Hdac1/2 Activator for Promoting And/Or Accelerating Myelination And/Or Remyelination

(19) TZZ¥_ _T

(11) EP 3 412 294 A1

(12) EUROPEAN PATENT APPLICATION

(43) Date of publication: (51) Int Cl.: 12.12.2018 Bulletin 2018/50 A61K 31/52 (2006.01) G01N 33/00 (2006.01)

(21) Application number: 17174916.1

(22) Date of filing: 08.06.2017

(84) Designated Contracting States: (72) Inventor: JACOB, Claire AL AT BE BG CH CY CZ DE DK EE ES FI FR GB 4051 Basel (CH) GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR (74) Representative: Schneiter, Sorin et al Designated Extension States: Omnis IP SA BA ME Ch. de Champ-Colomb 7 B Designated Validation States: 1024 Ecublens (VD) (CH) MA MD Remarks: (71) Applicant: Universite De Fribourg Claim 16. is deemed to be abandoned due to 1700 Fribourg (CH) non-payment of the claims fee (Rule 45(3) EPC).

(54) HDAC1/2 ACTIVATOR FOR PROMOTING AND/OR ACCELERATING MYELINATION AND/OR REMYELINATION

(57) The present invention is based on the surprising tems. The present inventor found that HDAC2 finding of the property of promoting myelination of acti- deacetylates eEFlAl and thereby prevents the latter from vators of histone deacetylase (HDAC) 1 and 2. In partic- removing outside the nucleus key inducers of myelin ular, such activators have the capacity of accelerating genes transcription. The HDAC1/2 activators are useful and increasing remyelination after lesions to the myelin in the treatment of diseases associated with demyelina- of nerve cells of the peripheral and central nervous sys- tion, such as Multiple Sclerosis. EP 3 412 294 A1

Printed by Jouve, 75001 PARIS (FR) 1 EP 3 412 294 A1 2

Description Schwann cells convert into repair cells that foster axonal regrowth, and redifferentiate to rebuild myelin. The au- Technical Field thors report that short-term HDAC1/2 inhibitor treatment early after lesion acceleration functional recovery and [0001] The present invention relates to an histone5 enhances regeneration of the crushed nerve cells of the deacetylase 1 and/or 2 (HDAC1/2) activator for promot- PNS. ing myelination, for accelerating remyelination after le- [0007] More specifically, Brügger et al, 2016 (ref. 1) sions to nerve cells, for treating diseases associated with have shown that HDAC2 interacts with the transcription demyelination, to methods for screening agents useful factor Sox10 and recruits histone H3 lysine 9 (H3K9) in the treatment of such diseases, and to methods of 10 demethylases (HDMs) to form a multifunctional protein treatment. complex that de-represses the Sox10 target genes Oct6 and Krox20 to allow their subsequent activation at differ- Background Art and Problems Solved by the Inven- ent time points of the regeneration process after lesion. tion Inactivating this mechanism was shown to result in earlier 15 conversion into repair Schwann cells after lesion and [0002] Multiple Sclerosis (MS) is a demyelinating dis- faster axonal regeneration. ease in which the insulating sheaths of nerve cells in the [0008] It is an objective underlying the present inven- brain, optic nerves and/or spinal cord are damaged. This tion to further elucidate the role of HDAC2 in the regen- damage disrupts the ability of parts of the nervous system eration process after lesion. It is an objective of the to communicate, resulting in a range of signs and symp- 20 present invention to elucidate the mechanisms control- toms, including physical and mental problems. MS is the ling Schwann cell conversion into repair cells and dedif- most frequent degenerative disease of the central nerv- ferentiation after lesion. ous system (CNS), the onset of which is often between [0009] It is a further objective underlying the present 20 and 50 years old. While there are treatments allowing invention to provide novel therapeutic strategies for the alleviating the symptoms of the disease, there is pres- 25 treatment of diseases and neural damages associated ently no cure for MS. There are several factors reported with demyelination in both, the PNS and the CNS. In par- to trigger the onset, such as viral infection, food, genetics ticular, it is an objective of the invention to provide a treat- and environmental factors. ment of MS and other demyelinating diseases. It is also [0003] On the cellular level, MS starts with attacks af- an objective of the invention to provide compounds ca- fecting oligodendrocytes, the cells that produce the my- 30 pable of promoting, accelerating and/or increasing remy- elin sheaths of axons in the CNS. These attacks are elination. It is an objective to provide novel assays for caused, at least in part, by a person’s own immune sys- screening compounds suitable to prevent or treat demy- tem, which is why MS is believed to be an immune-me- elination and for promoting remyelination. diated disorder. Following an attack of the myelin [0010] US 6,277,402 discloses a method for treating sheaths, new oligodendrocytes are generally recruited 35 multiple sclerosis, the method comprising administering to the site of lesion, where they regenerate the myelin a histamine H2 agonist and a phosphodiesterase inhib- sheath in a process referred to as remyelination. Recur- itor. The present invention is not concerned with hista- rent attacks and regeneration result in the pattern of suc- mine H2 agonists and phosphodiesterase inhibitors. cessive relapsing and remitting phases typical for early [0011] US2003/0134865 discloses a method for stages. MS evolves later into a progressive neurodegen- 40 screening comprising the step of exposing a xanthine erative disease where neurons are lost. Indeed, in this compound to a histone deacetylase. According to this later stage, remyelination does no longer occur or is not reference, screened compounds are useful in treating effective, resulting in neuronal loss. conditions caused by or exhibiting abnormal cellular pro- [0004] MS is one of a series of demyelination disor- liferation or differentiation, such as cancer, or inflamma- ders. The latter can affect the CNS as well as the periph- 45 tion, in particular asthma or other inflammatory airway eral nervous system (PNS). For example, demyelination disease, such as COPD (Chronic obstructive pulmonary occurs after traumatic lesions of the CNS and the PNS. disease). The present invention is not concerned with [0005] The myelin sheath, which serves to increase these conditions. nerve conduction velocities, is deposited around axons by specialized cells in the central and peripheral nervous 50 Summary of the Invention systems of higher vertebrates. Indeed, myelin is synthe- sized by oligodendrocytes in the CNS (as already men- [0012] Remarkably, the present inventor provides ev- tioned above) and by Schwann cells in the PNS. idence for the biological activity of activators of HDAC1/2, [0006] Brügger et al, 2016 (ref. 1) have reported that, in particular HDAC2, with respect to the myelination and after sciatic nerve crush lesion, HDAC2 coordinates the 55 remyelination of nerve cells in both, the PNS and the action of other chromatin-remodelling enzymes to induce CNS. the upregulation of Oct6, a key transcription factor for the [0013] In an aspect, the invention provides an activator development of Schwann cells. After injury, mature of HDAC (histone deacetylase) 1 or 2 (increasing

