WO2009063241A1

WO2009063241A1 - 3-hydroxyanthranilic acid or salts thereof1 for treating cancer or infections

Info

Publication number: WO2009063241A1
Application number: PCT/GB2008/051059
Authority: WO
Inventors: Christopher Joseph Schofield; Vincenzo Cerundolo
Original assignee: Ludwig Institut Fur Krebsforschung Ag; The Chanceller, Masters And Scholars Of The University Of Oxford
Priority date: 2007-11-13
Filing date: 2008-11-13
Publication date: 2009-05-22
Also published as: GB0722274D0

Abstract

24 Abstract This invention relates to a novel inhibitor of indoleamine 2,3-dioxygenase (IDO) and its use in the treatment of cancer or infections, either alone or in combination with additional therapeutic agents.

Description

3-HYDROXYANTHRANILIC ACID OR SALTS THERE0F1 FOR TREATING CANCER OR INFECTIONS

This invention relates to a novel inhibitor of indoleamine 2,3-dioxygenase (IDO) and its use in the treatment of cancer or infections, either alone or in combination with additional therapeutic agents.

Acquired tolerance of tumours is undesirable and harmful to the host, but acquired tolerance of other classes of antigens (such as fetal antigens and harmless foreign antigens at mucosal surfaces) is beneficial and necessary. IDO has been implicated as a normal, endogenous mechanism of peripheral tolerance and immunosuppression in a number of settings. It was originally described as contributing to maternal tolerance toward the fetus, as shown by the fact that mice treated early in pregnancy with 1- methyl-tryptophan (IMT), which is an inhibitor of IDO, underwent immune mediated rejection of allogeneic concepti. The fetus represents an example of a set of foreign antigens to which the immune system is forced to remain tolerant and therefore is conceptually analogous to tumours in this regard. More generally, it has now been observed that mice treated with IDO inhibitors become refractory to acquired tolerance induction in a number of settings. For example, blocking IDO with IMT prevents the induction of tolerance to islet cell allografts by the fusion protein cytotoxic T lymphocyte-associated antigen 4— Ig (CTLA4-Ig), and IMT blocks the tolerance that normally occurs when foreign antigens are introduced into the anterior chamber of the eye (an immunologically privileged site). In settings where self tolerance has already been disrupted (for example, autoimmune disorders), pharmacologic inhibition of IDO causes marked exacerbation of inflammation and worsened symptoms of disease, as shown in models as diverse as inflammatory bowel disease, EAE, and experimental allergic asthma. Conversely, ectopic overexpression of IDO by gene transfer results in suppression of immune responses. For example, MHC mismatched lung allografts transfected with IDO are protected from rejection without further immunosuppression, and similar results have been reported in corneal transplants. Therefore, in vivo, IDO functions as a molecular mechanism contributing to acquired peripheral tolerance.

However, IDO does not seem to be required for the constitutive maintenance of tolerance to self. This is shown by the fact that mice genetically modified to lack IDO (Ido-/- mice) do not develop lethal autoimmune or lymphoproliferative disorders and mice treated systemically for up to 28 days with pharmacologic IDO inhibitors have not been observed to develop spontaneous autoimmunity. Therefore, in certain settings, IDO can be very important for tolerance, but the effects of IDO are selective and are narrowly focused on specific forms of acquired peripheral tolerance. This specificity is potentially an advantage when contemplating the clinical use of pharmacologic IDO inhibitors since these would not be predicted to have severe spontaneous autoimmunity as a limiting side effect.

We have now found a novel inhibitor of IDO.

According to the invention, we provide a method of treating a subject with a cancer or an infection, the method comprising administering to the subject a therapeutically effective amount of 3 hydroxyanthranilic acid or a pharmaceutically acceptable salt thereof.

The method may comprise at least one additional therapeutic agent.

The at least one additional therapeutic agent may be an antineoplastic chemotherapy agent. Suitable antineoplastic chemotherapeutic agent is selected from the group consisting of cyclophosphamide, methotrexate, fiuorouracil, doxorubicin, vincristine, ifosfamide, cisplatin, gemcytabine, busulfan, ara-C, and combinations thereof.

Alternatively, the at least one additional therapeutic agent may be radiation therapy. The radiation therapy may be localized radiation therapy delivered to the tumour or may be total body irradiation.

