Chromanyl derivatives for use in the treatment of bone disorders

Abstract

The present invention relates to the field of medicine. The invention in particular relates to amide-derivatives of 2-hydroxy-2-methyl-4-(3,5,6-trimethyl-1,4-benzoquinon-2-yl)-butanoic acid and related compounds for treating or preventing bone disorders. It was found that the compounds can for instance promote bone formation. The compounds can increase expression of specific genes associated with bone disorders.

Description

[0001] CHROMANYL DERIVATIVES FOR USE IN THE TREATMENT OF BONE DISORDERS

[0002] Field of the invention

[0003] The present invention relates to the field of medicine. The invention in particular relates to amidederivatives of 2-hydroxy-2-methyl-4-(3,5,6-trimethyl-1 ,4-benzoquinon-2-yl)-butanoic acid and related compounds for treating or preventing bone disorders. It was found that the compounds can for instance promote bone formation. The compounds can increase expression of specific genes associated with bone disorders.

[0004] Background art

[0005] Balance between bone formation and bone resorption is important for bone homeostasis. The continuous process of bone degradation by bone-resorbing osteoclasts followed by the formation of bone matrix by bone-forming osteoblasts, a process known as bone remodeling, requires high amounts of energy in the form of adenosine triphosphate (ATP). ATP can be generated via mitochondrial oxidative phosphorylation (OXPHOS) and / or cytoplasmatic glycolysis. OXPHOS is a more efficient means of generating ATP, as glycolysis generates two ATP molecules per glucose molecule, whereas OXPHOS generates about 36 ATPs per glucose. Mitochondrial proteins are encoded by nuclear DNA (nDNA) and mitochondrial DNA (mDNA), that encodes 13 OXPHOS proteins and the mitochondrial translation machinery.

[0006] The most common pathogenic mDNA variant m.3243A>G in the MT-TL1 gene that leads to defective translation of mDNA encoded proteins can result in impairment of OXPHOS. Accordingly, patients carrying a m.3243A>G mutation demonstrate a metabolic shift, wherein a larger fraction of their total ATP levels is produced by glycolysis rather than oxidative phosphorylation (OXPHOS) in comparison to healthy controls. Besides ATP production, mitochondria are multifunctional organelles, and thus, impaired OXPHOS function can affect other mitochondrial functions than ATP production, e.g. substrates production for epigenetic reactions such as tricarboxylic acid (TCA) cycle intermediates, regeneration of NAD+for other reactions like malate dehydrogenase (MDH2), regulation of mitochondrial stress responses (MSR).

[0007] Although mitochondria have emerged as regulators of stem cell function and fate, the cellular and molecular mechanisms underlying the changes in bone that occur upon mitochondrial dysfunction remain unknown. Furthermore, it is unknown how the bone homeostasis can be maintained in patients suffering from mitochondrial dysfunction.

[0008] WO2014 / 011047 and WO2017 / 060432 disclose amide-derivatives of 2-hydroxy-2-methyl-4- (3,5,6-trimethyl-1 ,4-benzoquinon-2-yl)-butanoic acid for treating or preventing mitochondrial disorders.

[0009] There is a need for improved methods of treatment of subjects suffering from bone disease. There is a need for small molecule therapies for treatment of subjects suffering from bone disease, and for small molecule therapies that can prevent and / or treat bone disease. Furthermore, there is a need for therapies with long-lasting effects on preventing and / or treating bone disease. Summary of the invention

[0010] The inventors have surprisingly found that chromanyl derivatives as described herein can be used to treat or prevent bone disorders, for instance by increasing bone formation capacity of osteoprogenitor cells. The compounds promote a bone-forming transcriptional program with upregulation of key osteogenic genes, e.g., RUNX2 and LRP5, which enhances the mineralization capacity of cultured marrow stromal cells. Accordingly the invention provides a compound represented by general structure (la) or (lb): wherein L is a linker comprising 1 to 10 optionally substituted backbone atoms selected from carbon, nitrogen and oxygen; R1and R2are each independently selected from H, Ci - Ce alkyl, or Ci - Ce alkenyl, or R1and R2together form a bridging moiety that is a further linker L, or R1is joined with a backbone atom of the linker L to form a bridging moiety that is a further linker L which forms a cyclic structure and / or R2is joined with a backbone atom of the linker L to form a bridging moiety that is a further linker L which forms a cyclic structure; R3is selected from H, Ci - Ce alkyl, or Ci - Ce alkenyl, wherein the alkyl or alkenyl may be substituted with one or more halogen atoms, hydroxyl moieties, or (halo)alkoxy moieties, or R3is absent when the nitrogen atom to which it is connected is connected to L via a double bond; or R3is joined with a backbone atom of the linker L to form a bridging moiety that is a further linker L which forms a cyclic structure; and R4is selected from H or Ci - Ce alkyl, wherein the alkyl may be substituted with one or more halogen atoms, hydroxyl moieties, or (halo)alkoxy moieties; or R4is absent; R7is in each instance individually a Ci - Ce alkyl; X is an anion when R4is not absent and is absent when R4is absent; for use in a method of treating or preventing a bone disorder.

[0011] In preferred embodiments each R7is methyl. Preferably X is a pharmaceutically acceptable anion. Preferably linker L is selected from L1-L28as shown herein, wherein R1together with R1forms a bridging moiety that is a further linker L; R2together with R2forms a bridging moiety that is a further linker L; R3together with R3forms a bridging moiety that is a further linker L; and R5together with R5forms a bridging moiety that is a further linker L. In preferred embodiments the further linker L is -CH2- or - (CH2)2- or -(CH2)3- or -(CH2)4-. Preferably linker L together with to at least one of R1or R2forms a bridging moiety that is a further linker L which forms a cyclic structure, wherein that cyclic structure is a 4-10 membered heterocycle. Preferably that cyclic structure is a 6 membered heterocycle. In preferred embodiments the compound is represented by structure (Vila), (VII b) , (Vile), (Vlld), (Vile), or (Vllf):

[0012] Preferably the bone disorder is associated with aberrant expression of LRP5 or RUNX2. Preferably the bone disorder is not associated with aberrant expression of GDF5. Preferably the method is for decreasing bone fracture risk, for treating osteoporosis, for treating Type-1 diabetic bone disease, for increasing the osteogenic potential of marrow stromal cells (MSC), for decreasing glycolytic ATP production in MSC, for increasing proliferation of MSC, for increasing sternness of MSC, for increasing bone formation capacity, or for increasing the mineralization capacity of MSC. In preferred embodiments the subject has a (primary) mitochondrial disease, more preferably the subject carries a mitochondrial DNA m.3243A>G mutation. Preferably the subject does not suffer from LRP5-linked osteoporosis- pseudoglioma syndrome. Preferably the subject does not undergo concomitant therapy using hormones for increasing bone formation. Also provided is a method for treating or preventing a bone disorder, the method comprising the step of administering a compound as defined herein to a subject.