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HDAC1/2 activity and/or expression) for increasing, ac- Brief Description of the Drawings celerating and/or promoting myelination. [0014] In an aspect, the invention provides an activator [0022] of HDAC (histone deacetylase) 1 or 2 (increasing HDAC1/2 activity and/or expression) for increasing, ac- 5 Figure 1A shows immunofluorescence of acetylated celerating and/or promoting remyelination. eEF1A (left images), acetylated eEF1A and S100 [0015] In an aspect, the invention provides an activator proteins (center images) and the combined fluores- of HDAC (histone deacetylase) 1 or 2 (increasing cence of DAPI, acetylated eEF1A and S100 proteins HDAC1/2 activity and/or expression) for increasing, ac- (right side images), in sciatic nerve preparations ob- celerating and/or promoting remyelination after lesions 10 tained from four-day old control mice (top row) and to myelin of nerve cells. HDAC1/2 conditional (Schwann cell-specific) knock- [0016] In an aspect, the invention provides an activator out mice (dKO). The bottom left image shows accu- of HDAC (histone deacetylase) 1 or 2 (increasing mulation of acetylated eEF1A in the knock-out mice, HDAC1/2 activity and/or expression) for treating and/or demonstrating that HDAC1/2 deacetylate eEF1A preventing a demyelinating disease. 15 during developmental myelination. [0017] In an aspect, the invention provides an activator of HDAC (histone deacetylase) 1 or 2 (increasing Figure 1B shows an IP (immunoprecipitation) of HDAC1/2 activity and/or expression) for treating and/or eEF1A1 and Western blot (WB) image obtained from preventing one or more condition selected from the group sciatic nerve lysates showing the accumulation of consisting of: (1) traumatic injury of the PNS, (2) multiple 20 acetylated eEF1A1 (Ace-eEF1A1) in the dKO mice sclerosis, (3) Charcot-Marie-Tooth disease, (4) mentioned with respect to Fig. 1A, compared to con- Waardenburg syndrome, (5) Guillain-Barré syndrome, trol. The IP confirms the result shown in Fig. 1A. (6) chronic inflammatory demyelination polyneuropathy, (7) demyelination due to aging, diabetes or due to toxic Figures 2A, 2B and 2C show WB images of Ace- agents, (8) demyelination and hypomyelination due to 25 eEFIA (presumably Ace-eEF1A1) following IP of other diseases, such as Acute disseminated encephalo- Sox10 or eEF1A1 (Fig. 2A) or JMJD2C (Fig. 2C), or myelitis, transverse myelitis, Leukodystrophy, Central of HDAC2 following IP of eEF1A1 (Fig. 2B), obtained pontine myelinolysis, Glioma, (9) schizophrenia, (10) de- from de-differentiated rat Schwann cells cultured in myelination after traumatic lesion of the CNS. the presence of the HDAC1/2 inhibitor Mocetinostat [0018] In an aspect, the invention provides a method 30 orits vehicle.Nuclear and cytoplasmic fractions were for screening for agents suitable in the treatment and/or separately analysed. Sox10, eEF1A1, GAPDH and prevention of a disease or condition associated with de- Lamin A/C WB (Fig. 2A) on cell lysates show the IP myelination, and/or for screening for agents suitable for inputs. GFP IP is used as a negative control. promoting myelination and/or for increasing and/or accelerating remyelination, the method comprising: 35 Figures 3A and 3B show WB images obtained from lysates of sciatic nerves after crush lesion (crush) - assessing the capacity of a candidate agent to in- and unlesioned contralateral samples (contra) taken crease the HDAC1/2 activity and/or expression, from mice treated with the HDAC2 activator theophylline or its vehicle, 14 (Fig. 3A) and 30 (Fig. 3B) wherein presence of capacity to increase HDAC1/2 ac- 40 days post lesion (dpl). The results show that treat- tivity and/or expression indicates that said candidate ment with the activator results in increased levels of agent is an agent suitable for the treatment of said dis- HDAC2 at 14dpl (Fig. 3A), and increased levels of eases and for promoting myelination. P0 (myelin protein zero), Sox10, Krox20, and [0019] In an aspect, the invention provides a method JMJD2C at 14 and/or 30dpl. for treating and/or preventing a disease or condition as- 45 sociated with demyelination, the method comprising the Figures 4A and 4B are electron microscope images step of administrating, to a subject in need thereof, an of cross-sections of injured sciatic nerves at 14 (Fig. activator of HDAC1 and/or HDAC2. 4A) and 30 (Fig. 4B) dpl, following administration of [0020] In an aspect, the invention provides a method the HDAC2 activator theophylline or its vehicle. for increasing, accelerating and/or promoting the remy- 50 When the activator was administered, the myelin elination of nerve cells, the method comprising the step sheaths (black rings) appeared thicker compared to of administrating,to a subject in need thereof,an activator images obtained from control samples. of HDAC1 and/or HDAC2. [0021] Further aspects and preferred embodiments of Figures5A and 5Bshow the g-ratiodetermined from the invention are defined herein below and in the append- 55 samples as described with respect to Figs. 4A and ed claims. Further features and advantages of the inven- 4B, at 14 and 30 dpl, respectively. When the tion will become apparent to the skilled person from the HDAC1/2 activator was administered, the g-ratio description of the preferred embodiments given below. was significantly lower, demonstrating increased

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myelin thickness. Detailed Description of the Preferred Embodiments

Figure 5C illustrates the parameters used for deter- [0024] The present invention relates to activators of mining the g-ratio mentioned with respect to Figs 5A HDAC (histone deacetylase) 1 or 2 of HDAC1/2 enzy- and 5B. 5 matic activity and/or HDAC1/2 expression and their med- ical uses, as well as methods of treatment and/or pre- Figures 6A and 6B show performance of mice at vention comprising administering the activators. The in- 14dpl and 30dpl, respectively, on a Rotarod, while vention also relates to methods of screening for candi- comparing mice treated with the HDAC2 activator or date agents that are suitable in the treatment and/or pre- the vehicle control treatment. At 14dpl, mice having 10 vention of a demyelinating condition. received the HDAC2 activator exhibit significantly [0025] Forthe purposeof the present invention, an "ac- higher motor function and coordination. At 30dpl, the tivator of HDAC1/2", an "activator of HDAC1/2 activity" vehicle group has recovered enough function to per- and/or an "HDAC1/2 activator" is an agent that increases form the test for the entire duration, and there is no the enzymatic activity of HDAC1 and/or HDAC2, and/or more difference detectable compared to mice treat- 15 which increases expression of HDAC1 and/or HDAC2, ed with the HDAC2 activator. in particular in cells selected from Schwann cells, other glia cells, in particular oligodendrocytes, and nerve cells. Figures 7A and 7B show performance of mice at Said enzymatic activity is preferably the deacetylase ac- 14dpl and 30dpl, respectively, in a toe pinch test. As tivity of HDAC1 and/or HDAC2. Allosteric modulators are in Figs 6A and 6B, mice treated with the HDAC220 also considered as an HDAC1/2 activator, in as far as activator are compared to mice having received the they contribute to increasing the activity and/or the ex- vehicle control treatment. At 14dpl and 30dpl, mice pression of HDAC1 and/or HDAC2. having received the HDAC2 activator show signifi- [0026] In an embodiment, the activator increases cantly higher sensory function recovery. HDAC1 and/or HDAC2 deacetylase activity and/or ex- 25 pression in vivo and/or in vitro. Deacetylase activity Figures 8A, 8B and 8C show immunofluorescence and/or expression in vitro includes activity and/or expres- images obtained 14dpl from samples of mice that sion determined in living cells, for example in primary have received a lesion in the dorsal funiculus of the Schwann cell cultures, or in cells in a sample taken from spinal cord. Figs 8A and 8B show the lesion site, an organism, for example taken from an organism before whereas Fig. 8C shows the unlesioned contralateral 30 determining said activity and/or expression. The site of the spinal cord. The effect of the HDAC2 ac- HDAC1/2 used in in vitro assays may or may not be re- tivator is compared to control vehicle. The images combinant. Deacetylase activity in vitro also includes ac- show higher amounts of HDAC2 (Fig. 8A), higher tivity determined in a sample containing an isolated Sox10 and higher myelin content (MBP, Fig. 8B) in polypeptide susceptible of exhibiting HDAC1 or HDAC2 the lesion site when the activator was administered. 35 deacetylase activity, for example a sample comprising No effect of the activator was observed in the unle- isolated and/or recombinant HDAC1/2. sioned contralateral site of the spinal cord (Fig. 8C). [0027] HDAC1 and HDAC2 are two different types of deacetylases of class I histone deacetylases. They have Figure 9 shows electron microscopy images of spi- partially the same and partially different substrates. It has nal cord samples as described for Figs 8A-8C. When 40 been observed that one of these HDACs can compen- an HDAC2 activator was administered, remyelina- sate for the loss of activity of the other. For example, in tion of the lesioned spinal cord takes place more rap- case of knock out of HDAC2, HDAC1 was observed to idly, as can be seen from the image on the bottom at least partially assume the function of HDAC2. There- right compared to bottom left (vehicle). fore, the two HDAC members are generally mentioned 45 together in the present specification. In a preferred em- Figure 10 shows immunofluorescence images ob- bodiment, said HDAC is HDAC2. In an embodiment, said tained from the spinal cord samples of mice treated activator is at least an activator of HDAC2, and optionally with the HDAC1/2 inhibitor Mocetinostat (bottom im- also an activator of HDAC1. In case of activators of only ages) or with vehicle control (top images). The image one of HDAC1 or HDAC2, activators of HDAC2 are pre- in the left column show that the inhibitor results in 50 ferred. In an embodiment, said activator is an activator accumulation of Ace-eEF1A in the nuclei. The posi- of HDAC2 or of both HDAC1 and HDAC2. Preferably, tion of the latter is revealed by DAPI staining (right said activator is not an activator of HDAC1 exclusively, images). without being an activator of HDAC2. [0028] HDAC1/2 activators have been reported in the [0023] Hereinafter, preferred embodiments of the de- 55 literature. In particular, WO2010/011318 A2, published vice of the invention are described, in order to illustrate on January 28, 2010 and filed on July 23, 2009 under the invention, without any intention to limit the scope of application number PCT/US2009/04267, discloses acti- the present invention. vators of HDAC1 and other HDACs. WO2010/011318 is