The cancer according to the invention may be selected from the group consisting of melanoma, colon cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer, leukemia, brain tumours, lymphoma, sarcoma, ovarian cancer, and Kaposi's sarcoma.

The method be also further comprise bone marrow transplantation or peripheral blood stem cell transplantation. Where the method relates to the treatment of an infection, the infection may be selected from the group consisting of a viral infection, infection with an intracellular parasite, and infection with an intracellular bacteria.

Particular viral infections include human immunodeficiency virus or cytomegalovirus.

Particular intracellular parasite infections may be selected from the group consisting of Leishmania donovani, Leishmania tropica, Leishmania major, Leishmania aethiopica, Leishmania mexicana, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae.

Particular intracellular bacterial infections may be selected from the group consisting of Mycobacterium leprae, Mycobacterium tuberculosis, Listeria monocytogenes, and Toxplasma gondii.

In the method, the at least one additional therapeutic agent may be a vaccine, for example, an anti-viral vaccine, a vaccine against HIV, a vaccine against tuberculosis, a vaccine against malaria. The vaccine may also be a tumour vaccine or a melanoma vaccine. Preferably, the tumour vaccine comprises genetically modified tumour cells or genetically modified cell lines. In such cases, preferably the genetically modified tumour cells or genetically modified cell line has been transfected to express granulocyte-macrophage stimulating factor (GM-CSF).

Alternatively, the vaccine may comprise one or more immunogenic peptides.

Further, the tumour vaccine may comprise dendritic cells.

Further, the additional therapeutic agent may be a cytokine, for example a granulocyte - macrophage colony stimulating factor (GM-CSF) or flt3-ligand.

According to the invention we further provide a method of treating a subject receiving a bone marrow transplant or peripheral blood stem cell transplant comprising administering a therapeutically effective amount of 3 -hydroxy anthranilic acid or a pharmaceutically acceptable salt thereof to such a subject.

Preferably, the 3 -hydroxy anthranilic acid or a pharmaceutically acceptable salt thereof is administered in an amount effective to increase the delayed type hypersensitivity reaction to tumour antigen, delay the time to relapse of post-transplant malignancy, increase relapse free survival time post-transplant, and/or increase long-term post- transplant survival.

Preferably, the 3 -hydroxy anthranilic acid or a pharmaceutically acceptable salt thereof is administered prior to full hematopoetic reconstitution.

Salts of 3-hydroxyanthranilic acid may be formed by reacting the free acid, or a salt thereof, with one or more equivalents of the appropriate base. The reaction may be carried out in a solvent or medium in which the salt is insoluble or in a solvent in which the salt is soluble, e.g. ethanol, tetrahydrofuran or diethyl ether, which may be removed in vacuo, or by freeze drying. The reaction may also be a metathetical process or it may be carried out on an ion exchange resin.

Pharmaceutically acceptable salts of 3-hydroxyanthranilic acid alkali metal salts, e.g. sodium and potassium salts; alkaline earth metal salts, e.g. calcium and magnesium salts; salts of the Group III elements, e.g. aluminium salts; and ammonium salts. Salts with suitable organic bases, for example, salts with hydroxylamine; lower alkylamines, e.g. methylamine or ethylamine; with substituted lower alkylamines, e.g. hydroxy substituted alkylamines; or with monocyclic nitrogen heterocyclic compounds, e.g. piperidine or morpholine; and salts with amino acids, e.g. with arginine, lysine etc, or an N-alkyl derivative thereof; or with an aminosugar, e.g. N-methyl-D-glucamine or glucosamine. The non-toxic physiologically acceptable salts are preferred, although other salts are also useful, e.g. in isolating or purifying the product.

3-hydroxyanthranilic acid or pharmaceutically acceptable salt thereof for use in the method will generally be administered in the form of a pharmaceutical composition. Thus, according to a further aspect of the invention there is provided a pharmaceutical composition including preferably less than 80% w/w, more preferably less than 50% w/w, e.g. 0.1 to 20%, of 3 -hydroxy anthranilic acid or a pharmaceutically acceptable salt thereof, in admixture with a pharmaceutically acceptable diluent or carrier.

We also provide a process for the production of such a pharmaceutical composition which comprises mixing the ingredients. Examples of pharmaceutical formulations which may be used, and suitable diluents or carriers, are as follows: for intravenous injection or infusion - purified water or saline solution; for inhalation compositions - coarse lactose; for tablets, capsules and dragees - microcrystalline cellulose, calcium phosphate, diatomaceous earth, a sugar such as lactose, dextrose or mannitol, talc, stearic acid, starch, sodium bicarbonate and/or gelatin; for suppositories - natural or hardened oils or waxes.