[0013] Description of the invention

[0014] The inventors have surprisingly found that chromanyl derivatives as described herein can be used to treat or prevent bone disorders, for instance by increasing bone formation capacity of osteoprogenitor cells. The compounds promote a bone-forming transcriptional program with upregulation of key osteogenic genes, e.g., RUNX2 and LRP5, which enhances the mineralization capacity of cultured marrow stromal cells. Compounds

[0015] The invention relates to compounds useful for practicing methods of treating or preventing a bone disorder. The compounds for use according to the invention are represented by general structure (la) or (lb): wherein,

[0016] - L is a linker comprising 1 to 10 optionally substituted backbone atoms selected from carbon, nitrogen and oxygen;

[0017] - R1and R2are each independently selected from H, Ci - Ce alkyl, or Ci - Ce alkenyl, or R1and R2together form a bridging moiety that is a further linker L, or R1is joined with a backbone atom of the linker L to form a bridging moiety that is a further linker L which forms a cyclic structure and / or R2is joined with a backbone atom of the linker L to form a bridging moiety that is a further linker L which forms a cyclic structure;

[0018] - R3is selected from H, Ci - Ce alkyl, or Ci - Ce alkenyl, wherein the alkyl or alkenyl moiety may be substituted with one or more halogen atoms, hydroxyl moieties, or (halo)alkoxy moieties, or R3is absent when the nitrogen atom to which it is connected is connected to L via a double bond; or R3is joined with a backbone atom of the linker L to form a bridging moiety that is a further linker L which forms a cyclic structure; and

[0019] - R4is selected from H or Ci - Ce alkyl, wherein the alkyl moiety may be substituted with one or more halogen atoms, hydroxyl moieties, or (halo)alkoxy moieties; or R4is absent;

[0020] - R7is in each instance individually a Ci - Ce alkyl moiety;

[0021] - X is an anion when R4is not absent and is absent when R4is absent.

[0022] The 2-carboxy variant of vitamin E is also known as Trolox™ (6-hydroxy-2, 5,7,8- tetramethylchroman-2-carboxylic acid). When R7is methyl, compounds of general structure la or lb can be seen as amide derivatives thereof. The compounds as such are known in the art, for instance WO2014 / 011047 and WO2017 / 060432 disclose amide-derivatives of 2-hydroxy-2-methyl-4-(3,5,6- trimethyl-1 ,4-benzoquinon-2-yl)-butanoic acid for treating or preventing mitochondrial disorders and / or conditions associated with mitochondrial dysfunction. Compounds of general structure la can be described as “closed form” and compounds of general structure lb can be described as “open form”. The open form is an oxidized variant of the closed form. The open form is found as metabolite of the closed form when the latter is administered. After 24h treatment of a P4 cell line with compound l-IVa- X (a compound of general structure (la) wherein as per compound X the following apply: L = L19; R1= H; R2-R2’ = L3; R3= H, in the S,R-configuration), about 48% (±10%) of closed compound was converted into the open form. About 15% (±3%) was converted during the same period when incubated in medium only. Such conversion is also disclosed in Beyrath et al., DOI: 10.1038 / s41598-018-24900-3 , and in Koene et al., DOI: 10.1186 / s13023-017-0715-0.

[0023] R7is in each instance individually a Ci - Ce alkyl. In preferred embodiments -R7is in each instance individually a Ci - C4 alkyl, more preferably a Ci - C2 alkyl, most preferably methyl. In preferred embodiments the same choice is made for each R7, most preferably each R7is methyl. In a preferred embodiment, the compound is represented by structure (IVa) or (IVb). In other words, structure (IVa) is a preferred embodiment of structure (la), and structure (IVb) is a preferred embodiment of structure (lb).

[0024] (IVa) (IVb)

[0025] The compound identified by general structure (la) or (lb) comprises at least one chiral carbon atom (stereocenter), i.e. the atom marked C* in the structures below.

[0026] Both the compound having an S-configuration as the compound having an R-configuration of the carbon atom marked C* are encompassed in the present invention, as well as mixtures of the different stereoisomers. Such a mixture may have one of the configurations in enantiomeric excess, or may be racemic. Whenever one or more additional stereocenters are present in the compound according to the invention, for example in linker L, each may individually exist in the S-configuration, in the R- configuration, or as a mixture of both configurations. Such a mixture may have one of the configurations in enantiomeric excess, or may be racemic. In case additional stereocenters are present, all diastereomers of the compound of general structure (la) or (lb), in each possible ratio, are encompassed in the present invention.

[0027] R1and R2are each independently selected from H, Ci - Ce alkyl or Ci - Ce alkenyl, or one or both of R1and R2are embedded in a cyclic structure as described here below. Preferably, R1is H or Ci - C2 alkyl or R1and R2are joined together and thus form a second linker between the nitrogen atom bearing R1and the nitrogen atom bearing R2, or R1is joined with a backbone atom of the linker L in a cyclic structure, more preferably R1is H or Ci - C2 alkyl, even more preferably R1is H or methyl (Me), most preferably R1is H. Preferably, R2is H or Ci - C2 alkyl or R1and R2are joined together and thus form a second linker between their two nitrogen atoms, or R2is joined with a backbone atom of the linker L in a cyclic structure, more preferably R2is H, Ci - C2 alkyl or joined with a backbone atom of the linker L in a cyclic structure, even more preferably R2is H, methyl (Me) or joined with a backbone atom of the linker L in a cyclic structure. In one embodiment, R2is H, methyl (Me), preferably R2is H. In an especially preferred embodiment, R2is joined with a backbone atom of the linker L in a cyclic structure, as further defined below, preferably a saturated cyclic structure, most preferably a piperidine ring.

[0028] In preferred embodiments R1and R2are each independently selected from H, Ci - Ce alkyl, or Ci - Ce alkenyl, or R1and R2together form a bridging moiety that is a further linker L, or R1is joined with a backbone atom of the linker L to form a bridging moiety that is a further linker L which forms a cyclic structure and / or R2is joined with a backbone atom of the linker L to form a bridging moiety that is a further linker L which forms a cyclic structure. Whenever a further linker L is present, this can also be referred to as a second linker. When more than one linker is present, a cyclic structure is formed. More preferably R1and R2are each independently selected from H or Ci - Ce alkyl, or R1and R2together form a bridging moiety that is a further linker L, or R1is joined with a backbone atom of the linker L to form a bridging moiety that is a further linker L which forms a cyclic structure and / or R2is joined with a backbone atom of the linker L to form a bridging moiety that is a further linker L which forms a cyclic structure. In R1 , R2, and R3, Ci - Ce alkyl is preferably Ci - C4 alkyl, more preferably Ci - C3 alkyl, even more preferably Ci - C2 alkyl.

[0029] For compounds as described herein, any further linker L is independently selected from the linker L as depicted in general structure (la) or (lb). In other words, when a second linker is present, the first and the second linker do not need to be the same. In some embodiments the same linker is selected for linker L and for the further linker L. In some embodiments different linkers are selected for linker L and for the further linker L.

[0030] In one embodiment, the nitrogen atom bearing R1is connected to the nitrogen atom bearing R2via a second linker. This second linker is defined by joining together R1and R2. Thus, the nitrogen atom bearing R1, the nitrogen atom bearing R2, the linker L and the further linker L together form a cyclic structure, which is preferably a 4 - 10-membered cyclic structure, more preferably a 5 - 8-membered cyclic structure, most preferably a 6-membered cyclic structure. In a preferred embodiment, the second linker is -CH2-CH2- or -CH2-CH2-CH2-, most preferably -CH2-CH2-.