4 7 EP 3 412 294 A1 8 entirely and expressly incorporated herein by reference. DPCPX (8-Cyclopentyl-1,3-dipropylxanthine or PD- The HDAC activators disclosed starting on page 2, line 116,948), a salt of any one of the aforementioned, an 26 through page 10, line 4 are expressly incorporated alkylated form of any one of the aforementioned and com- herein by reference. Furthermore, flavonoids as dis- binations comprising two or more of the aforementioned closed on page 21, line 17 through page 21, line 26 are 5 compounds. Salts include pharmaceutically acceptable expressly incorporated herein by reference. Further salts. HDAC activators disclosed on page 21, line 27 through [0032] In an embodiment, the HDAC1/2 activator is se- page 48, line 5 are expressly incorporated herein by ref- lected from the group consisting of: a xanthene com- erence. The definitions of terms in these paragraphs giv- pound, xanthydrol, xanthone, and combinations of two en elsewhere in the specification of WO2010/011318 are 10 or more of the aforementioned. also expressly incorporated herein by reference. Such [0033] In an embodiment, the HDAC1/2 activator is definitions are given, for example, on page 50, line 6 theophylline or a pharmaceutically acceptable salt there- through page 63, line 22 and are expressly incorporated of. hereinby reference. HDAC1 activators disclosed in Table [0034] In an embodiment, the HDAC1/2 activator is for 5 (pages 114-115) of WO2010/011318 are also express- 15 treating and/or preventing a disease or condition asso- ly incorporated herein by reference. Furthermore, HDAC ciated with demyelination. Preferably, the HDAC1/2 ac- activators and embodiments as disclosed in the claims tivator is used for accelerating, increasing and/or promot- of WO2010/011318 and as defined in the specification ing myelination and in particular remyelination in such are expressly incorporated herein by reference. diseases and conditions. [0029] In an embodiment, the HDAC1/2 activator is se- 20 [0035] In an embodiment, the HDAC1/2 activator is for lected from the group consisting of: an alkaloid com- accelerating regeneration of nerve cells of the peripheral pound, an iron chelator, deferoxamine, a flavonoid, for nervous system (PNS) and/or of the central nervous sys- example ginkgetin K, a compound comprising a catechol tem (CNS). moity, Chembridge 5104434, sciadopilysin, tetrahydro- [0036] In an embodiment, the HDAC1/2 activator is for gamboic acid, TAM-11, gambogic acid, or a derivative 25 treatment, prophylaxis and/or for increasing remyelina- thereof, LY235959, CGS19755, SKF 97541, etidronic tion in the context of: acid, levonordefrin, methyldopa, ampicillin trihydrate, D- aspartic acid, gamma-D-glutamylaminomethylsulfonic (i) demyelination due to traumatic injury of the pe- acid, phenazopyridine hydrochloride, oxalamine citrate ripheral nervous system; salt, podophyllotoxin, (+-)-4-amino-3-(5-chloro-2-30 (ii) demyelination due to Multiple Sclerosis; thienyl)-butanoic acid, (RS)- (tetrazol-5-yl) glycine, or (iii) peripheralneuropathies where demyelinationoc- R(+)-SKF-81297. The structures of the aforementioned curs, such as subtypes of the Charcot-Marie Tooth compounds are disclosed in WO2010/011318, and are diseases where gene mutation leads to hypomyeli- incorporated herein by reference, in particular page 44, nation or demyelination, Waardenburg syndrome, line 4 through page 18, line 5. 35 Guillain-Barré syndrome, chronic inflammatory de- [0030] In an embodiment, the HDAC1/2 activator is an myelinating polyneuropathy or neurofibromatosis; alkaloid compound. Preferably, the HDAC1/2 activator (iv) demyelination due to aging, diabetes or toxic is a xanthine compound. In an embodiment, the agents; HDAC1/2 activator is a xanthine compound as defined (v) demyelination and/or hypomyelination due to oth- in US2003/0134865, which is entirely and expressly in- 40 er diseases of the CNS, such as Acute disseminated corporated herein by reference. In an embodiment, the encephalomyelitis, transverse myelitis, Leukodys- xanthine compound is xanthine, a derivative of xanthine trophy, Central pontine myelinolysis, Glioma, schiz- or a salt of any one of the aforementioned. In an embod- ophrenia; iment, xanthine compound is alkylated xanthine, prefer- (vi) demyelination after traumatic lesion of the CNS. ably a methylated xanthine (methylxanthine). Said45 alkylated xanthine may be mono-, di-, or trialkylated xan- [0037] In an embodiment, the HDAC1/2 activator of thine, for example. In an embodiment, said HDAC1/2 ac- the invention is provided, for promoting, increasing tivator is a dialkylated xanthine, for example a dimethyl- and/or accelerating remyelination of nerve cells of the xanthine. PNS after lesion, and which activator is administered at [0031] In an embodiment, the xanthine compound is 50 the earliest 5 days after occurrence of the lesion, prefer- selected from the group consisting of theophylline, caf- ably at the earliest 7 days after lesion. It has previously feine, acepifylline (etaphylline), bamifylline, bufylline, ca- been show that HDAC2 activity immediately after a lesion faminol, cafedrine, diprophylline, doxofylline, enprofyl- affecting nerve cells results in an upregulation of Oct6, line, etamiphylline, etofylline, proxyphylline, suxamid- which slows down the upregulation of cJun. cJun in turn ofylline, theobromine, paraxanthine, 8-chlorotheophyl- 55 induces the conversion of mature Schwann cells into re- line, 8-phenyltheophylline, IBMX (1-Methyl-3-(2-methyl- pair Schwann cells and thereby promotes axonal re- propyl)-7H-purine-2,6-dione), DMPX (3,7-dimethyl-1- growth. Therefore, in case of a lesion of peripheral propargylxanthine), CPX (8-Cyclopentyltheophylline), nerves, HDAC2 activity immediately after a lesion neg-

5 9 EP 3 412 294 A1 10 atively impacts axonal regrowth and should preferably HDAC1/2 activity and/or expression indicates that said be avoided. However, the present inventor has shown candidate agent is an agent suitable for the treatment of that after the conversion of mature Schwann cells into said diseases and for promoting myelination. repair Schwann cells, HDAC2 activity contributes to re- [0041] HDAC1/2 activity and/or expression may be demyelination. Therefore, the invention provides adminis- 5 termined in any suitable way. In vitro, the HDAC1/2 ac- tering an HDAC1/2 activator with some delay after dam- tivity is preferably determined by assessing the HDAC1/2 age to the PNS. This delay is preferably 5 days, more deacetylase activity with respect to a suitable substrate. preferably 7 days, and most preferably 8 or 9 days (days In an embodiment, the substrate comprises a marker post lesion, dpl). In accordance with an embodiment, the molecule, for example marker peptides, suitable for as- HDAC1/2 activator is administered during the period of 10 sessing, preferably quantifying, the consumption of the 5 to 25 dpl, preferably 6 to 20 dpl, more preferably 7 to substrate and/or the production of the product. The term 17 dpl and most preferably 9 to 15 dpl. "substrate" for the present invention encompasses [0038] In another embodiment, the HDAC1/2 activator dipeptides, tripeptides, oligopeptides and polypeptides of the invention is provided, for promoting, increasing as well as derivatives thereof. and/or accelerating remyelination of nerve cells of the 15 [0042] In an embodiment, said substrate is or compris- CNS after lesion or after a demyelinating event. In an es a Eukaryotic elongation factor (eEF) peptide, prefer- embodiment, the invention provides the HDAC1/2 acti- ably a eEF1A peptide, more preferably a eEF1A1 pep- vator for increasing and/or accelerating remyelination of tide. Said peptide may be comprised in the sample nat- nerve cells of the CNS after an attack to glia cells, in urally, for example expressed by cells contained in the particular oligodendrocytes. For treating any demyelinat- 20 sample, or may be added in the form of an isolated and/or ing condition or event occurring in the CNS, the HDAC1/2 recombinant polypeptide. Said eEF peptide is preferably activator is preferably administered rapidly and/or imme- of human or animal origin, preferably of vertebrate origin, diately after the demyelinating event. Furthermore, in most preferably said eEF peptide is a mammal eEF, such case of a demyelinating condition in the CNS, the as a human or rodent eEF. HDAC1/2 activator may be administered at time of diag- 25 [0043] Assessing or determining said HDAC1/2 activ- nosis or rapidly thereafter. In an embodiment, the ity preferably refers to one or more activities related to HDAC1/2 activator is administered within 30 days from the qualitative finding of presence or absence of an en- a demyelinating event in the CNS or said diagnosis, more zymatic activity as specified, the quantitative approxima- preferably within 15 days, even more preferably within 9 tionof such activity, forexample in terms ofrate of product days and most preferably within 7 days after the occur- 30 consumption, relative comparison of activities, and/or al- rence of a demyelinating event or after the diagnosis of so the quantitative measurement of such activity, for ex- a demyelinating event or condition. In other embodi- ample. ments, the HDAC1/2 activator is administered within 6 [0044] For example, suitable marking systems could days from a demyelinating event in the CNS or said di- be substrates in which deacetylation induces the loss of agnosis, more preferably within 4 days, even more pref- 35 fluorescence activated energy transfer (FRET) between erably within 2 days and most preferably within 1 day. suitable donor/acceptor pairs that would be added on [0039] The present invention also relates to methods suitable sites within the substrate which are preceding of screening. In an embodiment, the method of screening and following the cleavage site (for example the N- and comprises the step of assessing, in an in vitro sample C-terminus of a substrate peptide). This would allow to exposed to the candidate compound, whether HDAC1/2 40 assess a loss of FRET upon cleavage because of the activity and/or expression is increased compared from a resulting separation of acceptor and donor moieties. A sample which is devoid of said candidate compound. similar principle could be used to change (for example, Said in vitro sample may comprise cells or an isolated destroy or obtain) any physical, chemical, or biological and/or recombinant polypeptide exhibiting HDAC1/2 ac- (e.g. enzymatical)property depending on proximity of two tivity. Said polypeptide is preferably human or animal 45 parts for functionality. For example, it is possible for a HDAC1, or human or animal HDAC2, and/or said cells marker of HDAC1/2 activity to obtain measurable phys- preferably express human or animal HDAC1, or human ical, chemical or biological property, such as fluores- or animal HDAC2. In some embodiments, said polypep- cence, enzymatical or other biological activity through tide and/or said cells comprise HDAC1 or 2 from a ver- deacetylation of the substrate. tebrate animal, preferably from a mammal, such as a 50 [0045] In an embodiment, expression of HDAC1/2, rodent.Preferably, saidsample comprises cellsthat have preferably HDAC2 may be assessed by Western blot, been taken previously from a subject, preferably before preferably following immunoprecipitation with an anti- the step of assessing HDAC1/2 activity and/or expres- body capable of binding to the starting product or an end sion. product of HDAC1/2 activity (e.g. Acet-eEFlAl or [0040] In an embodiment, the method for screening 55 eEF1A1), or an antibody binding to another protein con- comprises the step of evaluating the capacity of a can- nected to said starting and/or end product of HDAC1/2 didate agent to increase the HDAC1/2 activity and/or ex- activity. pression, wherein presence of capacity to increase [0046] In an embodiment, HDAC1/2 activity is as-