When the 3 -hydroxy anthranilic acid or a pharmaceutically acceptable salt thereof is to be used in aqueous solution, e.g. for infusion, it may be necessary to incorporate other excipients. In particular there may be mentioned chelating or sequestering agents, antioxidants, tonicity adjusting agents, pH-modifying agents and buffering agents.

Solutions containing 3 -hydroxy anthranilic acid or a pharmaceutically acceptable salt thereof may, if desired, be evaporated, e.g. by freeze drying or spray drying, to give a solid composition, which may be reconstituted prior to use.

When not in solution, the 3 -hydroxy anthranilic acid or a pharmaceutically acceptable salt thereof preferably is in a form having a mass median diameter of from 0.01 to lOμm. The compositions may also contain suitable preserving, stabilising and wetting agents, solubilisers, e.g. a water-soluble cellulose polymer such as hydroxypropyl methylcellulose, or a water-soluble glycol such as propylene glycol, sweetening and colouring agents and flavourings. Where appropriate, the compositions may be formulated in sustained release form.

The content of 3 -hydroxy anthranilic acid or a pharmaceutically acceptable salt thereof in a pharmaceutical composition is generally about 0.01-about 99.9wt%, preferably about 0.1 -about 50wt%, relative to the entire preparation.

The dose of 3-hydroxyanthranilic acid or a pharmaceutically acceptable salt thereof is determined in consideration of age, body weight, general health condition, diet, administration time, administration method, clearance rate, combination of drugs, the level of disease for which the patient is under treatment then, and other factors.

While the dose varies depending on the target disease, condition, subject of administration, administration method and the like, for oral administration as a therapeutic agent for the treatment of cancer in a patient suffering from such a disease is from 0.01 mg - 1O g, preferably 0.1 - 100 mg, is preferably administered in a single dose or in 2 or 3 portions per day. Where 3-hydroxyanthranilic acid or a pharmaceutically acceptable salt thereof is used in combination with other therapeutic agents, these may be used at their normal therapeutic doses, e.g., as set out in pharmacopoeias or prescribing guides, such as the Physicians' Desk Reference (PDR).

In certain cases, 3-hydroxyanthranilic acid or a pharmaceutically acceptable salt thereof supplements the activity of the additional therapeutic agent(s) in a synergistic fashion, such that the additional therapeutic agent(s) can be administered at a lower dose than is normally used.

The potential activity of 3-hydroxyanthranilic acid or a pharmaceutically acceptable salt thereof in the treatment of cancer or infections has been demonstrated in the following predictive experiments, which demonstrate that 3-hydroxyanthranilic acid is an IDO inhibitor. Brief Description of the Drawings

Figure 1: Fast Protein liquid chromatography profile of recombinant human IDO protein IDO- pET21 a expression vector was used to transform E CoIi strains (BL21 and HMS 174) . These were cultured to appropriate density at 370C. Protein expression was then induced using IPTG in the presence of the heme precursor at 300C. The bacterial lysate was passed through a His -Trap Column and the purified protein ran through an FPLC column. The pure IDO protein is eluted after 150ml. Figure 2: Recombinant human IDO protein is enzymatically active; A. Western blotting for human recombinant IDO protein purified from E CoIi BL21 and E CoIi HMS 174. Various amounts of the protein (0, 2.5, 5, 10 ug) were stained using rabbit anti human IDO antibody (Chemicon), as a primary antibody, then with anti-rabbit HRP antibody. B: IDO enzymatic activity assay. Various amounts of recombinant human IDO protein were added to tryptophan-containing incubation medium. The concentration of kynurenine after one hour was measured by HPLC. Figure 3: Comparison between IMT isomers using human recombinant IDO protein. A: Effect of DMSO on activity of human recombinant IDO protein. IDO enzymatic activity was measured in the presence of various percentages of DMSO in the assay. B: Comparison between IMT isomers. Activity of human recombinant IDO protein was measured at various substrate concentrations in the presence of 0.8mM of IMT(D), IMT(L) or IMT(DL). Kynurenine production was measured by HPLC.