[0031] In another embodiment, the nitrogen atom bearing R1is connected to a backbone atom of the linker via a second linker, thereby forming a cyclic structure, preferably a 4 - 10-membered cyclic structure, more preferably a 5 - 8-membered cyclic structure, most preferably a 6-membered cyclic structure. The backbone atom of the linker to which the nitrogen atom is connected in this respect has a substituent R1, which is joined together with R1. Thus, nitrogen atom bearing R1, part of first linker located between the nitrogen atom bearing R1and the atom bearing R1, the backbone atom bearing R1and the second linker together form the cyclic structure. In this embodiment, the nitrogen atom bearing R2is not included in this cyclic structure, only part of the backbone of linker L is included (along with the further linker L that is formed by R1and R1’). In a preferred embodiment, this connection between R1and R1’ is a -CH2-CH2- or -CH2-CH2-CH2- bridge, most preferably a -CH2-CH2- bridge. Most preferably, the cyclic structure containing the nitrogen atom bearing R1is a fully saturated ring, preferably selected from a piperidine ring, a pyrrolidine ring, a piperazine ring, an imidazolidine ring, a pyrazolidine ring and an azepane ring, more preferably a piperazine ring, a piperidine ring or a pyrrolidine ring, most preferably a piperidine ring.

[0032] In another embodiment, the nitrogen atom bearing R2is connected to a backbone atom of the linker via a second linker, thereby forming a cyclic structure, preferably a 4 - 10-membered cyclic structure, more preferably a 5 - 8-membered cyclic structure, most preferably a 6-membered cyclic structure. The backbone atom of the linker to which the nitrogen atom is connected in this respect has a substituent R2, which is joined together with R2. Thus, the nitrogen atom bearing R2, part of first linker located between that nitrogen atom and the atom bearing R2, the backbone atom bearing R2and the second linker together form the cyclic structure. In this embodiment, the nitrogen atom bearing R1is not included in this cyclic structure. In a preferred embodiment, this connection between the nitrogen atom bearing R2and a backbone atom of the linker is -CH2-CH2- or -CH2-CH2-CH2-, most preferably- CH2-CH2-. Most preferably, the cyclic structure containing the nitrogen atom bearing R2is a fully saturated ring, preferably selected from a piperidine ring, a pyrrolidine ring, a piperazine ring, an imidazolidine ring, a pyrazolidine ring and an azepane ring, more preferably a piperidine ring or a pyrrolidine ring, most preferably a piperidine ring. It is also possible that a connection exists between R1and an R1substituent on the linker and between R2and an R2substituent on the linker.

[0033] In another embodiment, the nitrogen atom bearing R2is connected to a backbone atom of the linker via a second and a third further linker L, thereby forming a bicyclic structure, preferably a 6 - 12- membered cyclic structure, more preferably a 6 - 9-membered cyclic structure such as a bicyclooctane- like structure, most preferably a [2.2.2]bicyclooctane-like structure. The backbone atom of the linker to which the nitrogen atom is connected in this respect has a substituent R2and R3’ which are joined together with R2and R3, respectively. Thus, the nitrogen atom bearing R2, part of first linker located between that nitrogen atom and the atom bearing R2, the backbone atom bearing R2and the second linker together form one cycle of the bicyclic structure, and the part of the first linker located between the nitrogen atom bearing R2and the atom bearing R3’, and the third linker form a second cycle of the bicyclic structure. In a preferred embodiment, this connection between the nitrogen atom bearing R2and a backbone atom of the linker is a -CH2-, -CH2-CH2- or -CH2-CH2-CH2- bridge, most preferably a -CH2-CH2- bridge, wherein two or three, preferably two, carbon atoms are present between the nitrogen atom bearing R2and the backbone atom of the linker. Most preferably, the cyclic structure containing the nitrogen atom bearing R2is a fully saturated structure. Among the above-mentioned possibilities for R2it is most preferred that R2is joined with R2.

[0034] The linker L can be connected to either neighboring nitrogen atom via a double bond. Preferably the linker L is not connected via a double bond to the nitrogen atom bearing R1. When the nitrogen atom bearing R2is connected to L via a double bond, that nitrogen atom is part of an imine moiety. That nitrogen atom can also be an imine when R2 is connected to it via a double bond. (e.g. when R2= Ci - Ce alkenyl). In such instances, R3is absent. Preferred moieties comprising an imine moiety include guanidine, amidine and pyridine. For guanidine and amidine, one of the nitrogen atoms is substituted to form the connection with the nitrogen atom bearing R1via linker L. For pyridine, one of the carbon atoms is substituted. When the nitrogen atom bearing R2is part of an amine moiety, it is connected to the linker and R2via two single bonds, and R3is present. It is preferred that the nitrogen atom bearing R2is part of an amine moiety, i.e. having three or four single bonds to each of R1, R2, R3and optionally R4.

[0035] In the instance that R3is present, R3is selected from H, Ci - Ce alkyl, or Ci - Ce alkenyl, wherein the alkyl or alkenyl moiety may be substituted with one or more halogen atoms, hydroxyl groups or (halo)alkoxy moieties, preferably R3is H, Ci - Ce alkyl, more preferably R3is H or Ci - C4 alkyl, even more preferably R3is H or Ci - C2 alkyl, wherein the alkyl moiety may be substituted with one or more halogen atoms, hydroxyl groups or (halo)alkoxy moieties. Halogen atoms include fluorine (F), chlorine (Cl), bromine (Br), iodine (I), and astatine (At), preferably the halogen atom is fluorine (F). Preferred alkoxy moieties include methoxy and ethoxy. In haloalkoxy moieties, at least one hydrogen atom of an alkoxy moiety is replaced by a halogen atom, preferably by F. Preferred substituents for the alkyl moieties are halogen atoms and alkoxy moieties. Suitable moieties for R3include, preferably are limited to, H, methyl (Me), trifluoromethyl (-CF3), ethyl (Et), isopropyl (iPr), cyclopropyl (-cPr), methylene cyclopropyl (-CH2cPr), n-propyl (n-Pr), 2,2,2-trifluoroethyl (-CH2CF3), 2-hydroxy-ethyl (-CH2CH2OH), and methoxymethyl (-CH2OCH3), more preferably R3is H or methyl (Me), most preferably R3is H. Alternatively, R3is preferably Ci - C4 alkyl, wherein the alkyl moiety may be substituted with one or more halogen atoms or (halo)alkoxy moieties, more preferably R3is Ci - C2 alkyl, wherein the alkyl moiety may be substituted with one or more halogen atoms or (halo)alkoxy moieties. It is particularly preferred that R3is H or -CH2CH2OH. In some embodiments R3is H. In some embodiments R3is - CH2CH2OH.

[0036] R4is either absent or is selected from H or Ci - Ce alkyl, wherein the alkyl may be substituted with one or more halogen atoms or (halo)alkoxy moieties, preferably R4is H or Ci - C4 alkyl, wherein the alkyl moiety may be substituted with one or more halogen atoms or (halo)alkoxy moieties, more preferably R4is H or Ci - C2 alkyl, wherein the alkyl moiety may be substituted with one or more halogen atoms or (halo)alkoxy moieties. Halogen atoms include fluorine (F), chlorine (Cl), bromine (Br), iodine (I), and astatine (At), preferably the halogen atom is fluorine (F). Preferred alkoxy moieties include methoxy and ethoxy. In haloalkoxy moieties, at least one hydrogen atom of an alkoxy moiety is replaced by a halogen atom, preferably by F. Suitable moieties for R4include, preferably are limited to, H, methyl (Me), trifluoromethyl (-CF3), ethyl (Et), isopropyl (iPr), cyclopropyl (-cPr), methylene cyclopropyl (- CH2cPr), n-propyl (n-Pr), 2,2,2-trifluoroethyl (-CH2CF3), methoxymethyl (-CH2OCH3). Even more preferably R4is H or methyl (Me), most preferably R4is H.