6 11 EP 3 412 294 A1 12 sessed by assessing the quantity of the present of the of HDAC1/2 responsible for their function during the re- acetylated or deacetylated substrate of HDAC1/2. This myelination process, the inventor treated adult mice with may be made, for example, by immunofluorescence, for the HDAC1/2 inhibitor Mocetinostat or its vehicle imme- example by exposing the substrate to an antibody spe- diately after a sciatic nerve crush lesion and collected cific for either the acetylated or the deacetylated variant 5 sciatic nerves 24 h later. of the substrate. [0047] In an embodiment, the step of assessing the Materials and Methods capacity of a candidate agent to increase the HDAC1/2 activity and/or expression comprises: exposing a sample [0051] Sciatic nerve crush lesions were carried out in comprising a polypeptide exhibiting HDAC1/2 deacety- 10 adult mice (3-4 month old) with the following procedure: lase activity to a said candidate compound, and, deter- Isoflurane (3% for induction, 1.5-2% for narcosis during mining whether in said sample said HDAC1/2 deacety- operation) was used for anesthesia. For analgesia, 0.1 lase activity and/or expression is increased compared to mg/kg buprenorphine (Temgesic; Essex Chemie) was a comparative sample devoid of said candidate com- administered by i.p. injection 1 h before lesion and 12 pound. 15 hours after the operation. An incision was made at the [0048] The present invention comprises methods of height of the hip and the sciatic nerve was exposed on treatment and/or prophylaxis, said methods comprising one side. The nerve was crushed (5 x 10 sec with crush administering an HDAC1/2 activator, including com- forceps: Ref. FST 00632-11) and the wound was closed pound increasing the expression of HDAC1/2. In these using Histoacryl Tissue Glue (BBraun). After the opera- methods, the compound if preferably administered in a 20 tion, mice were wrapped in paper towels and placed on form as appropriate for the particular activator. Oral and a warming pad until recovery from anesthesia. The parenteral administration are preferred for administrating HDAC1/2 inhibitor Mocetinostat (Selleckchem) or its ve- the HDAC1/2 activator. For example, theophylline may hicle was injected in the pelvic cavity at 10 mg/kg after be administered orally or parenterally. Preferably, a ther- wound closure and mice were sacrificed 24 h later. apeutically and/or pharmaceutically effective amount of 25 [0052] For immunoprecipitation (IP), the injured sciatic the activator is administered. Preferably, the activator is nerve was collected from the lesion site to around 12 mm administered together with a pharmaceutically accepta- distal to the lesion site (region where the nerve splits into ble carrier. Preferably, the HDAC1/2 activator is admin- the three branches of tibial, sural and common peroneal istered in the form of a pharmaceutical composition. The nerves). After perineurium removal, sciatic nerves were invention also provides pharmaceutical compositions for 30 frozen in liquid nitrogen, pulverized with a chilled mortar treatment and/or prophylaxis of the diseases, conditions and pestle, lysed in radioIP assay (RIPA) buffer (10 mM and/or disorders disclosed in the present specification. Tris/HCl, pH 7.4, 150 mM NaCl, 50 mM NaF, 100 mM [0049] While certain of the preferred embodiments of NaVO4, 1 mM EDTA, 0.5% wt/vol sodium deoxycholate, the present invention have been described and specifi- and0.5% Nonidet P-40) for 15 minon ice, and centrifuged cally exemplified above, it is not intended that the inven- 35 to pellet debris. Supernatants were collected and pre- tion be limited to such embodiments. Various modifica- cleared for 1 h with 30m l protein A/G PLUS agarose tions may be made thereto without departing from the beads (Santa Cruz Biotechnology). One liliter mil of scope and spirit of the present invention, as set forth in cleared lysates was rotated overnight at 4° C with immu- the following claims. Herein below, examples of the in- noprecipitating antibodies: 2 mg of Acetyl-lysine (rabbit, vention are disclosed. These examples are for illustration 40 Abcam, cat. # ab21623) or GFP (rabbit, Abcam, cat. # only and are not intended to limit the scope of the present ab290) antibodies were used per nerve and 2 nerves invention. were used per IP. Rabbit anti-GFP antibody was used as negative control IP. Forty microliters of agarose beads Examples were added, and samples were rotated for 1h at 4°C. 45 Immunoprecipitates were pelleted, washed four times Example 1: Identification of the deacetylation target with RIPA buffer, and another four times with 50 mM Tris of HDAC1/2 buffer, pH 6.8. [0053] Proteins from these agarose pull-downs were [0050] It was previously found that the two highly ho- then in-solution digested with 100 ng sequencing grade mologous histone deacetylases HDAC1 and HDAC250 trypsin (Promega) for 6 hours at 37°C after the following (HDAC1/2) were robustly upregulated in Schwann cells treatment: the dry beads were suspended in 30 ml of 8 (myelinating glia of the peripheral nervous system) al- M urea in 50 mM Tris/HCl, pH 8.0, followed by reduction ready one day (for HDAC2) after a sciatic nerve crush of the proteins with 3 ml 0.1 M DTT for 30 min at 37°C lesion in adult mice (ref. 1). It was shown that ablation of and alkylation by addition of 3 ml 0.5 M iodoacetamide these two HDACs in adult Schwann cells impairs remy- 55 for 30 min at 37°C in the dark, and urea dilution to 2 M elination after a sciatic nerve crush lesion (ref. 1) and that by addition of 20 mM Tris/HCl pH 8.0 containing 2 mM this HDAC1/2-dependent mechanism occurs already at CaCl2. Digestion was stopped by adding 1/20 of volume one day post lesion (ref. 1). To identify the direct target of 20% (v/v) TFA. An aliquot of 5 ml of each digest was

