US 20110318271A1 (19) United States (12) Patent Application Publication (10) Pub. N0.: US 2011/0318271 A1 Volke et a]. (43) Pub. Date: Dec. 29, 2011

(54) INHIBITORS FOR THE C07C 229/26 (2006.01) TREATMENT OF DEPRESSION A61K 49/00 (2006.01) (75) Inventors: Vallo Volke’ Taml (BE); Maarja (52) US. Cl...... 424/92, 562/561, 56521/i/553é15; Krass, Tartu (EE); Annika Volke, Tartu (EE); Eero Vasar, Tartu (EB) (57) ABSTRACT . The sub'ects of the current invention are com ounds, Which (73) Asslgnee: UENEIVERSITY 0F TARTU’ Tam exhibit eirginase inhibiting activity (including gi?uoromethy ( ) lornithine (DFMO) and L-norvaline, but not limited to them), _ and Which can be used as therapeutically active agents for the (21) Appl' NO" 13/141’992 treatment and prevention of depression and/or depression . related conditions. Other sub'ects of the resent invention are (22) PCT Flled: Dec‘ 29’ 2008 the use of said arginase inhiliiting comprounds as therapeuti cally active agents for the manufacture of pharmaceutical (86) PCT NO‘: PCT/EE2008/000027 compositions for human and veterinary application, pharma § 371 (0X1) ceutical composition comprising said arginase inhibiting (2) (4) Date; Se 14 2011 compound and a method for treatment and prevention of ’ ' P‘ ’ depression and/or depression-related conditions. Also, a _ _ _ _ method for identifying compounds suitable for use as thera Pubhcatlon Classl?catlon peutically active agents for treatment and/or prevention of (51) Int, Cl, depression and/or depression-related conditions is disclosed. A61K 31/197 (200601) The invention further comprises a kit for selecting com C12Q 1/34 (200601) pounds for treatment and/or prevention of depression and/or A611) 25/24 (200601) depression-related conditions. Patent Application Publication Dec. 29, 2011 Sheet 1 of2 US 2011/0318271 A1

Fig 1

250 001 250

200 'k 200 lmmobility(s) 150 T lmmobility(s) 150 100 H % i VI 100 50

Sal lmi 20 100 500mg/kg Sal DFMO 400 mg/kg L-norvaline Patent Application Publication Dec. 29, 2011 Sheet 2 of2 US 2011/0318271 A1

* p<0.01 vs. saline group # p<0.001 vs. L-norvaline 250 200- T lmmobility(s) 150— 100