[0037] X is an anion when R4is not absent and is absent when R4is absent. When X is an anion it can be any anion, preferably a physiologically or pharmaceutically acceptable anion, more preferably a monovalent anion. In preferred embodiments X is a pharmaceutically acceptable anion. X is preferably selected from F, Cl, Br, I, HSO4, NO3, CF3CO2, formate, acetate, propionate, glycolate, pyruvate, oxalate, maleate, malonate, succinate, fumarate, tartarate, citrate, benzoate, cinnamate, mandelate, sulfonate and salicylate. More preferably, X is Cl, I, TFA or formate, even more preferably Cl, I, TFA or formate, still more preferably X is Cl or formate, most preferably X is Cl.

[0038] When R4is H the compound can be seen as originating from formal protonation. This protonation is preferably accomplished with an acid such as hydrogen chloride (HCI), trifluoroacetic acid (CF3COOH), or formic acid (HCOOH), more preferably with HCI or formic acid. It follows that in such cases X is the conjugate base of that acid. When R4is methyl, the compound can be seen as originating from formal methylation. This is preferably accomplished with methyl iodide (CH3I). Thus, in a preferred embodiment, R4= -CH3 when X = I, and R4= H when X = Cl, CF3COO, or HCOO.

[0039] Pharmaceutically acceptable salts are those salts that are suitable to be administered as drugs or pharmaceuticals to humans and / or animals. The pharmaceutically acceptable salts of the amine or imine moiety of the compound according to the invention are known to those skilled in the art, and can originate from formal treatment of the compound with an acid (protonation agent) or an alkylating agent. Suitable acids include organic acids or inorganic acids. Examples of inorganic acids include, but are not limited to, hydrochloric acid (HCI), hydrobromic acid (HBr), hydroiodic acid (HI), sulphuric acid (H2SO4), nitric acid (HNO3), trifluoroacetic acid (TFAH or CF3CO2H) and phosphoric acid (H3PO4). Examples of organic acids include, but are not limited to, formic acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, sulfonic acids and salicylic acid. When an acid as exemplified here is used to formally prepare the salt, R4is hydrogen, and the type of acid determines counter ion X. Alternatively, the salt can be formed by formal treatment with an alkylating agent. Suitable alkylating agents include, but are not limited to, Ci - Ce alkyl halides (such as methyl iodide, ethyl iodide, propyl iodide, butyl chloride, butyl fluoride, butyl bromide), dimethyl sulphate, dimethyl carbonate, methyl triflate, methyl fluorosulfonate, methyl chlorosulfonate, methyl methanesulfonate and methyl benzenesulfonate. The salt may be prepared by actual treatment of the non-salt compound with an acid or alkylation agent, as indicated above, or via other means known in the art and / or exemplified further below.

[0040] Appropriate linkers L are linkers comprising 1 to 10 optionally substituted backbone atoms selected from carbon, nitrogen and oxygen, more preferably comprising 1 to 8 optionally substituted backbone atoms. L may thus comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 optionally substituted backbone atoms. Herein, backbone atoms are those atoms that make up the shortest chain between the two nitrogen atoms bearing either R1or R2. The backbone may be a linear structure, but (part of) the backbone may also be part of a cyclic structure. When the backbone is part a cyclic structure, the backbone is defined as the shortest chain between the two nitrogen atoms bearing either R1or R2. In one embodiment, one of the backbone atoms comprises a substituent R5, and one of the backbone atoms comprises a substituent R5, preferably two different backbone atoms comprise the substituents R5and R5, wherein R5together with R5forms a bridging moiety that is a further linker L which preferably forms a 4 - 10-membered cyclic structure, more preferably a 5 - 8-membered cyclic structure, most preferably a 6-membered cyclic structure. In this embodiment, the two nitrogen atoms bearing either R1or R2are not included in the cyclic structure, but instead only part of the backbone of the linker is included. In a preferred embodiment, this connection between the backbone atom(s) of the linker, bearing the R5and R5substituents, is a -(CH2)n- bridge, wherein n = 1 - 6, preferably a -CH2-CH2- or -CH2-CH2-CH2- bridge, wherein one to six, preferably two or three, carbon atoms are present between the substituted backbone atom(s) of the linker.

[0041] To fulfil their valence requirements, the carbon and nitrogen backbone atoms of the linker may bear hydrogen atoms, may be substituted, or double or triple bonds may be present between adjacent backbone atoms, as will be understood by the skilled person. In the context of the invention, hydrogen is not regarded a substituent. Whenever an oxygen atom is present as backbone atom in the linker, the skilled person will understand that the oxygen backbone atom bears no hydrogen atoms, substituents or double or triple bonds. Triple bonds may be present between two carbon atoms of the backbone. The backbone atoms, together with the hydrogen atoms and / or the substituents, constitute the linker. In the context of the present invention, “optionally substituted” is used to indicate that an (backbone) atom may bear one or more substituents, or may bear no substituents and sufficient hydrogen atoms may be present instead, to fulfil the valence requirements of said (backbone) atom.

[0042] Suitable substituents include but are not limited to halogen, NH2, NHR6, N(R6)2, NHNH2, N3, NHC(=O)R6, NHC(=O)NHR6, NHC(=O)NH2, NHC(=O)OR6, OH, OR6, OC(=O)R6, R6(e.g. alkyl, cycloalkyl), aralkyl, alkenyl, alkynyl, aryl, heteroaryl, OC(=O)OR6, OC(=O)NHR6, O(SO2)R6, O(SO2)OH, O(PO2)OH, SH, SR6, C(=O)R6, alkyl-NH2, alkyl-OH, alkyl-SH, C(=O)CF3, C(=O)OR6, C(=O)OH, C(=O)H, C(=O)OR6, C(=O)NH2, C(=O)NMe2, C(=O)N(R6)2, C(=S)NH2C(=S)SH, ON, NO, CNO, ONC, OCN, SON, SNC, CNS, S(=O)R6, S(=O)2R6, S(=O)2(OH), P(=O)(OH)2or P(=O)(OH)(OR6). Atoms having two or more remaining valencies, such as carbon backbone atoms, may bear a double bonded substituent, such as oxo (=0), imino (=NH or =NR6), thioxo (=S), alkylidene (=CH2 or =CHR6or =C(R6)2). Herein, each R6is independently an alkyl moiety, preferably a Ci - Ce alkyl moiety, more preferably a Ci - C2 alkyl moiety. Within R6, one or more CH2 moieties may each independently be replaced by one of O, S or NH, and / or one or more CH moieties may be replaced by N. In addition, two substituents on the same atom or on different atoms may be joined to form cyclic structures. If two substituents on a single backbone atom are joined in a cyclic structure, this cyclic structure may be regarded as being connected via a spiro junction to the backbone. If two substituents on different backbone atoms are joined in a cyclic structure, part of this cyclic structure is (part of) the backbone, and the backbone is considered to be the shortest chain of atoms between the two nitrogen atoms bearing either R1or R2. The cyclic structures formed as such may be all-carbon or may comprise 0 - 3 heteroatoms (e.g. N, O, S and / or P), and may comprise 0 - 3 double bonds. All atoms in these cyclic structures may optionally be substituted. Examples of suitable cyclic structures are optionally substituted cycloalkyl, optionally substituted cycloheteroalkyl, optionally substituted aryl or optionally substituted heteroaryl. As further indicated below, a cyclic structure may also be formed by joining one substituent on a backbone atom with R1or with R2.

