS buffer (with Ca2+), Isoluminol and HRP.
Isoluminol (10 mg/ml) should be kept dark at RT. HRP should be kept in the fridge, or can be frozen if stored for longer periods. The Isoluminol Assay buffer is quite stable but should be prepared fresh every day or twice daily.
Immediately after addition of cell suspension initiate Luminescence measurement, the reaction starts at once when the cells are added to the assay plate.
Measurement points are taken at 1-minute intervals for 25 minutes.
Trolox was also tested in this system.
Results are shown in
The graphs shown in
From
The following protocol was followed:
All incubation are done in 37° C. heating cabinet
Let the plate reach room temperature and measure the fluorescence using a plate reader (Ex 485 and Em 545)
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2010/064767 | 10/4/2010 | WO | 00 | 6/21/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61248332 | Oct 2009 | US |