Figure 4: Screening of several compounds using recombinant human IDO protein. IDO enzymatic activity was measured at 37C for 1 hour in the presence of 0.4mM of tryptophan and various concentrations of test compounds. IMT(L) was used as a positive control for IDO inhibition. Compound C3 displayed very high efficacy of inhibition. Figure 5: Screening of several compounds for inhibition of tryptophan uptake by human THP-I cells. A: Effect of DMSO on the rate of tryptophan uptake in THP-I cells. The rate of 3H-L-tryptophan uptake by THP-I cells was measured in the presence of various concentrations of DMSO. In subsequent uptake assays, DMSO was used to dissolve inhibitory compounds. The final concentration of DMSO was typically 5%. B: Tryptophan uptake by THP-I cells in the presence of various test compounds. The rate of 3H-L-tryptophan uptake by THP-I cells was measured in the presence of ImM of unlabeled tryptophan, IMT (L) or other test compounds. Compounds Cl, C4, C6, C7 and ClO could not be tested because of their toxicity to cells.

Figure 6: Compound C3 is a competitive inhibitorof IDO. Activity of human recombinant IDO protein was measured at various concentrations of IMT (L), compound C3 and compound C2. Compound C2 was selected as a negative control since it did not display any inhibition in previous screening (figure 4). This assays was conducted in competition with 33uM tryptophan (A), lOOuM tryptophan (B) and 30OuM tryptophan (C). Kynurenine production was measured by HPLC. Figure 7: Compound C3 also inhibits cellular IDO activity. A: Inhibition of IDO activity in culture of IDO-lentivirus transduced THP-I cells. These cells were cultured RPMI medium for 48 hours in the presence of 0.5mM of IMT(L) or compound C3. Supernatants were harvested and concentrations of tryptophan and kynurenine measured by HPLC. B: Inhibition of IDO activity in lysates of IDO-lentivirus transduced THP-I cells. Cells were lysed and lysates added to tryptophan-containing medium containing no inhibitor or 0.4mM of IMT(L) or compound C3. The final concentration of tryptophan in the assay was also 0.4mM. Figure 8: Compound C3 is not a generic inhibitor of heme-containing enzymes. Human recombinant cytochrome p450 enzyme was added at 0.25uM to NADPH-containing solution in the presence or absence of 20OuM Compound C3, then the level of NADPH usage measured over time. Compound C3 did not reduce NADPH usage by cytochrome p450.

Figure 9: The kynurenine pathway. Compound C3 is 3 Hydroxy-enthranillic acid (3HAA) is downstream of IDO in the kynurenine pathway. Other metabolites in the kynurenine pathway with similar chemical structures include kynurenic acid (KA), 3 hydroxykynurenine (3HK) and quinolininc acid (QA). Figure 10: Inhibition of IDO activity by metabolites of the kynurenine pathway. A: Inhibition of recombinant human IDO activity by kynurenine metabolites. Activity of recombinant human IDO was measured in the presence of 0.4mM tryptophan and various concentrations of IMT (L), 3HAA, QA, KA and 3HK. Kynurenine production was measured by HPLC. B: Inhibition of IDO activity in lysates of IDO-lentivirus transduced THP-I cells. Cells lysed and lysates added to tryptophan containing incubation medium in the presence of various concentrations of IMT (L), 3HAA, QA, KA and 3HK. The final concentration of tryptophan in the assays was 0.4mM. Kynurenine production was measured by HPLC. Figure 11: Inhibition of IDO activity in culture and of tryptophan uptake. A: Inhibition of IDO enzymatic activity in IDO-lentivirus transduced THP-I cells. These cells were cultured in RPMI medium in the presence of various concentrations of IMT(L), 3HAA, QA and KA. 3HK was not used in this assay because of its toxicity to cells in culture. Kynurenine was measured in the supernatants after 48 hours by HPLC. B: Inhibition of tryptophan uptake by kynurenine metabolites. 3H-L-tryptophan uptake by THP-I cells was measured in the presence of each compound. Recombinant human IDO protein:

The Pet 21a expression vector was used for human His-tagged IDO expression in BL21 and HMS 174 strains of E CoIi. After an initial culture at 37⁰C, protein expression was induced at 3O⁰C, in the presence of a heme precursor. This is to enhance cytosolic expression of the IDO protein and minimize the amount of protein in inclusion bodies. This allows the purification of an already folded protein, with a heme group at its active site.

Figure IA is a Fast Protein liquid chromatography profile of the protein after purification thorough a His- Trap column. The protein was eluted at about 150 minutes, and the amount of protein purified from the initial 6 L bacterial culture was estimated at 5 mg.