7 13 EP 3 412 294 A1 14 analyzed by LC-MS/MS on an EASY-nLC1000 chroma- Materials and Methods tograph connected to a QExactive HF mass spectrome- ter (Thermo Fisher Scientific) using three replicate injec- [0057] To generate HDAC1/2 conditional knockout tions in case of the in-solution digests. Peptides were mice, mice homozygous for Hdac1 and Hdac2 floxed al- trapped on a Pepmap100 Trap C18 300m m x 5 mm 5 leles (ref. 3) were crossed with mice expressing Cre re- (Thermo Fisher Scientific) and separated by backflush combinase under control of the Dhh promoter (Dhh-Cre, onto the analytical column (C18 Aqua Magic, 3 mm, 100 ref. 4) to ablate HDAC1 and HDAC2 in Schwann cell Å, 75 mm 3 150 mm) with a 20 or 40 min gradient from precursors. Genotypes were determined by PCR on ge- 5% to 40% solution B (95% acetonitrile, 0.1% formic acid) nomic DNA. at a flow rate of 300 nl/min. Full MS (resolution 60000, 10 [0058] For immunofluorescence, P4 mouse pups were automatic gain control target of 1e6, maximum injection killed by decapitation and their sciatic nerves were fixed time of 50 ms) and top15 MS/MS (resolution 15000, tar- in situ with 4% paraformaldehyde (PFA) for 10 min, dis- get of 1e5, 110 ms) scans were recorded alternatively in sected, embedded in O.C.T. Compound (VWR chemi- the range of 400 to 1400 m/z, with an inclusion window cals), and frozen at -80°C. Sciatic nerve cryosections of 1.6 m/z, relative collision energy of 27, and dynamic 15 (5mm thick) were first incubated with acetone for 10 min exclusion for 20 s. at -20°C, washed in PBS/0.1% Tween 20, blocked for 30 [0054] Fragment spectra data was converted to mgf min at room temperature (RT) in blocking buffer (0.3% with ProteomeDiscoverer 2.0 and peptide identification Triton X-100/ 10% Goat serum/PBS), and incubated with made with EasyProt software, and processed with Max- primary antibodies overnight at 4°C in blocking buffer. Quant/Andromeda version 1.5.0.0 (MQ) searching20 Sections were then washed 3 times in blocking buffer against the forward and reversed UniprotKB SwissProt and secondary antibodies were incubated for 1 h at RT mouse protein database (Release 2014_01) with the fol- in the dark. Sections were washed again, incubated with lowing parameters: parent mass error tolerance of 10 DAPI for 5 min at RT, washed and mounted in Citifluor ppm, trypsin cleavage mode with 2 missed cleavages, (Agar Scientific). Primary antibodies: EEF1A-pan static carbamidomethylation on Cys, variable oxidation 25 (Acetyl-Lys41) (rabbit, 1:200, Assay Biotech, cat. # on Met and acetylation on Lys. Based on reversed data- D12106)and S100 beta antibody [SH-B1] (mouse, 1:300, base peptide spectrum matches, a 1% false discovery Gene Tex, cat. # GTX11178). Secondary antibodies rate (FDR) was set for acceptance of target database were from Jackson ImmunoResearch. Photos were ac- matches. quired using a Leica TCS SP-II confocal microscope. Z- 30 series projections are shown. Results [0059] For IP, the entire sciatic nerve of P4 mice was collected. Then followed the same procedure for IP as [0055] Mass spectrometry analyses on the nerves of described above in Example 1 was followed, with the mice treated with Mocetinostat revealed increased abun- following modifications: 2 mg of eEF1A1 antibody (rabbit, dance of Eukaryotic elongation factor (eEF)1A1 peptides 35 Abcam, cat. # ab157455) were used per nerve and one (and of other peptides such as histone peptides, which nerve per IP; after the fourth wash with RIPA buffer, pro- are known HDAC targets) compared to vehicle, after im- teins were eluted from the agarose beads with 15 ml 0.1% munoprecipitation with an anti-acetyl-lysine antibody, formic acid and neutralized with 1.5 M Tris, pH 8.0. Six suggesting that HDAC1/2 deacetylate eEF1A1 after le- microliters of Laemmli buffer were added and samples sion. 40 were boiled for 10 min. Analysis of acetylated eEF1A1 levels was then done by Western blot using EEF1A-pan Example 2: HDAC1/2 and eEF1A1 during develop- (Acetyl-Lys41) primary antibody at a 1:1000 dilution and ment a light chain-specific goat anti-rabbit-HRP (horse radish peroxidase) secondary antibody (Jackson ImmunoRe- [0056] In order to determine whether acetylated45 search). Detection was done with Immobilon Western eEF1A1 is also deacetylated by HDAC1/2 during devel- Chemiluminescent HRP Substrate (Millipore). opmental myelination, HDAC1/2 was specifically ablated in Schwann cells during embryonic development at the Results Schwann cell precursor stage by crossingHdac1 and Hdac2 floxed mice with Dhh-Cre mice until obtention of 50 [0060] The results of immunofluorescence and IP are Dhh-Cre+/-, HDAC1 flox/flox, HDAC2flox/flox, generating ho- shown in Figures 1A and 1B, respectively. In the column mozygous HDAC1/2 double knockout mice (dKO) in "Acetylated eEF1A" of Fig. 1A, the control does not show Schwann cells. Dhh-Cre-/-littermate mice were used as any color staining, whereas in the HDAC1/2 knock-out control (ctrl). It was previously shown that HDAC1/2 are mice the regular staining in green shows accumulation strongly upregulated after birth in mouse Schwann cells 55 of acetylated eEF1A (Ace-eEFIA). In the middle column when the myelination process starts (ref. 2). "Acetylated eEF1A/S100", both images show the red staining by S100 beta antibody, marking Schwann cells. In the right-side images, the staining by DAPI of DNA