[0043] In the context of the present invention, the term "alkyl" refers to saturated aliphatic groups including straight-chain, branched-chain, cyclic groups, and combinations thereof, having the number of carbon atoms specified, or if no number is specified, preferably having up to 12 carbon atoms. "Straight-chain alkyl" or "linear alkyl" group refers to alkyl groups that are neither cyclic nor branched, commonly designated as "n-alkyl" groups. One subset of alkyl groups is Ci - Ce alkyl, which includes groups such as methyl, ethyl, n-propyl, isopropyl, butyl, n-butyl, isobutyl, sec-butyl, t-butyl, pentyl, n- pentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and any other alkyl group containing between one and six carbon atoms, where the Ci - Ce alkyl groups can be attached via any valence on the Ci - Ce alkyl groups. Preferred Ci - Ce alkyl groups are linear or branched, more preferably linear.

[0044] In one embodiment, the backbone atoms are optionally substituted with one or more substituents selected from the group consisting of R6, carboxy, oxo, and primary amino or a backbone atom may be joined with R1to form a 4-10-membered cyclic structure and / or a backbone atom may be joined with R2to form a 4-10-membered cyclic structure, or two backbone atoms may be joined to form a cyclic structure, wherein R6is as defined above, preferably R6is Ci - Ce alkyl, more preferably Ci - C2 alkyl. Preferred substituents of the backbone atoms are alkyl, such as methyl (Me or -CH3), carboxyl (- C(=O)OH), oxo (=0) and primary amino (-NH2).

[0045] Preferred linkers L are identified here below as L1to L28. More preferred are L1to L26:, even more preferred are L1, L3, L16, L17, L19, L21, L22, L24, and L26; still more preferred are L1, L3, L16, L19, L21, L22, and L24; even more preferred are L1, L3, L19, L21, L22, and L24; still even more preferred are L1, L3, L19, and L24. Most preferred in general is L19. L1and L3are most preferred for further linkers L. When L is L1, any further linker L is preferably also L1. When L is L19, the further linker L bridging R2and R2’ is preferably L1, L3, L16, or L17, most preferably L3.

[0046]

[0047] In the above linkers, R1' together with R1forms a bridging moiety that is a further linker L;

[0048] R2together with R2forms a bridging moiety that is a further linker L;

[0049] R3together with R3forms a bridging moiety that is a further linker L; and

[0050] R5together with R5forms a bridging moiety that is a further linker L.

[0051] Herein, it is preferred that the dashed bond at the left side of each of the structures for L1to L28indicates the bond between the linker L and the nitrogen atom bearing R1, and the dashed bond at the right side of each of the structures for L1to L28indicates the bond between the linker L and the nitrogen atom bearing R2.

[0052] Each occurrence of R1represents the connection of a further linker L between the linker and the nitrogen atom bearing R1, wherein R1is joined with R1via this further linker, thus forming a cyclic structure that is preferably a 4 - 10-membered cyclic structure, preferably a 5 - 8-membered cyclic structure, most preferably a 6-membered cyclic structure, which is built up from the nitrogen atom bearing R1, atoms of the backbone of the linker, and atoms which make up the bridge joining R1and R1. Likewise, each occurrence of R2represents the connection of a further linker L between the linker and the nitrogen atom bearing R2, wherein R2is joined with R2via this further linker, thus forming a 4 - 10-membered cyclic structure, preferably a 5 - 8-membered cyclic structure, most preferably a 6- membered cyclic structure, which is built up from the cationic nitrogen atom, 1 - 4 atoms of the backbone of the linker, and 1 - 4 atoms which make up the bridge joining R2and R2. Likewise, each occurrence of R5and R5represent the connection of a further linker L between one backbone atom of the linker, bearing R5, and another backbone atom of the linker, bearing R5, wherein R5is joined with R5via that further linker, thus forming a cyclic structure that is preferably 4 - 10-membered, preferably a 5 - 8-membered cyclic structure, most preferably a 6-membered cyclic structure, which is built up from 2 - 5 atoms of the backbone of the linker, and 1 - 5 atoms which make up the bridge joining R5and R5. Thus, in linkers L10, L22, L23, L24and L25, R1is joined to R1via a second linker, preferably a - CH2-CH2- or -CH2-CH2-CH2- bridge, more preferably a -CH2-CH2- bridge. Thus, in a compound comprising linker L10, wherein R1and R1are joined via a -CH2-CH2- bridge, the nitrogen atom bearing R1is embedded in a six-membered cyclic structure, which is built up from the nitrogen atom bearing R1, two carbon atoms and one nitrogen atom of the backbone of the linker, and two more carbon atoms which make up the bridge of R1and R1. This -CH2-CH2- bridge between the nitrogen atom bearing R= and the central nitrogen atom in the backbone of linker L10may be represented as L1. Likewise, in linkers L18, L19and L21, R2is joined to R2via a second linker, preferably a -CH2-CH2- or -CH2-CH2- CH2- bridge, more preferably a -CH2-CH2-CH2- bridge. Likewise, in linker L20and L26, R5is joined to R5via a second linker, preferably a -CH2-CH2- or -CH2-CH2-CH2- bridge, more preferably a -CH2- CH2- bridge.

[0053] In some embodiments, a further linker L in compounds that comprise a further linker L is -CH2- or - (CH2)2- or -(CH2)3- or -(CH2)4-. In preferred embodiments, a further linker L in compounds that comprise a further linker L is-(CH2)2- or -(CH2)3- or -(CH2)4-. In more preferred embodiments, a further linker L in compounds that comprise a further linker L is-(CH2)2- or -(CH2)3-

[0054] Linker L26comprises a disubstituted cycloalkyl moiety, preferably a disubstituted cyclohexyl moiety, and may thus occur in either the c / s-form or the trans-form, preferably in the trans-form.

[0055] Linker L27comprises a bicyclic cycloalkyl moiety, preferably a bicyclic cyclooctyl moiety. When L = L27it is highly preferred that L, R2, and R3together comprise 7, 8, 9, 10, 1 1 , or 12 carbon atoms. Most preferably L27is comprised in an azabicyclooctane such as azabicyclo[2.2.2]octane.

[0056] Linkers L11, L12, L13, L14, L15, L18(as long as R2-R2’ is not -C(O)-), L19(as long as R2-R2’ is not - CH2-), L20(as long as R5-R5is not -CH2-), L21(as long as R2-R2is not -CH2-CH2-), L22(as long as R1-R1is not -CH2-CH2-), L23(as long as R1-R1is not -CH2-CH2-), L24(as long as R1-R1is not - CH2-) and L25(as long as R1-R1is not -CH2-) comprise an additional stereocenter. The stereoisomer, when indicated in the structures of those linkers, above is meant as illustrative, not as limiting. As indicated further above, each stereocenter present in the compounds according to the invention may individually be present in each of its stereoisomeric forms, either S or R, or as a mixture of both isomers in any ratio. In view of the stereocenter already present at the carbon atom indicated as C*, the compounds having these linkers may be ( / ?, / ?); (S,R); (R,S); or (S,S). Throughout the description, the first designator (R or S) of the configuration is for the carbon atom indicated as C*, and the second designator thereof defines the configuration of the additional stereocenter that may be present in the compound according to the invention.