50‘

c Sal L-ornit Sal L-Ornit Sal Sal L-norv L-norv US 2011/0318271A1 Dec. 29, 2011

ARGINASE INHIBITORS FOR THE 1-8 .) Thus, arginase inhibitors may increase the production of TREATMENT OF DEPRESSION NO. As NO synthase inhibitors possess antidepressant and anxiolytic like properties (Volke V, Wegener G, Bourin M, FIELD OF INVENTION Vasar E. Antidepressant- and anxiolytic-like effects of selec tive neuronal NOS inhibitor 1-(2-tri?uoromethylphenyl) [0001] The present invention relates to compositions useful imidaZole in mice. Behavioural Brain Research 2003; 140: in the therapy of depression and depression-related condi 141 -147), arginase inhibitors should have the opposite effect. tions. More particularly the invention relates to compounds, Surprisingly, as We demonstrate in the folloWing invention, Which exhibit arginase inhibiting activity. arginase inhibitors possess antidepressant properties. [0009] Hereby a neW potent and surprising effect of tWo BACKGROUND OF THE INVENTION structurally distinct arginase inhibitors DFMO and L-norva [0002] Arginase (EC Nr 3.5.3.1) is an enzyme, activity of line has been disclosed leading to the perspective to use Which results in converting the compounds With arginase inhibiting activity in the treatment [0003] L- into L- and urea, being an and/or prevention of depression and/or depression-related essential part of the urea cycle. In addition to being metabo conditions. liZed to L-ornithine, L-arginine is also a precursor of NO, a free radical molecule involved in a Wide range of biological BRIEF DESCRIPTION OF THE DRAWINGS processes. [0010] FIG. 1 Effect of imipramine (15 mg/kg), L-norval [0004] Several compounds useful for the treatment of a ine, and DFMO in the forced sWimming test (n:8 per group). variety of diseases and disorders have been also shoWn as Results are expressed as mean:S.E.M. *iP<0.01, * *iP<0. possessing arginase inhibiting activity. E.g. arginase inhibi 001, versus saline. tors N(omega)-hydroxy-L-arginine (NOHA), N'-hydroxy [0011] FIG. 2 Effect of L-ornithine (500 mg/kg), L-norva nor-L-arginine (nor-NOHA), 2 (S)-amino-6-boronohexanoic line (500 mg/kg), and their combination in the forced sWim acid (ABH), S-(+)-Amino-6-iodoacetamidohexanoic acid, ming test (n:10 per group). Results are expressed as 1116211118. S-(+)-Amino-5-iodoacetamidopentanoic acid, L-norvaline, E.M. *iP<0.01 vs. saline+saline group; #iP<0.001, vs. or L-HOArg have been shoWn as a possible means for treat Sal+L-norvaline group. ment of asthma, pulmonary hypertension and sickle cell dis ease (WO/2004/073623 Treatment of conditions associated DISCLOSURE OF THE INVENTION With decreased bioavailability, including elevated arginase conditions). [0012] It has noW been found that arginase inhibitors, e. g., [0005] L-norvaline is considered to be a potent arginase L-norvaline and alpha-DFMO are therapeutically promising inhibitor (Rognstad R. Sources of ammonia for urea synthesis as antidepressant agents. in isolated rat liver cells. Biochim Biophys Acla 1977; 496: [0013] The subjects of the current invention are com 249-254; Chang C I, Liao J C, Kuo L. Arginase modulates pounds, Which exhibit arginase inhibiting activity (including nitric oxide production in activated macrophages. Am J di?uoromethylomithine (DFMO) and L-norvaline, but not Physiol 1998; 274: H342-H348). Alpha-DFMO is Well limited to them), and Which therefore can be used as thera knoWn as an inhibitor of omithine decarboxylase, but is also peutically active agents for the treatment and prevention of a potent inhibitor of arginase (Selamnia M, Mayeur C, Robert depression and/ or depression-related conditions. V, Blachier F. Alpha-di?uoromethylomithine (DFMO) as a [0014] The compounds inhibiting arginase activity can be potent arginase activity inhibitor in human colon carcinoma di?uoromethylomithine (DFMO), L-norvaline, but not lim cells. Biochem Pharmacol 1998; 55: 1241-12453) ited to them. [0006] The use of alfa-DFMO (e?ornithine, sometimes [0015] Other subjects of the present invention are: the use called “e?ornithine”) is knoWn and has been described alone of said arginase inhibiting compounds as therapeutically or in combination With other compounds in US. Pat. No. active agents for the manufacture of pharmaceutical compo 6,573,290 for the treatment or prevention of cancer (DFMO sitions for human and veterinary application; and pharmaceu and celecoxib in combination for cancer chemoprevention tical compositions comprising said compounds and pharma and therapy), US. Pat. No. 6,258,845 (DFMO and sulindac ceutically acceptable carrier. Representatives of such carriers combination in cancer chemoprevention) and US. Pat. No. are generally knoWn in the human and veterinary pharmaceu 4,925,835 (AZiridinyl putrescine containing compositions tical ?eld. Examples of such carriers are starch, alginates, and theiruses in treating prostate cancer), US. Pat. No. 6,277, stearates, gelatin, lactose, microcrystalline cellulose, etc. The 411 (Pharmaceutical formulation containing DFMO for the pharmaceutical compositions may have any form suitable for treatment of cancer). its application, for instance they may be in the form of cap [0007] Norvaline is an analog of the branched chain amino sule, poWder, tablet, solution, suspension, lotion, etc. acid and is not present among the 20 common natural [0016] Thus, di?uoromethylomithine (DFMO), L-norval amino acid compounds of proteins. It has been used in various ine or other arginase inhibitors can be used as therapeutically combinations in research Work being included into peptides, active agents for manufacturing pharmaceutical composi as Well as in nutritional compositions, eg in WO/2008/ tions for human and veterinary application. The pharmaceu 067641 (Composition for improving blood How in Working tical compositions comprising di?uoromethylomithine muscles comprising L-arginine, Cralaegus extract and arti (DFMO), L-norvaline or other arginase inhibitors can be used choke ?avonoids). for treatment and/or prevention of depression and/or depres [0008] It has been demonstrated, that increased activity of sion-related conditions. arginase results in a diminished output of NO (Que L G, [0017] Also, a method for identifying compounds suitable George S E, Gotoh T, Mori M, HuangY C. Effects of arginase for use as therapeutically active agents for treatment and/or isoforms on NO production by nNOS. Nitric Oxide 2002; 6: prevention of depression and/or depression-related condi US 2011/0318271A1 Dec. 29, 2011