8 15 EP 3 412 294 A1 16 shows, in addition to the red and green fluorescences cat. # ab157455), Sox10 (rabbit, DCS Innovative Diag- where present, in blue the nuclei in the sections. Fig. 1B nostik-Systeme, cat. # SI058C01), JMJD2C (rabbit, Ab- shows high amount of Ace-eEF1A1 in the sciatic nerves cam, cat. # ab85454) or GFP (rabbit, Abcam, cat. # of the knock-out mice at postnatal day (P)4. ab290) antibodies were used per IP. Rabbit anti-GFP [0061] Its is shown here by immunofluorescence and 5 antibody was used as negative control IP. Acetylation of IP using P4 sciatic nerves that acetylated eEF1A (pre- eEF1A1 and co-immunoprecipitation of acetylated sumably eEF1A1) levels were strongly increased in eEF1A with Sox10 or JMJD2C were detected using Schwann cells (S100-positive) of dKO mice compared EEF1A-pan (Acetyl-Lys41) primary antibody (rabbit, As- to Ctrl mice, indicating that HDAC1/2 deacetylate eEF1A say Biotech, 1:1000, cat. # D12106). Co-immunoprecip- (presumably eEF1A1) during developmental myelination 10 itation of HDAC2 with eEF1A1 was detected using an in Schwann cells. HDAC2 antibody (mouse, Sigma, 1:1000, cat. # H2663). Light chain-specific goat anti-rabbit-HRP or anti-mouse- Example 3: IP of Sox10 and detection of eEF1A HRP secondary antibodies (Jackson ImmunoResearch) were used. Detection was done with Immobilon Western [0062] Sox10 is a transcription factor recruited to the 15 Chemiluminescent HRP Substrate (Millipore). Inputs of Oct6 SCE HR2 ( Oct6 enhancer) in Schwann cells follow- the IPs were assayed on lysates used for IPs. GAPDH ing lesion of peripheral nerves, thereby inducing the up- (mouse, Gene Tex, 1:5000, cat. # GTX28245) and Lam- regulation of the transcription factor Oct6. Oct6 upregu- inA/C (mouse, Sigma, 1:2000, cat. # SAB4200236) an- lation results in the slowing down of the conversion of tibodies were used to evaluate the efficiency of cytoplas- differentiated Schwann cells into repair Schwann cells 20 mic and nuclear fractionation. afterlesion and thereby slows down the process ofaxonal regrowth, but eventually induces remyelination through Results the upregulation of the transcription factor Krox20, a major inducer of myelination and remyelination. In addition, [0065] While eEF1A1 is mainly localized in the cyto- Sox10 also upregulates Krox20 expression by direct ac- 25 plasm, it was first found by immunofluorescence in puri- tivation of the Krox20 MSE, a critical enhancer of the fied rat Schwann cells cultured under de-differentiating Krox20 gene for its activation. The following experiments conditions in the presence of the HDAC1/2 inhibitor aim at determining the presence or absence of an inter- Mocetinostat that acetylated eEF1A is mainly localized action between Sox10 and eEF1A1. in the nucleus, although a fraction is also found in the 30 cytoplasm (data not shown). Here it is shown by subcel- Materials and Methods lular fractionation, immunoprecipitation and Western blot analyses that indeed inhibition of HDAC1/2 activity leads [0063] Purified primary rat Schwann cell cultures were to a strong increase of acetylated eEF1A1 levels, which obtained as described (ref. 5). Rat Schwann cells were is predominantly localized in the nucleus of these cells then grown in proliferation medium: DMEM containing 35 (Figure 2A, right panel). Under these conditions, it was 10% Fetal calf serum (FCS, Gibco), 1:500 penicil- also found that the major transcription factor of differen- lin/streptomycin (Invitrogen), 4 mg/ml crude GGF (bovine tiation and myelination Sox10 was partially re-localized pituitary extract, Bioconcept), and 2 mM forskolin (Sig- from the nuclear to the cytoplasmic compartment and ma), at 37°C and 5% CO2/95% air. Schwann cell de- that acetylated eEF1A (presumably eEF1A1) co-immu- differentiation culture protocol was previously described 40 noprecipitated with Sox10 in both compartments ( Figure (ref. 6). Briefly, Schwann cells were first growth-arrested 2A, left side panels). This interaction was potentiated by in defined medium (DM, ref. 5) for 8 to 15 h, then 1 mM HDAC1/2 inhibition, which also led to a strong decrease dbcAMP (Sigma) was added to induce differentiation. of Sox10 expression in the nuclear compartment, sug- Cells were incubated in this medium for another 3 days. gesting that acetylated eEF1A1 interacts with Sox10 to The medium was then changed to DM only without db- 45 drag it out of the nucleus. The results also show that cAMP, and incubated in this medium for 3 days (differ- HDAC2 co-immunoprecipitates with eEF1A1 in the nu- entiation mimicking adult Schwann cell stage). To induce cleus (Figure 2B), most likely to deacetylate it and there- de-differentiation, cells were then changed to prolifera- by send it back to the cytoplasm to protect Sox10 from tion medium and incubated in this medium for 3 days. cytoplasmic re-localization and likely from degradation. [0064] Two 15-cm culture plates of confluent de-differ- 50 [0066] Figure 2C shows that JMJD2C co-immunopre- entiated rat Schwann cells were used per IP. Cells were cipitates with acetylated eEF1A (presumably eEF1A1) in either treated with 0.6 mM Mocetinostat (HDAC1/2 inhib- presence of the HDAC1/2 inhibitor, also predominantly itor) or its vehicle for 3 days in de-differentiation medium. in the nucleus. Cells were washed with PBS and submitted to subcellular fractionationto separate the nuclear from the cytoplasmic 55 Example 4: The effect of theophylline on HDAC2, fractions. IPs were then carried out on each fraction nu- Sox10, JMJD2C, Krox20 and P0 expression clear and cytoplasmic, as described above. Immunopre- cipitating antibodies: 2.5 mg of eEF1A1 (rabbit, Abcam, [0067] Because HDAC1/2 inhibition strongly increases

9 17 EP 3 412 294 A1 18 acetylated eEF1A1 levels and leads to Sox10 re-locali- Example 5: HDAC2 activator increases myelin thick- zation to the cytoplasm and to an overall decrease of ness Sox10 expression levels, the inventor hypothesized that increasing HDAC2 expression and activity may prevent [0070] This example evaluates the effect of the HDAC2 Sox10 re-localization to the cytoplasm and thereby in- 5 activator theophylline on myelin thickness of crushed sci- crease Sox10 expression and activity on its target genes atic nerves in the course of remyelination. Krox20 and myelin protein zero (P0). To enhance remyelination after a sciatic nerve crush lesion, the effect of Materials and Methods theophylline was tested, a drug currently used to treat asthma for its bronchodilator properties, and to treat10 [0071] Adult mice were treated with theophylline or ve- chronic obstructive pulmonary disease by its antiinflam- hicle as described above in Example 4 from 9 to 13 days matory properties due to its ability to increase HDAC2 post sciatic nerve crush lesion. Injured sciatic nerves expression and activity at low doses. were collected for morphological analysis by electron microscopy. Briefly, mice were killed with 150 mg/kg pento- Materials and Methods 15 barbital i.p. (Esconarkon; Streuli Pharma AG) and sciatic nerves were fixed in situ with 3% paraformaldehyde and [0068] Adult mice were treated with intraperitoneal in- 0.15% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. jection of 10 mg/kg/day theophylline or its vehicle from 9 Fixed tissues were post-fixed in 2% osmium tetroxide, to 13 days post sciatic nerve crush lesion. At 14 days dehydrated through a graded acetone series as de- post lesion (dpl) the injured sciatic nerve ("crush") was 20 scribed previously (ref. 7), and embedded in Spurr’s resin collected, as well as the same region of the contralateral (Electron Microscopy Sciences). Ultrathin cross-sections nerve ("contra") as internal control for each animal. After (90-nm thick) were made, as described (ref. 7). All anal- perineurium removal, sciatic nerves were frozen in liquid yses were done at 5 mm distal to the lesion site. No con- nitrogen, pulverized with a chilled mortar and pestle, trasting reagent was applied. Images were acquired us- lysed in RIPA buffer for 15 min on ice, and centrifuged 25 ing a Philips CM 100 BIOTWIN equipped with a Morada to pellet debris. Supernatants were collected, and protein side-mounted digital camera (Olympus). The g-ratio of concentration was determined by Lowry Protein assay at least 50 axons was measured per animal and 3 animals (Bio-Rad Laboratories). Sciatic nerves were submitted per group (theophylline or vehicle) were used at 14 dpl. to SDS-PAGE and analysed by Western blot. Primary At 30 dpl, only 2 animals were used (more animals will antibodies: HDAC2 (mouse, 1:1000, Sigma, cat. 30 # be added at 30 days post lesion for statistical analyses). H2663), Sox10 (rabbit, 1:250, DCS Innovative Diagnos- tik-Systeme, cat. # SI058C01), GAPDH (glyceraldehyde- Results 3-phosphate-dehydrogenase, mouse, 1:5000, Genetex, cat. # GTX28245), P0 (chicken, 1:1000, Aves Labs, cat. [0072] The results are shown in Figures 4A, 4B, 5A # PZO), Krox20 (rabbit, 1:500, Axxora, cat. # CO-PRB- 35 and 5B. The two images in Fig. 4A are cross sections of 236P-100), JMJD2C (rabbit, 1:500, Abcam, cat. # crushed nerves 14 dpl of vehicle-treated and theophyl- ab85454). All secondary antibodies were from Jackson line-treated mice. Fig. 4B shows the same as Fig. 4A, ImmunoResearch: light chain-specific goat anti-mouse- but at 30 dpl. In Figs. 5A and 5B, the g-ratio of nerves at HRP (horse radish peroxidase) and goat anti-rabbit- 14 dpl and 30 dpl, respectively, is shown. The g-ratio is HRP, and heavy chain-specific goat anti-chicken-HRP. 40 a parameter inversely related to the thickness of the myelin layer. Figure 5C illustrates the parameters d and D Results used for calculating the g-ratio used in the axis of ordinate in Figs 5A and 5B. Figs 5A and 5B show that in animals [0069] The results show that a short treatment with the- treated with the HDAC2 activator, the g-ratio is signifi- ophylline for 4 consecutive days starting from 9 days post 45 cantly lower, these nerves thus having overall thicker my- sciatic nerve crush lesion, when axons have already re- elin sheaths. Figs 5A and 5B thus confirm statistically the grown and when Schwann cells start to re-differentiate visual impression given by Figs 4A and 4B. to rebuild myelin, resulted indeed in increased HDAC2 Treatment with theophylline resulted in a thicker myelin expression levels compared to vehicle-treated mice, and sheath already at 14 dpl and presumably also at 30 dpl. also in a high increase of Sox10, JMJD2C, Krox20 and 50 Myelin sheath thickness was measured by the g-ratio P0 at 14 and/or 30 dpl ( Figures 3A and 3B). This strongly (axon diameter: [axon+myelin] diameter): the lower the supports the hypothesis that increasing HDAC2 expres- g-ratio is, the thicker the myelin sheath is. These results sion and/or activity accelerates the remyelination proc- indicate that theophylline treatment leads to faster remy- ess after lesion. elination after sciatic nerve crush lesion. 55