[0057] In some embodiments preferred linkers are L5, L8, L11, L12, L16, L17, L19, L21, L26, L27, and L28. Especially preferred linkers are L5, L8, L11, L12, L16, L17, L19, L21and L26. Even more preferred linkers are l_ii |_16 |_19anc| |_26anc|most preferably the linker is L19. Preferably, L19is combined with R2-R2= L1or L3, most preferably with R2-R2= L3. Preferably, L21is combined with R2-R2= L1or L3, most preferably with R2-R2’ = L1. Preferably, L26is combined with R5-R5= L1or L3, more preferably with R5-R5= L1, most preferably wherein the cyclohexyl is trans- ,4-disubstituted. Especially preferred is the combination of linker L19with R2-R2= L3and R3= H, Me, Et, iPr, CH2OCH3 or CH2CF3, more preferably R3= Me, Et, iPr or CH2CF3, most preferably R3= H.

[0058] It is preferred that linker L contains 1 - 5 optionally substituted backbone atoms and / or linker L contains at least one backbone atom other than carbon. It is especially preferred that the nitrogen atom bearing R2is connected to a backbone atom of the linker via a second linker wherein R2is joined with R2’, more preferably wherein the cyclic structure thus formed is a piperidine ring, a pyrrolidine ring, an imidazolidine ring, a pyrazolidine ring or an azepane ring, most preferably a piperidine ring, and / or at least one of the backbone atoms is substituted with a carboxylic acid moiety. It is preferred that L is any one of L2, L4- L21, L23, L25, L26, L27, and L28especially preferred that L is any one of L2, L4- L21, L23, L25and L26, more preferably one of L5, L8, L11, L12, L16, L17, L19, L21and L26. When X and R4are present, it is preferred that linker L contains 3 - 10 backbone atoms, or 2 backbone atoms of which one is connected to the nitrogen atom bearing R2via a second linker. In such cases it is preferred that L is any one of L2-L28, especially preferred that L is any one of L2- L26, more preferably one of L5, L8, L11, L12, [_16,L17;L19, |_21a n d[_26

[0059] In one embodiment, linker L is L1and R1and R2are joined together in a cyclic structure via a second linker L1, thus forming a six-membered piperazine ring including in total four carbon atoms from the two linkers, the nitrogen atom bearing R1and the nitrogen atom bearing R2. For this embodiment, R3is preferably Ci-Ce alkyl substituted with a hydroxyl group, more preferably -CH2CH2OH. In one embodiment, linker L is L19and R2and R2are joined together in a cyclic structure via a second linker which is L3, thus forming a six-membered piperidene ring including in total five carbon atoms from the linkers and also including the nitrogen atom bearing R2.

[0060] In a preferred embodiment, the compound is represented by general structure (la) or (lb), wherein:

[0061] - R1is selected from H, Ci - Ce alkyl or Ci - Ce alkenyl, or R1is joined with a backbone atom of the linker L in a cyclic structure;

[0062] - R2is joined with a backbone atom of the linker L to form a cyclic structure selected from a piperidine ring, a pyrrolidine ring, an imidazolidine ring, a pyrazolidine ring or an azepane ring; and

[0063] - R3is selected from H, Ci - Ce alkyl or Ci - Ce alkenyl, wherein the alkyl or alkenyl moiety may be substituted with one or more halogen atoms, hydroxyl moieties or (halo)alkoxy moieties, or R3is absent when the distal nitrogen atom is part of an imine moiety.

[0064] In preferred embodiments linker L together with to at least one of R1or R2forms a bridging moiety that is a further linker L which forms a cyclic structure, wherein that cyclic structure is a 4-10 membered heterocycle. Preferably that cyclic structure is a 4-9, more preferably 4-8, still more preferably 4-7, even more preferably 5-7, most preferably 5-6 membered heterocycle. For example, when linker L is -CHR2’- CH2-, and R2and R2’ together form -CH2CH2CH2-, a six-membered ring may be formed, in this case a piperidinyl ring.

[0065] In a preferred embodiment, the compound of general structure (la) or (lb) is represented by structure (Vila), (Vllb), (Vile), (Vlld), (Vile), or (Vllf):

[0066] More preferred are structure (Vila), (Vllb), (Vile), and (Vllf), most preferred are structure (Vila) and (Vllb). In structure (Vila), (Vllb), (Vile), (Vlld), (Vile), and (Vllf) it is preferred that each R7is C1-C2 alkyl, more preferably methyl.

[0067] Herein, R2is joined with a backbone atom via a second linker forming a cyclic structure. Herein, R3, R4, X and R7are as defined above. In these compounds the carbon atom indicated with C* above may be in R-configuration or in S-configuration, preferably it is in S-configuration. Likewise, the carbon atom at the 2-position of the piperidine ring may be in R-configuration or in S-configuration, preferably it is in R-configuration. Thus, the configuration of the compounds according to structure (Vila) or (Vllb) may be (R,R); (S,R); (R,S); or (S,S), preferably it is (S,R). In highly preferred embodiments, the invention provides a compound for use as described above, wherein the compound is represented by structure (Vllb), wherein each R7is methyl, X is as defined above and is preferably Ch; R3is as defined above and is preferably hydrogen; and R4is as defined above and is preferably hydrogen. It is even more preferred for this compound to be of the S,R configuration.

[0068] In a preferred embodiment, the compound of general structure (I) is represented by structure (Villa) or (VIII b) :

[0069] Herein, R2is joined with a backbone atom via a second linker forming a cyclic structure. Herein, R3, R4and X are as defined above. In the compound according to structure (Villa) or (Vlllb), R3is preferably H or Ci - C2 alkyl, most preferably R3is H. In the compound according to structure (Villa) or (Vlllb), R4is preferably H or Ci - C2 alkyl, most preferably R4is H. In the compound according to structure (Villa) or (VIII b) , X is preferably Cl, I, TFA or formate, most preferably X is Cl. In the compound according to structure (Villa) or (Vlllb), the carbon atom indicated as C* above may be in R- configuration or in S-configuration, preferably it is in S-configuration. Likewise, the carbon atom at the 2-position of the piperidine ring may be in R-configuration or in S-configuration, preferably it is in R- configuration. Thus, the configuration of the compounds according to structure (Villa) or (Vlllb) may be (R,R); (S,R); (R,S); or (S,S), preferably it is (S,R). In one embodiment, the compound of general structure (I) is represented by structure (Villa). In an alternative embodiment, the compound of general structure (I) is represented by structure (Vlllb).

[0070] In a preferred embodiment, the compound of general structure (la) or (lb) is represented by structure

[0071] (IXa) (IXb)

[0072] Herein, R2is joined with a backbone atom via a second linker forming a cyclic structure. Herein, R3is as defined above. In the compound according to structure (IXa) or (IXb), R3is preferably H or Ci - C2 alkyl, most preferably R3is H. In the compound according to structure (IXa) or (IXb), the chiral carbon indicated as C* above may be in R-configuration or in S-configuration, preferably it is in S- configuration. Likewise, the carbon atom at the 2-position of the piperidine ring may be in R- configuration or in S-configuration, preferably it is in R-configuration. Thus, the configuration of the compounds according to structure (IXa) or (IXb) may be (R,R); (S,R); (R,S); or (S,S), preferably it is (S,R). In one embodiment, the compound of general structure (la) is represented by structure (IXa). In an alternative embodiment, the compound of general structure (lb) is represented by structure (IXb).