tions and/ or anxiety is disclosed, Which comprises determin Brain Research 2003; 140: 141 -147. In a separate experiment ing Whether the compound under investigation exhibits argi arginase product L-ornithine or saline Was injected 15 min nase inhibiting activity, and if the named compound exhibits prior to L-norvaline. arginase inhibiting activity, then the named compound is [0025] The Forced SWimming test Was performed as selected as candidate for a compound suitable for use as described by Porsolt et al Porsolt R D, Bertin A, Jalfre M. therapeutically active agent for treatment and/or prevention Behavioral despair in mice: a primary screening test for anti the named diseases and/ or conditions. depressants. Arch Int Pharmacodyn Ther 1977; 229: 327 [0018] Also included in the invention is a method for treat 336. ment and prevention of depression and/ or depression-related [0026] A glass cylinder With a diameter of 12 cm Was ?lled conditions, Which comprises administering to a mammal suf With 8 cm Water at 25° C. An animal Was put into the Water and fering from depression and/ or depression-related conditions its behaviour Was videotaped during 6 min. An observer blind or a mammal supposed to gain depression or depression to treatment protocol counted the immobility time during the related disorder a therapeutically effective amount of a com last 4 min of the 6 min test. pound Which exhibits arginase inhibiting activity. [0019] Moreover, a kit for selecting novel compounds for Results: treatment and/or prevention of depression and/or depression [0027] The immobility time Was 220 sec in control animals. related conditions is disclosed. This kind of kit comprises at L-norvaline given intraperitoneally (i.p.) in the doses of 20, least a means for determining a compound’s arginase inhib 100 and 500 mg/kg decreased the immobility time to 146, 107 iting activity by a method knoWn to the person skilled in the and 96 sec, respectively (FIG. 1). art. If arginase inhibiting activity is detected, then the com [0028] In a separate experiment DFMO (400 mg/kg, i.p.) pound is selected for a further screening of its properties as a decreased the immobility time from 219 sec to 191 sec. LoWer drug candidate for the treatment and/ or prevention of depres doses of DFMO (10-100 mg/kg, i.p.) Were ineffective. sion and/or depression-related conditions. [0029] Both L-norvaline and DFMO reduce the immobility time, thus exhibiting an antidepressant-like effect. DESCRIPTION OF EMBODIMENTS EXAMPLE 2 [0020] The antidepressant properties of arginase inhibiting Effect of L-ornithine, L-norvaline, and Their Combi agents L-norvaline and DFMO Were investigated by means of nation in Forced SWimming Test a set of behavioural tests in animals conventionally employed in pharmacology and generally considered predictive of anti [0030] In a separate experiment L-omithine (500 mg/kg) depressant activity in man (Porsolt R D, Bertin A, Jalfre M. did not in?uence the immobility time (saline 199 sec, L-or Behavioral despair in mice: a primary screening test for anti nithine 214 sec, FIG. 2). HoWever, pre-treatment With L-or depressants. Arch Int Pharmacodyn Ther 1977; 229: 327 nithine antagonised the effect of L-norvaline on immobility 336; CraWley J, GoodWin F K. L-norvaline, DFMO and L-or time (L-norvaline 127 sec, L-ornithine +L-norvaline 215 sec; nithine have been tested in the folloWing tests: Forced p<0.001 vs. L-norvaline group). SWimming Test in mice, (Example 1, Example 2), Locomotor [0031] Thus, tWo structurally distinct arginase inhibitors Activity Test (Example 3). induced antidepressant-like effect, indicating that both mol [0021] Locomotor activity of animals Was measured using ecules, as Well as arginase inhibitors in general, are of interest an automated system as described previously (VolkeV, Wege as therapeutic agents in the treatment and/or prevention of ner G, Bourin M, Vasar E. Antidepres sant- and anxiolytic-like depression and/ or depression-related conditions. effects of selective neuronal NOS inhibitor 1-(2-tri?uorom [0032] Moreover, the arginase product L-ornithine Was ethylphenyl)-imidaZole in mice. Behavioural Brain Research able to block the effect of L-norvaline, further supporting that 2003; 140: 141-147). the antidepressant effect of study compounds are depending on arginase inhibition. [0022] Data Were analyZed With one-Way or tWo-Way analysis of variance (ANOVA) When appropriate. Post hoc EXAMPLE 3 comparisons betWeen individual groups Were performed by NeWman-Keuls test. Effect of L-ornithine, L-norvaline, and Their Combi nation on locomotor Activity EXAMPLE 1 [0033] Locomotor activity Was measured using an auto mated system With six chambers (45><45><45 cm) made from Forced SWimming Test transparent acrylic (MOTI, Technical & Scienti?c Equipment GMBH, Germany; Volke V, Wegener G, Bourin M, Vasar E. [0023] Male C57BL/6/Bkl mice (Scanbur BK AB, SWe Antidepressant- and anxiolytic-like effects of selective neu den) Weighing 20-25 g Were used. Mice Were kept 10 per cage ronal NOS inhibitor 1-(2-tri?uoromethylphenyl)-imidaZole (21><37><15 cm) in an animal house at 200 C. in a 12 h light/ in mice. Behavioural Brain Research 2003; 140: 141-147). dark cycle (light on at 7.00 a.m.). Tap Water and food pellets The apparatus-naive mice Were put into the chamber, and Were available ad libitum. The animals Were kept for at least vertical and horiZontal activity Was counted during a 10 min. tWo Weeks in the animal colony before entering experiments. test period. [0024] The measurement of locomotor activity, and forced sWimming test Were carried out consecutively 45 and 55 min Results: after treatment With study compounds, according to Volke V, [0034] The study compounds either alone or in combina Wegener G, Bourin M, Vasar E. Antidepressant- and anxi tion did not change the ambulation of animals, excluding the olytic-like effects of selective neuronal NOS inhibitor 1-(2 possibility that the drug effects in the forced sWimming test tri?uoromethylphenyl)-imidaZole in mice. Behavioural Were of non-speci?c origin. US 2011/0318271A1 Dec. 29, 2011