Several fractions were eluted between 140 min and 180 min. Aliquots of fractions 8 to 12 were loaded separately, or after pooling, onto 4% acrylamide gel together with bacterial protein suspension that had not been ran through a His trap column (Pre- column). The stained protein gel, showed that fractions 8 to 12 as well as the pooled fraction contained a single band with an approximate size of 45KDa, similar to known molecular weight of human IDO protein (Figure IB).

To ascertain the identity of this purified protein, western blotting with a rabbit polyclonal anti-human antibody (Chemicon) was used. First, the purified protein was concentrated from FPLC fractions to lmg/ml using a Qiagen spin column. Various amounts of this concentrates were then loaded and tested by WB. This showed positive and dose-dependent staining of these samples with IDO-specific antibody. This confirmed the identity of the purified protein (Figure 2A).

Next, the purified IDO protein was examined for enzymatical activity. The use of lysate-based assay to test for the activity in protein samples was adapted. A similar incubation medium (containing tryptophan, catalase, methylene blue and ascorbic acid) was added to 10 fold dilutions of the purified proteins. The final amounts of protein in the reaction ranged between Ing and lμg. Kynurenine production in the reaction was measured by HPLC . This showed a positive and linear relationship between the amount of IDO protein in the reaction and the level of kynurenine production, confirming that the purified recombinant IDO protein is enzymatically active (Figure 2B). From these data, the specific activity of the purified enzyme was calculated at 120 μmole kynurenine/mg protein/ hour. This specific activity is within the range published by other investigators

Use of recombinant human IDO protein in inhibition assays:

Next, the use of IDO-protein based assay was extended to: a) confirm differences between IMT isomers, and b)screen for new inhibitors. IMT isomers and many of the compounds in to be screened were of poor solubility, so DMSO was used to dissolve these compounds before they were added in the IDO protein assay. However, the final concentration of DMSO that can be used to dissolve test compounds without directly reducing IDO specific activity was determined. IDO specific activity (lOOng or lμg of IDO protein) was measured in the presence of different percentages of DMSO in the assay (Figure 3A). From this, it was determined that 2.5% DMSO did not affect IDO specific activity. Therefore, in all subsequent IDO protein assays where DMSO was used to dissolve inhibitors, its final concentration did not exceed 2.5%.

To confirm previously observed data, activity of recombinant IDO protein at various substrate concentrations in the presence of 0.8mM of IMT(D), L or DL (Figure 3B) was measured. In the absence of any inhibitor, IDO specific activity increased with increased substrate concentration and plateaus at 70 μmole/mg protein/hour at O. ImM of tryptophan, due to excess substrate concentrations and the amount of enzyme being rate-limiting. In the presence of IMT(L) or IMT(DL), the substrate-dependent increase in IDO activity was slower than in the absence of inhibitor and activity plateaus at 20 μumole/mg protein/hour (70% inhibition). Thus, IMT(DL) and IMT(L) showed similar inhibitory efficacy of the recombinant IDO protein activity. The D isomer also slowed the rate of IDO activity and reduced the plateau level by 25%.

The use of human IDO recombinant assay has allowed us to confirm observation obtained in cell culture and in lysate-based assay; the L isomer is a more efficacious inhibitor of IDO activity the the D isomer of IMT.

Screening of novel IDO inhibitors:

A mini library of compounds was screened. The chemical structures, names and molecular weights of these compounds are described in table I:

Thereafter, these compounds are referred to as Cl to ClO. IMT(L) was also used as a positive control for IDO inhibition. Again, these sparingly soluble compounds were initially dissolved in DMSO so that final DMSO concentration is the assay was 2.5%. Also, at various dilutions of these inhibitors, DMSO concentration was kept constant. Compound 4 could not be used in this assay since it could not be dissolved within the threshold concentration of DMSO.

Compounds C2, C5, C7 and C9 showed little or no inhibition of IDO activity even at excess concentrations of 5mM. Compounds Cl, C6, C8 and ClO showed moderate efficacy, with 50% inhibition of IDO activity reached in the millimolar range of these compounds. Expectedly, IMT(L) inhibited IDO, reaching 50% inhibition at 312μM.

Surprisingly, compound C3 (3- Hydroxyanthranilic acid, also referred to herein as 3-

HAA) was a very potent inhibitor of IDO enzymatic activity with 95% inhibition reached at the lowest assayed concentration of 79μM (Figure 4).