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Example 6: Functional recovery by Rotarod and Toe sheaths on axons of neurons. Such damages are char- pinch test acteristic of multiple sclerosis. To model multiple sclerosis demyelinating lesions, the lysolecithin model was [0073] In this example, it was tested whether theophyl- used, where focal demyelination occurs within 3 days line treatment also leads to faster functional recovery af- 5 following lysolecithin injection, due to death of mature ter sciatic nerve crush lesion. oligodendrocytes. In this model, oligodendrocyte precursor cells (OLP) are recruited in the lesion site at 5 dpl Materials and Methods and start re-differentiating at 10 dpl. Remyelination starts at 14 dpl (ref. 8). [0074] Adult mice were treated with theophylline or ve- 10 hicle as described above in Example 4 from 9 to 13 days Materials and Methods post sciatic nerve crush lesion (carried out as described above in Example 1). Mice were placed three times on [0077] A focal demyelinating lesion was induced in the the Rotarod apparatus at a fixed speed of 15 rpm to test dorsal funiculus of the spinal cord of adult mice by injec- balance and motor coordination, at 14 and 30 dpl. The 15 tion of lysolecithin (1 ml at 1 %), as described (ref. 9). duration of each trial was limited to 600 s, and trials were This demyelination model is used as a model of multiple separated by a 30 min recovery period. Latency to fall sclerosis lesion and is well characterized, the kinetics of from the rotating beam was recorded and the average of demyelination and remyelination being well known. Mice the three trials was used for quantification. Recovery of were treated as described above in Example 4 with the- sensory function was tested at 14 and 30 dpl by toe pinch 20 ophylline or vehicle for 4 days from 9 to 13 days post test: each toe of the rear foot on the right side (lesioned lysolecithin lesion, when OLP start to re-differentiate to side) was pinched with equal pressure applied by the remyelinate demyelinated axons. Mice were killed with sameexperimenter using flat tipforceps. Immediatewith- 150 mg/kg pentobarbital i.p. (Esconarkon; Streuli Phar- drawal was recorded as functional sensitivity of the ma AG) and perfused with 4% PFA. Spinal cords were pinched toe. In case no toe exhibited sensitivity, the same 25 collected, post-fixed for 2 h in 4% PFA, incubated in 30% test was applied to toes of the contralateral side (unin- sucrose overnight at 4°C, embedded in OCT compound jured side), which always resulted in immediate with- and stored at -80°C. Spinal cords were then processed drawal. All mice were between 3 and 4 months old. The as sciatic nerves for immunofluorescence, as described experimenter was blinded regarding the treatment that in Example 2. Primary antibodies: HDAC2 (rabbit, 1:200, mice received. 30 Santa Cruz Biotechnology, cat. # sc-7899), APC (mouse, 1:200, Calbiochem/Millipore, cat. # OP80), MBP (rat, Results 1:500, Serotec, cat. # MCA409S), Sox10 (rabbit, 1:250, DCS Innovative Diagnostik-Systeme, cat. # SI058C01). [0075] Figures 6A, 6B, and 7A, 7C show the results at 14 and 30 dpl of the Rotarod and the Toe pinch test, 35 Results respectively. These figures show that mice treated with theophylline exhibited higher performances on the Ro- [0078] The results are shown in Figures 8A, 8B and tarod (motor function and coordination) and had recov- 8C. It is shown here that theophylline treatment strongly ered the sensitivity of more toes at 14 dpl, as compared increases the levels of HDAC2 expression in the lesion with vehicle-treated mice (Fig. 6A and 7A). The periph- 40 site of the spinal cord, including in differentiated oli- eral nervous system can spontaneously regenerate after godendrocytes (APC-positive), as compared to vehicle lesion, and at 30 dpl vehicle-treated mice had already treatment, as can be seen from the green fluorescence recovered enough of their motor and coordination func- in the bottom images on the left and in the middle of Fig. tion to perform as well as theophylline-treated mice on 8A. Only low-intensity green fluorescence is seen in the the Rotarod (Fig. 6B) However, vehicle-treated mice had 45 sample of the top left image of Fig. 8A, where no HDAC2 not yet recovered their sensory function as well as the- activator was used. Consistent with the findings in the ophylline-treated mice at 30 dpl (Fig. 7B). These data PNS, theophylline treatment also increases the levels of show that theophylline accelerates motor and sensory Sox10 and of myelin basic protein (MBP) in the lesion functional recovery after sciatic nerve crush lesion. site at 14 dpl, as compared to vehicle treatment (Fig. 8B). 50 Indeed, the top image in Fig. 8B under "MBP" shows only Example 7: Effect of the HDAC2 activator on remy- very little fluorescence, compared to the corresponding elination of neurons of the central nervous system image of mice treated with the activator. At 14 dpl, MBP (CNS) levels in the lesion site of mice treated with the HDAC2 activator are similar to the levels found in the unlesioned [0076] The present example has the purpose of eval- 55 contralateral part of the dorsal funiculus (Fig. 8C). These uating whether the HDAC2 activator also positively af- data indicate that theophylline is also efficient to increase fects the remyelination of neurons in the CNS, after dam- HDAC2 expression levels and to accelerate remyelina- ages resulting in destruction of oligodendrocyte myelin tion in the CNS after a demyelinating lesion.

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Example 8: The HDAC2 activator increases remyeli- 1 Brügger, V. et al. Delaying histone deacetylase nation in the CNS early response to injury accelerates conversion into repair Schwann cells and nerve regeneration. Nat. [0079] This example aims at showing the effect of the Comm. 8, 14272 (2017). HDAC2 activator on the myelin recovery in CNS neurons 5 2 Jacob, C. et al. Schwann cell survival and myeli- that were subjected to a demyelinating lesion as de- nation are critically dependent on HDAC1 and scribed in Example 7 above. Furthermore, the effect of HDAC2 function. Nat. Neurosci. 14, 429-436 (2011) an inhibitor on the levels of acetylated eEF1A in CNS is 3 Yamaguchi, T. et al. Histone deacetylases 1 and evaluated. 2 act in concert to promote the G1-to-S progression. 10 Genes Dev. 24, 455-469 (2010). Materials and Methods 4 Lindeboom, F. et al. A tissue-specific knockout re- veals that Gata1 is not essential for Sertoli cell func- [0080] For electron microscopy analyses, adult mice tion in the mouse. Nucleic Acids Res. 31, 5405-5412 were submitted to a lysolecithin lesion and treated with (2003). theophylline or vehicle, as described above in Example 15 5 Jacob, C., Grabner, H., Atanasoski, S. & Suter, U. 7. Mice were killed at 14 dpl with 150 mg/kg pentobarbital Expression and localization of Ski determine cell i.p. (Esconarkon; Streuli Pharma AG) and perfused with type-specific TGFbeta signaling effects on the cell 3% PFA and 0.15% glutaraldehyde in 0.1 M phosphate cycle. J. Cell Biol. 182, 519-530 (2008). buffer, pH 7.4. Spinal cords were collected and proc- 6 Monje, P.V., Soto, J., Bacallao, K. & Wood, P.M. essed as described above in Example 5. 20 Schwann cell dedifferentiation is independent of mi- [0081] For immunofluorescence, adult mice were togenic signaling and uncoupled to proliferation: role treated with a single intratechal injection of 10 mg/kg of cAMP and JNK in the maintenance of the differ- Mocetinostat (HDAC1/2 inhibitor) or its vehicle and their entiated state. J. Biol. Chem. 285, 31024-31036 spinal cord was collected 24 h after injection. Mice were (2010). killed and perfused with 4% PFA and their spinal cord 25 7 Pereira, J.A. et al. Integrin-linked kinase is required was processed as described above in Example 7. Pri- for radial sorting of axons and Schwann cell remy- mary antibody:EEF1A-pan (Acetyl-Lys41)antibody (rab- elination in the peripheral nervous system. J. Cell bit, Assay Biotech, 1:1000, cat. # D12106). Biol. 185, 147-161 (2009). 8 Fancy, S.P. et al. Axin2 as regulatory and thera- Results 30 peutic target in newborn brain injury and remyelination. Nat. Neurosci. 14, 1009-1016 (2011). [0082] Consistent with strongly increased MBP levels 9 Zawadzka, M. et al. CNS-resident glial progeni- in the lesion site of theophylline-treated mice, remyelina- tor/stem cells produce Schwann cells as well as ol- tion was also strongly enhanced at 14 dpl, as compared igodendrocytes during repair of CNS demyelination. with vehicle-treated mice, indicating that theophylline35 Cell Stem Cell. 6, 578-590 (2010) treatment also accelerated remyelination of the CNS in the context of demyelinating lesions.In Figure 9, the bottom right image shows thicker myelin layers (dark) Claims around axons, compared to the bottom left image. The bottom right image reflects a situation between the top 40 1. An activator of HDAC (histone deacetylase) 1 or 2 right and the bottom left image. enzymatic activity and/or expression for increasing [0083] Furthermore, mice treated intratechally with the and/or promoting myelination. HDAC1/2 inhibitor Mocetinostat for 24 h showed robust increase of acetylated eEF1A, strongly suggesting that 2. The activator of HDAC1 or HDAC2 of claim 1, for eEF1A1 is also deacetylated by HDAC1/2 in the CNS 45 increasing and/or promoting remyelination after le- and that a similar mechanism controlling Sox10 expressions to myelin of nerve cells. sion and activity by acetylated eEF1A1 occurs in oligodendrocytes, such as in Schwann cells. In Figure 10, 3. The activator of HDAC1 or HDAC2 of claim 1 or 2, the image on the bottom left shows higher levels of green for accelerating regeneration of nerve cells of the fluorescence in the nuclei (can be derived from the po- 50 peripheral nervous system (PNS) and/or of the cen- sitions of the blue DAPI fluorescence in the images on tral nervous system (CNS). the right side, white arrows) than the image on the top left side. 4. The activator of HDAC1 or HDAC2 of any one of the preceding claims, for treating and/or preventing one References 55 or more diseases and/or conditions selected from the group consisting of: (1) traumatic injury of the [0084] PNS, (2) multiple sclerosis, (3) Charcot-Marie-Tooth disease, (4) Waardenburg syndrome, (5) Guillain-