[0035] The descriptions above lead to the conclusion that for a further screening of its properties as a drug candidate for compounds exposing arginase inhibiting activity can be used the treatment and/ or prevention of depression and/ or depres as pharmacological agents for treating and/or preventing of sion-related conditions. depression and/or depression-related conditions. Di?uorom 1. Compounds, Which exhibit arginase inhibiting activity ethylornithine (DFMO) has been shoWn as a candidate for the for use as therapeutically active agents for treatment and/or treatment and/or prevention of depression and/or depression prevention of depression and/or depression-related condi tions. related conditions. L-norvaline has been shoWn as a candidate 2. Compound according to claim 1, Which is di?uorom for the treatment and/ or prevention of depression and/ or ethylomithine (DFMO). depression-related conditions. Accordingly, L-norvaline, 3. Compound according to claim 1, Which is L-norvaline. DFMO and other compounds With arginase inhibiting activity 4. Use of a compound according to claim 1 as therapeuti can be considered as candidates for producing a human medi cally active agent for manufacturing a pharmaceutical com cine for treatment and/or prevention of depression and/or position for human application. depression-related conditions. Respectively, L-norvaline, 5. Use of a compound according to claim 1 as therapeuti DFMO and other compounds With arginase inhibiting activity cally active agent for manufacturing a pharmaceutical com can be considered as candidates for producing a veterinary position for veterinary application. medicine for treatment and/or prevention of depression and/ 6. A pharmaceutical composition for treatment and/or pre or depression-related conditions. vention of depression and/or depression-related conditions [0036] A method for treating or preventing depression and/ comprising a therapeutically active agent and a pharmaceu or depression-related conditions by administering a mammal tically acceptable carrier, characterized in that the therapeu a pharmaceutical composition comprising a compound, tically active agent is a compound according to claim 1. Which exhibits arginase inhibiting activity is hereby dis 7. Method for identifying compounds for use as therapeu closed. tically active agents for treatment and/or prevention of depression and/or depression-related conditions comprising [0037] It is noW obvious for one skilled in the art, that other a) determining Whether the compound under investigation compounds, Which exhibit arginase inhibiting activity, may exhibits arginase inhibiting activity, b) and if the named com also be useful for treatment and/or prevention of depression pound exhibits arginase inhibiting activity, then the named and/or depression-related conditions. Thus, a method for compound is identi?ed as a compound applicable for use as identifying a compound suitable for treatment and/ or preven therapeutically active agent for treatment and/or prevention tion of depression and/or depression-related conditions com prises determining Whether the compound under investiga of depression and/or depression-related conditions. tion exhibits arginase inhibiting activity, and if the named 8. Method for treating and/ or preventing of depression and/or depression-related conditions in a mammal in need of compound exhibits arginase inhibiting activity, then the such treatment and/or prevention, said method comprising named compound is selected as candidate for a compound administering to said mammal a therapeutically effective suitable for the treatment and/or prevention the named dis amount of a compound Which exhibits arginase inhibiting eases and/ or conditions. activity. [0038] One skilled in the art can also conclude, that a kit for 9. Method according to claim 8, Whereas said compound is selecting novel compounds for treatment and/ or prevention of di?uoromethylomithine (DFMO). depression and/or depression-related conditions comprises at 10. Method according to claim 8, Whereas said compound least a means for determining a compound’s arginase inhib is L-norvaline. iting activity by a method knoWn to a person skilled in the art. 11. A kit for identifying compounds for use as therapeuti This means involves a biochemical assay, Wherein the pro cally active agents for treatment and/or prevention of depres duction of sion and/or depression-related conditions, Which comprises [0039] L-ornithine or urea is measured, but is not limited to. at least a means for determining a compound’s arginase inhib The assay is performed in vitro on puri?ed enzyme arginase, iting activity. or in a cell culture or in another model system. If arginase inhibiting activity is detected, then the compound is selected