Testing for inhibition of tryptophan uptake:

Next, whether compounds tested above had any effect on tryptophan transport was examined. First, we established that 5% level of DMSO in the transport assay did not affect ³H-L-tryptophan uptake by THP-I cells. Thereafter, test compounds added in the transport assay were dissolved at this concentration of DMSO (Figure 5A).

Subsequently, we measured the rate of ³H-L-tryptophan uptake by THP-I cells in the presence of ImM of each compound in the library. Unlabeled tryptophan and IMT (L) were used as specific competitive inhibitors of tryptophan uptake. Compounds Cl, C4, C6, C7 and ClO were not tested since they induced cell lysis even at very low concentrations. As expected, unlabeled tryptophan and IMT(L) significantly reduced ³H-L-tryptophan uptake by THP-I cells by 70% and 63% respectively. Compounds C2, C5, C8 and C9 showed little or no inhibition of tryptophan uptake. Compound C3 showed a moderate 28% inhibition of ³H-L-tryptophan uptake (Figure 5B).

Relative to IMT(L), compound C3 showed four- fold higher inhibition of IDO activity and two-fold lower inhibition of tryptophan transport.

IDO inhibitory properties of Compound C3 (3-HAA):

The inhibitory properties of compound C3 was examined further by varying both inhibitor and substrate concentrations in IDO protein activity assay. In the same assay, IMT(L) and compound C2 were used as positive and negative controls for IDO inhibition respectively. Three- fold dilutions of the substrate were used, 33μM (Figure 6A), lOOμM (Figure 6B) and 300μM (Figure 6C). Inhibitor concentrations were at 0, 33, 100, and 300μM. In the absence of any inhibitor, IDO activity was dependent upon substrate concentration. Compound C2 showed no inhibition of IDO activity even at low substrate concentration and high inhibitor concentration. As expected, IMT(L) showed dose-dependent inhibition of IDO activity. The percentage of inhibition by IMT(L) was more significant at 33uM of tryptophan than at 300μM of tryptophan, due to the competitive nature of IMT(L) inhibitor. Compound C3 showed more significant inhibition of IDO activity that IMT(L) but following a similar pattern, indicating that it is also a competitive inhibitor of IDO activity. From these measurements, and using Michaelis-Menten equation, the inhibition constants for IMT (L) and compound C3 were calculated.

Compound C3 also inhibits cellular IDO activity:

It was also verified whether compound C3 also inhibited IDO activity in culture and in lysate assays. WT or IDO-lentivirus transduced THP-I cells were cultured alone or in the presence of IMT(L) or compound C3. In contrast to WT THP-I cells, IDO- lentivirus transduced THP-I cells produced kynurenine and consumed tryptophan accordingly. As expected, IMT(L) reduced kynurenine production by 45% and also decreased tryptophan consumption and by IDO-lentivirus transduced THP-I cells. Compound C3 reduced kynurenine production by 85% and also reduced tryptophan consumption by IDO-lentivirus transduced THP-I cells (Figure 7A). This result confirms that compound C3 is sufficiently taken up by cells in vitro to exert its inhibitory effect on human intracellular IDO.

Furthermore, the effect of adding compound C3 to lysates IDO-lentivirus transfected THP-I cells was examined. Again, compound C3 was able to inhibit IDO activity, and with higher efficacy than IMT(L). This experiment confirmed the potency of compound C3 at inhibiting activity IDO expressed in bacterial and mammalian systems (Figure 7B).

Compound C3 not a generic inhibitor of heme-containing enzymes: Further examination of the chemical structure of compound C3 raised the possibility that its inhibition of IDO activity could be the result of generic interaction with the heme group, at the active site of the IDO protein. Such interaction would reduce of abolish IDO enzymatic activity, since the heme group is crucial for its catalytic reaction. To examine this possibility, another heme-containing enzyme cytochrome P450 was used to verify whether its activity was also inhibited by compound C3.

In an assay to measure Cytochrome P450 activity, 200μM compound C3 was added and NADPH usage over time measured. Compound C3 did not inhibit Cytochrome P450 activity, indicating that it is not a generic inhibitor of heme-containing enzymes, and that its effect on IDO activity is specific (Figure 8).