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Barré syndrome, (6) chronic inflammatory demyeli- comprising two or more of the aforementioned com- nation polyneuropathy, (7) demyelination due to ag- pounds. ing, diabetes or due to toxic agents, (8) demyelination and hypomyelination due to other diseases, 10. The activator of any one of the preceding claims, for such as Acute disseminated encephalomyelitis,5 promoting and/or accelerating remyelination of transverse myelitis, Leukodystrophy, Central pon- nerve cells of the PNS after lesion, and which acti- tine myelinolysis, Glioma, (9) schizophrenia, (10) de- vator is administered at the earliest 5 days after oc- myelination after traumatic lesion of the CNS. currence of the lesion, preferably at the earliest 7 days after lesion. 5. The activator of any one of the preceding claims, 10 which is an activator of HDAC2 deacetylase activity. 11. The activator of any one of the preceding claims, which is for increasing and/or accelerating remyeli- 6. The activator of any one of the preceding claims, nation of nerve cells of the CNS after an attack to which increases HDAC1 and/or HDAC2 deacetylase glia cells, in particular oligodendrocytes. activity in vivo and /or in vitro. 15 12. A method for screening for agents suitable in the 7. The activator of any one of the preceding claims, treatment and/or prevention of the diseases men- which increases expressions of HDAC1 and/or tioned in claim 4, and/or for screening for agents suit- HDAC2 in cells, in particular in cells selected from able for promoting myelination and/or for increasing Schwann cells, other glia cells, in particular oli-20 remyelination, the method comprising: godendrocytes, and nerve cells. - assessing the capacity of a candidate agent to 8. The activator of any one of the preceding claims, increase the HDAC1/2 activity and/or expres- which is selected from the group consisting of: an sion, alkaloid compound, for example a xanthine com- 25 pound, an iron chelator, deferoxamine, a flavonoid, wherein presence of capacity to increase HDAC1/2 for example ginkgetin K, a compound comprising a activity and/or expression indicates that said candi- catechol moity, Chembridge 5104434, sciadopilysin, date agent is an agent suitable for the treatment of tetrahydrogamboic acid, TAM-11, gambogic acid, or said diseases and for promoting myelination. a derivative thereof, LY235959, CGS19755, SKF 30 97541, etidronic acid, levonordefrin, methyldopa, 13. The method of claim 12, comprising the step of as- ampicillin trihydrate, D-aspartic acid, gamma-D- sessing, in an in vitro sample of cells exposed to the glutamylaminomethylsulfonic acid, phenazopyridine candidate compound, said sample being obtained hydrochloride, oxalamine citrate salt, podophyllotox- from a subject, whether HDAC1/2 activity and/or ex- in, (+-)-4-amino-3-(5-chloro-2-thienyl)-butanoic ac- 35 pression is increased compared to a sample which id, (RS)- (tetrazol-5-yl) glycine, or R(+)-SKF-81297. is devoid of said candidate compound.

9. The activator of any one of the preceding claims, 14. The method of claim 12 or 13, wherein said step of which is selected from the group consisting of: a xan- assessing the capacity of a candidate agent to in- thine compound and from a xanthene compound, 40 crease the HDAC1/2 activity and/or expression com- wherein said xanthine compound may be selected prises: from theophylline, caffeine, acepifylline (etaphylline), bamifylline, bufylline, cafaminol, cafedrine, - exposing a sample comprising a polypeptide diprophylline, doxofylline, enprofylline, etamiphyl- exhibiting HDAC1/2 deacetylase activity to a line, etofylline, proxyphylline, suxamidofylline, theo- 45 said candidate compound, and, bromine, paraxanthine, 8-chlorotheophylline, 8-phe- - determining whether in said sample said nyltheophylline, IBMX (1-Methyl-3-(2-methylpro- HDAC1/2 deacetylase activity and/or expres- pyl)-7H-purine-2,6-dione), DMPX (3,7-dimethyl-1- sion is increased compared to a comparative propargylxanthine), CPX (8-Cyclopentyltheophyl- sample devoid of said candidate compound. line), DPCPX (8-Cyclopentyl-1,3-dipropylxanthine 50 or PD-116,948), a salt of any one of the aforemen- 15. A method for treating and/or preventing a disease tioned, an alkylated form of any one of the aforemen- selectedfrom the diseases listedin claim4, the meth- tioned and combinations comprising two or more of od comprising the step of administrating, to a subject the aforementioned compounds, and wherein said in need thereof, an activator of HDAC1 and/or xanthene compound may be selected from the group 55 HDAC2. consisting of xanthene, xanthydrol, xanthone, a salt of any one of the aforementioned, an alkylated form 16. A method for increasing and/or promoting the remy- of any one of the aforementioned and combinations elination of nerve cells, the method comprising the

13 25 EP 3 412 294 A1 26 step of administrating, to a subject in need thereof, an activator of HDAC1 and/or HDAC2.

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

• US 6277402 B [0010] • US 200904267 W [0028] • US 20030134865 A [0011] [0030] • WO 2010011318 A [0028] [0029] • WO 2010011318 A2 [0028]

Non-patent literature cited in the description

• BRÜGGER, V. et al. Delaying histone deacetylase • MONJE, P.V. ; SOTO, J. ; BACALLAO, K. ; WOOD, early response to injury accelerates conversion into P.M. Schwann cell dedifferentiation is independent repair Schwann cells and nerve regeneration.Nat. of mitogenic signaling and uncoupled to proliferation: Comm., 2017, vol. 8, 14272 [0084] role of cAMP and JNK in the maintenance of the dif- • JACOB, C. et al. Schwann cell survival and myeli- ferentiated state. J. Biol. Chem., 2010, vol. 285, nation are critically dependent on HDAC1 and 31024-31036 [0084] HDAC2 function. Nat. Neurosci., 2011, vol. 14, • PEREIRA, J.A. et al. Integrin-linked kinase is re- 429-436 [0084] quired for radial sorting of axons and Schwann cell • YAMAGUCHI, T. et al. Histone deacetylases 1 and remyelination in the peripheral nervous system.J. 2 act in concert to promote the G1-to-S progression. Cell Biol., 2009, vol. 185, 147-161 [0084] Genes Dev., 2010, vol. 24, 455-469 [0084] • FANCY, S.P. et al. Axin2 as regulatory and thera- • LINDEBOOM, F. et al. A tissue-specific knockout re- peutic target in newborn brain injury and remyelina- veals that Gata1 is not essential for Sertoli cell function. Nat. Neurosci., 2011, vol. 14, 1009-1016 [0084] tion in the mouse. Nucleic Acids Res., 2003, vol. 31, • ZAWADZKA, M. et al. CNS-resident glial progeni- 5405-5412 [0084] tor/stem cells produce Schwann cells as well as oli- • JACOB, C. ; GRABNER, H. ; ATANASOSKI, S. ; godendrocytes during repair of CNS demyelination. SUTER, U. Expression and localization of Ski deter- Cell Stem Cell, 2010, vol. 6, 578-590 [0084] mine cell type-specific TGFbeta signaling effects on the cell cycle. J. Cell Biol., 2008, vol. 182, 519-530 [0084]