[0073] In a preferred embodiment, the compound is according to general structure (IVa) or (IVb), wherein either R4= H and X = Cl or wherein R4and X are absent, and wherein:

[0074] (A) L = L1, R1-R2= L1, R3= H; (B) L = L1, R1= H, R2= H, R3= H;

[0075] (C) L = L2, R1= H, R2= H, R3= H;

[0076] (D) L = L3, R1= H, R2= H, R3= H;

[0077] (E) L = L4, R1= H, R2= H, R3= absent;

[0078] (F) L = L5, R1= H, R2= H, R3= absent;

[0079] (G) L = L6, R1= H, R2= H, R3= absent;

[0080] (H) L = L3, R1= H, R2= Me, R3= Me;

[0081] (I) L = L1, R1= H, R2= Me, R3= Me;

[0082] (J) L = L7, R1= H, R2= H, R3= absent;

[0083] (K) L = L8, R1= H, R2= H, R3= absent;

[0084] (L) L = L9, R1= H, R2= H, R3= absent;

[0085] (M) L = L10, R1-R1= L1, R2= H, R3= absent;

[0086] (N) L = L11, R1= H, R2= H, R3= H;

[0087] (O) L = L12, R1= H, R2= H, R3= absent;

[0088] (P) L = L13, R1= H, R2= H, R3= H;

[0089] (Q) L = L14, R1= H, R2= H, R3= H;

[0090] (R) L = L15, R1= H, R2= H, R3= H;

[0091] (S) L = L11, R1= H, R2= Me, R3= Me

[0092] (T) L = L16, R1= H, R2= H, R3= H;

[0093] (U) L = L17, R1= H, R2= H, R3= H;

[0094] (V) L = L16, R1= H, R2= Me, R3= Me;

[0095] (W) L = L18, R1= H, R2-R2= L3, R3= H;

[0096] (X) L = L19, R1= H, R2-R2= L3, R3= H;

[0097] (Y) L = L20, R1= H, R2= H, R5-R5’ = L3, R3= absent;

[0098] (Z) L = L21, R1= H, R2-R2= L1, R3= H;

[0099] (AA) L = L22, R1-R1= L1, R2= H, R3= H;

[0100] (AB) L = L23, R1-R1= L1, R2= H, R3= H;

[0101] (AC) L = L24, R1-R1= L3, R2= H, R3= H;

[0102] (AD) L = L25, R1-R1= L3, R2= H, R3= absent;

[0103] (AE) L = L26, R1= H, R2= H, R5-R5’ = L1, R3= H.

[0104] (AF) L = L19, R1= H, R2-R2= L3, R3= Me;

[0105] (AG) L = L19, R1= H, R2-R2= L1, R3= H;

[0106] (AH) L = L21, R1= H, R2-R2= L1, R3= Me;

[0107] (Al) L = L27, R1= H, R2-R2’ = -CH2-, R3-R3’ = L1, preferably R4= H, preferably X = Cl;

[0108] (AJ) L = L28, R1= H, R2= H, R3= H, preferably R4= H, preferably X = Cl;

[0109] (AK) L = L1, R1-R2= L1, R3= -CH2CH2OH, R4= H, X = Cl;

[0110] (AL) L = L1, R1-R2= L1, R3= -CH2CH2OH, R4= absent, X = absent.

[0111] It is thus preferred that the compound according to structure (la) or (lb) is selected from compounds A - AL defined above, more preferably from compounds A - AH defined above, even more preferably selected from compounds A - AK based on general strucuture (IVb), most preferably selected from compounds A - AH based on general strucuture (IVb). Especially preferred compounds are selected from F, K, N, O, U, V, T, X, Z, AE, AF, AG, AH, Al, and AJ, more preferred compounds are selected from F, K, N, O, U, V, T, X, Z, AE, AF, AG and AH, even more preferably N, T, X and AE, most preferably X. Herein, preferably R4= H and X = Cl, and the compound is preferably of general structure (IVb). In some embodiments, compounds X, AK, and AL are preferred.

[0112] Compound F may have the R-configuration, the S-configuration or a mixture thereof, preferably compound F is a mixture of the R- and S-enantiomers, more preferably a racemic mixture. Compound K may have the R-configuration, the S-configuration or a mixture thereof, preferably compound K is a mixture of the R- and S-enantiomers, more preferably a racemic mixture. Compound N may have the

[0113] R,R-configuration, R, S-configuration, S,R-configuration, the S, S-configuration or any mixture thereof, preferably compound N has the R,R-configuration or the S,R-configuration, most preferably the Reconfiguration. Compound O may have the R,R-configuration, R, S-configuration, S,R-configuration, the

[0114] S, S-configuration or any mixture thereof, preferably compound O is a mixture of the R,S- and S,S- diastereomers more preferably about 1 / 1 (mol / mol) mixture. Compound U may have the R- configuration, the S-configuration or a mixture thereof, preferably compound U has the R-configuration or the S-configuration. Compound V may have the R-configuration, the S-configuration or a mixture thereof, preferably compound V has the R-configuration. Compound T may have the R-configuration, the S-configuration or a mixture thereof, preferably compound T has the R-configuration or the S- configuration, most preferably the R-configuration. Compound X may have the R, R-configuration, Reconfiguration, S, R-configuration, the S, S-configuration or any mixture thereof, preferably compound X has the R, S-configuration or the S, R-configuration, most preferably the S, R-configuration. Compound Z may have the R-configuration, the S-configuration or a mixture thereof, preferably compound Z is a mixture of the R- and S-enantiomers, more preferably a racemic mixture. Compound AE may have the R rans-configuration, R,c / s-configuration, S rans-configuration, the S,c / s-configuration or any mixture thereof, preferably compound AE has the R rans-configuration or the S rans-configuration, most preferably the R rans-configuration. Compound AF may have the R, R-configuration, R, S-configuration, S, R-configuration, the S, S-configuration or any mixture thereof, preferably compound AF has the S,R- configuration. Compound AG may have the R, R-configuration, R, S-configuration, S, R-configuration, the S, S-configuration or any mixture thereof, preferably compound AG has the S, S-configuration or the S, R-configuration. Compound AH may have the R-configuration, the S-configuration or a mixture thereof, preferably compound AH has the S-configuration. Herein, the first designator (R or S) of the configuration is for the carbon atom indicated as C*, and in case an additional stereocenter is present in the compound according to the invention, the second designator thereof defines the configuration thereof. Compound AJ may have the R, R-configuration, the R, S-configuration, the S, R-configuration, the S, S-configuration, or a mixture thereof, preferably compound AJ has the S, R-configuration or the R, R-configuration or a mixture thereof, most preferably compound AJ has the R,R-confuguration.

[0115] Highly preferred compounds include compound N in the R, R-configuration (R,R-N), compound T in the R-configuration (R-T), compound AE in the R rans-configuration (R rans-AE), compound AJ in the R -configuration (R-AJ), and compound X in any configuration. The most preferred compounds include compound N in the R, R-configuration (R,R-N), compound T in the R-configuration (R-T), compound AE in the R rans-configuration (R,trans-AE) and compound X in any configuration, most preferably the compound according to the invention is compound X in the S, R-configuration (S,R-X). In one embodiment, these most preferred compounds according to the invention are compound N in the Reconfiguration (R,R-N), compound T in the R-configuration (R-T), compound AE in the R,trans- configuration (R,trans-AE) and compound X in any configuration, and optionally compound AJ preferably as R,R-AJ, wherein R4= H and X = Cl, more preferably the compound according to the invention is compound X in the S, R-configuration (S,R-X), wherein R4= H and X = Cl. In one embodiment, these most preferred compounds according to the invention are compound N in the Reconfiguration (R,R-N), compound T in the R-configuration (R-T), compound AE in the R,trans- configuration (R,trans- / E) and compound X in any configuration, and optionally compound AJ preferably as R,R-AJ, wherein most preferably the compound according to the invention is compound X in the S, R-configuration (S,R-X).