Screening kynurenine pathway metabolites:

Experiments described above clearly demonstrate that compound C3 is a potent and specific inhibitor of IDO activity. Compound C3 is 3-hydroxy anthranillic acid (3HAA). Coincidently, 3HAA is the product of metabolism of 3-hydroxy kynurenine by the enzyme kynureninase, downstream of IDO in the kynurenine pathway (Figure 9). Therefore, whether other metabolites of kynurenine pathway, with similar structures to 3HAA were able to inhibit IDO activity was examined. Compounds tested were kynurenic acid (KA), 3 hydroxy kynurenine (3HK), quinolinic acid (QA) as well as 3HAA.

Using recombinant IDO protein and various concentrations of these compounds, we observed that, unlike 3HAA, none of the other metabolites of the kynurenine pathway inhibited IDO activity even at high concentrations (Figure 1OA). These data were confirmed using lysates of IDO-lentivirus transduced THP-I cells. IDO activity in these lysates was dose-dependently inhibited by 3HAA, and to a lesser extent by IMT(L), but none of the other kynurenine pathway metabolites showed any IDO inhibition (Figure lOB).

When added in culture with IDO-lentivirus transduced THP-I cells, we observed a similar pattern with regards to IMT(L) and 3HAA, the latter being more potent. 3HK could not be tested in culture, since it was toxic to cells even at the lowest concentrations. KA showed very moderate some inhibition of kynurenine production in culture. Surprisingly, QA showed significant inhibition of kynuenine production by IDO-lentivirus transduced THP-I, but this inhibition is most likely IDO-independent, since QA had no effect on IDO activity in recombinant protein and in lysate-based assays (Figure HA)

Next, the effect of these kynurenine pathway metabolites on ³H-L-tryptophan uptake by THP-I cells was examined. Again 3HK could not be tested in this assay because of its toxicity. Expectedly, IMT(L) and more moderately 3HAA inhibited ³H-L-tryptophan uptake by THP-I cells. KA did not inhibit tryptophan uptake, suggesting that the moderate IDO inhibition observed in figure HA was independent of tryptophan transport. QA in contrast, showed 50% inhibition of ³H-L-tryptophan uptake by THP-I cells suggesting that its inhibition of kynurenine production in IDO-lentivirus transduced THP-I supernatants was dependent on inhibition of tryptophan uptake (Figure HB). Discussion:

The screen described above takes into consideration potency of the test compound at the active site of the target protein, its uptake by cells and whether it interferes with other tryptophan cellular pathways such as tryptophan uptake. Potency at the active site of protein using a recombinant human IDO protein was measured. This demonstrated that this protein was catalytically active and could be used for the screening of inhibitors. It confirmed previous observations with IMT isomers in cell culture and using cell lysate. This also allowed fast-streaming of inhibitor screening by testing many compounds at various concentrations in the same experiment.

Using this system, it was possible to screen a mini-library of compounds and to demonstrate that compound 3 Hydroxy anthranillic Acid (3HAA): a) strongly inhibits IDO catalytic activity in protein-based assay; b) is sufficiently transported across the cell membrane to exert its effect on IDO in cultured cells; c) only moderately inhibits tryptophan uptake.

3HAA is one of the metabolites downstream of IDO in the kynurenine pathway. We have demonstrated that other metabolites in this pathway did not inhibit IDO catalytic activity. However, a number of studies support the role of kynurenine pathway metabolites in IDO-dependent immuno-suppression (Okuda et al 1998, Fallarino et al

2003). For instrance, Fallarino and coworkers demonstrated that 3HAA and QA induced apoptosis of murine thymocytes in a manner that was dependent upon casapase-8 activation and Cytochrome C release from the mithochondria. However, in our settings, 3HAA did not reduce cell viability when added to cultured human and murine cells and IDO was inhibited by 3HAA at far lower concentrations than those reported as toxic by Fallarino and co-workers. Moreover, a number of studies reported that tryptophan supplementation to MLRs stimulated by IDO+ APCs was sufficient to restore T cell proliferation, despite the presence of IDO metabolites, including 3HAA, in the medium (Mellor 2003, Manpalat 2007). So, 3HAA is a promising compound with potential for use in tumour immunotherapy. Further studies, non-inventive studies are warranted to verify its toxicity, stability, bioavailability and efficacy in vivo.

It is surprising that one of the metabolites downstream of IDO has such a strong inhibitory effect on IDO. However, this potentially constitutes a physiologically relevant regulatory loop, whereby IDO activity is tightly regulated by its downstream metabolites. Since tryptophan is a scarce essential amino acid, its intracellular depletion by IDO may have detrimental effects on the IDO+cells, it is likely that the kynurenine pathway is regulated within these cells, and 3HAA may be the key regulatory element in the kynrenine pathway.

References

Mellor (2003) : J. Immunol 171(4) 1652 (2003)

Manlapat (2007) Eur. J. Immunol. 37(4) 1064 (2007)

Okuda (1998) : J. Neurochem. 70(1) 299-307 (1998)

Fallarino (2003) : Adv. Exp. Med. Biol 527, 183 (2003)

Claims

We claim:

1. A method of treating a subject with a cancer or an infection, the method comprising administering to the subject a therapeutically effective amount of 3 hydroxyanthranilic acid or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, comprising at least one additional therapeutic agent.

3. The method of claim 2, wherein at least one additional therapeutic agent is an antineoplastic chemotherapy agent.

4. The method of claim 3, wherein the antineoplastic chemo therapeutic agent is selected from the group consisting of cyclophosphamide, methotrexate, fluorouracil, doxorubicin, vincristine, ifosfamide, cisplatin, gemcytabine, busulfan, ara-C, and combinations thereof.

5. The method of claim 2, wherein the at least one additional therapeutic agent is radiation therapy.

6. The method of claim 5 wherein the radiation therapy is localized radiation therapy delivered to the tumour.

7. The method of claim 5 wherein the radiation therapy is total body irradiation.

8. The method of claim 1, wherein the cancer is selected from the group consisting of melanoma, colon cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer, leukemia, brain tumours, lymphoma, sarcoma, ovarian cancer, and Kaposi's sarcoma.

9. The method of claim 1, further comprising bone marrow transplantation or peripheral blood stem cell transplantation.

10. The method of claim 1, wherein the infection is selected from the group consisting of a viral infection, infection with an intracellular parasite, and infection with an intracellular bacteria.

11. The method of claim 10 wherein the viral infection is human immunodeficiency virus or cytomegalovirus.

12. The method of claim 10 wherein the intracellular parasite is selected from the group consisting of Leishmania donovani, Leishmania tropica, Leishmania major, Leishmania aethiopica, Leishmania mexicana, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae.

13. The method of claim 10 wherein the intracellular bacteria is selected from the group consisting of Mycobacterium leprae, Mycobacterium tuberculosis, Listeria monocytogenes, and Toxplasma gondii.

14. The method of claim 1 wherein at least one additional therapeutic agent is a vaccine.

15. The method of claim 14 wherein the vaccine is an anti- viral vaccine.

16. The method of claim 15 wherein the vaccine is against HIV.

17. The method of claim 14 wherein the vaccine is against tuberculosis.

18. The method of claim 14 wherein the vaccine is against malaria.

19. The method of claim 14 wherein the vaccine is a tumour vaccine.

20. The method of claim 19 wherein the tumour vaccine is a melanoma vaccine.

21. The method of claim 19 wherein the tumour vaccine comprises genetically modified tumour cells or genetically modified cell lines.

22. The method of claim 21 wherein the genetically modified tumour cells or genetically modified cell line have been transfected to express granulocyte-macrophage stimulating factor (GM-CSF).

23. The method of claim 14 wherein the vaccine comprises one or more immunogenic peptides.

24. The method of claim 19 wherein the tumour vaccine comprises dendritic cells.

25. The method of claim 2 wherein the additional therapeutic agent is a cytokine.

26. The method of claim 25 wherein the cytokine is granulocyte-macrophage colony stimulating factor (GM-CSF) or flt3-ligand.

27. A method of treating a subject receiving a bone marrow transplant or peripheral blood stem cell transplant comprising administering a therapeutically effective amount of 3 -hydroxy anthranilic acid or a pharmaceutically acceptable salt thereof to such a subject.

28. The method of claim 27 wherein the 3 -hydroxy anthranilic acid or a pharmaceutically acceptable salt thereof is administered in an amount effective to increase the delayed type hypersensitivity reaction to tumour antigen, delay the time to relapse of post-transplant malignancy, increase relapse free survival time post- transplant, and/or increase long-term post-transplant survival.

29. The method of 27 wherein the 3-hydroxyanthranilic acid or a pharmaceutically acceptable salt thereof is administered prior to full hematopoetic reconstitution.