[0116] In one embodiment, these most preferred compounds according to the invention are compound N in the R, R-configuration (R,R-N), compound T in the R-configuration (R-T), compound AE in the

[0117] R,frans-configuration (R,trans- / E) and compound X in any configuration, and optionally compound AJ preferably as R,R-AJ, most preferably the compound according to the invention is compound X in the

[0118] S, R-configuration (S,R-X).

[0119] In one embodiment, these most preferred compounds according to the invention are compound N in the R, R-configuration (R,R-N), compound T in the R-configuration (R-T), compound AE in the R,frans-configuration (R,trans-AE) and compound X in any configuration, and optionally compound AJ preferably as R,R-AJ, wherein R4= H and X = Cl, most preferably the compound according to the invention is compound X in the S, R-configuration (S,R-X), wherein R4= H and X = Cl.

[0120] In one embodiment, these most preferred compounds according to the invention are compound N in the R, R-configuration (R,R-N), compound T in the R-configuration (R-T), compound AE in the

[0121] R,frans-configuration (R,trans-AE) and compound X in any configuration, and optionally compound AJ preferably as R,R-AJ, most preferably the compound according to the invention is compound X in the

[0122] S, R-configuration (S,R-X).

[0123] In one preferred embodiment, these most preferred compounds according to the invention are compound N in the R, R-configuration (R,R-N), compound T in the R-configuration (R-T), compound AE in the R,frans-configuration (R,trans-AE) and compound X in any configuration, and optionally compound AJ preferably as R,R-AJ, most preferably the compound according to the invention is compound X in the S, R-configuration (S,R-X), wherein the compound is of structure (lb).

[0124] In one highly preferred embodiment, these most preferred compounds according to the invention are compound N in the R, R-configuration (R,R-N), compound T in the R-configuration (R-T), compound AE in the R,frans-configuration (R,trans-AE) and compound X in any configuration, and optionally compound AJ preferably as R,R-AJ, wherein the compound is of structure (lb), wherein R4= H and X = Cl, most preferably the compound according to the invention is compound X in the S, R-configuration (S,R-X), wherein the compound is of structure (lb), wherein R4= H and X = Cl.

[0125] In another highly preferred embodiment, these most preferred compounds according to the invention are compound N in the R, R-configuration (R,R-N), compound T in the R-configuration (R-T), compound AE in the R rans-configuration (R rans-AE) and compound X in any configuration, and optionally compound AJ preferably as R,R-AJ, wherein the compound is of structure (lb), most preferably the compound according to the invention is compound X in the S,R-configuration (S,R-X), wherein the compound is of structure (lb).

[0126] Compounds of the open configuration have been found to be more potent than compounds of the closed configuration. However, compounds of the closed configuration have shown a higher oral bioavailability than compounds of the open configuration.

[0127] The invention also includes all stereoisomers and geometric isomers of the compounds, including diastereomers, enantiomers, and cis / trans (E / Z) isomers. The invention also includes mixtures of stereoisomers and / or geometric isomers in any ratio, including, but not limited to, racemic mixtures.

[0128] An “effective amount” of a compound is an amount of a compound which, when administered to a subject, is sufficient to reduce or eliminate either one or more symptoms of a disease, or to delay the progression of one or more symptoms of a disease, or to reduce the severity of one or more symptoms of a disease, or to suppress the manifestation of a disease, or to suppress the manifestation of adverse symptoms of a disease. An effective amount can be given in one or more administrations.

[0129] The “effective amount” of that may be combined with carrier materials to produce a single dosage form which may vary depending upon the host to which the active ingredient is administered and the particular mode of administration. The unit dosage chosen is usually fabricated and administered to provide a desired final concentration of the compound in the subject, such as in the blood.

[0130] The effective amount (i.e. the effective total daily dose), preferably for adults, is herein defined as a total daily dose of about 5 to 2000 mg, or about 10 to 1000 mg, or about 20 to 800 mg, or about 30 to 800 mg or about 30 to 700 mg, or about 20 to 700 mg or about 20 to 600 mg, or about 30 to 600 mg, or about 30 to 500 mg, about 30 to 450 mg or about 30 to 400 mg, or about 30 to 350 mg or about 30 to 300 mg or about 50 to 600 mg, or about 50 to 500 mg, or about 50 to 450 mg, or about 50 to 400 mg or about 50 to 300 mg, or about 50 to 250 mg, or about 100 to 250 mg or about 150 to 250 mg. In the most preferred embodiment, the effective amount is about 200 mg.

[0131] Alternatively, the effective amount of the compound, preferably for adults, preferably is administered per kg body weight. The total daily dose, preferably for adults, is therefore about 0.05 to about 40 mg / kg, about 0.1 to about 20 mg / kg, about 0.2 mg / kg to about 15 mg / kg, or about 0.3 mg / kg to about 15 mg / kg or about 0.4 mg / kg to about 15 mg / kg or about 0.5 mg / kg to about 14 mg / kg or about 0.3 mg / kg to about 14 mg / kg or about 0.3 mg / kg to about 13 mg / kg or about 0.5 mg / kg to about 13 mg / kg or about 0.5 mg / kg to about 1 1 mg / kg.

[0132] The total daily dose for children is preferably at most 200 mg. More preferably the total daily dose is about 5 to 200 mg, about 10 to 200 mg, about 20 to 200 mg about 30 to 200 mg about 40 to 200 mg, or about 50 to 200 mg. Preferably, the total daily dose for children is about 5 to 150 mg, about 10 to 150 mg, about 20 to 150 mg about 30 to 150 mg about 40 to 150 mg, or about 50 to 150 mg. More preferably, the total daily dose is about 5 to 100 mg, about 10 to 100 mg, about 20 to 100 mg about 30 to 100 mg about 40 to 100 mg, or about 50 to 100 mg. Even more preferably, the total daily dose is about 5 to 75 mg, about 10 to 75 mg, about 20 to 75 mg about 30 to 75 mg about 40 to 75 mg, or about 50 to 75 mg.

[0133] Alternative examples of dosages which can be used are an effective amount of the compounds of the invention within the dosage range of about 0.1 pg / kg to about 300 mg / kg, or within about 1.0 pg / kg to about 40 mg / kg body weight, or within about 1 .0 pg / kg to about 20 mg / kg body weight, or within about 1 .0 pg / kg to about 10 mg / kg body weight, or within about 10.0 pg / kg to about 10 mg / kg body weight, or within about 100 pg / kg to about 10 mg / kg body weight, or within about 1 .0 mg / kg to about 10 mg / kg body weight, or within about 10 mg / kg to about 100 mg / kg body weight, or within about 50 mg / kg to about 150 mg / kg body weight, or within about 100 mg / kg to about 200 mg / kg body weight, or within about 150 mg / kg to about 250 mg / kg body weight, or within about 200 mg / kg to about 300 mg / kg body weight, or within about 250 mg / kg to about 300 mg / kg body weight. Other dosages which can be used are about 0.01 mg / kg body weight, about 0.1 mg / kg body weight, about 1 mg / kg body weight, about 10 mg / kg body weight, about 20 mg / kg body weight, about 30 mg / kg body weight, about 40 mg / kg body weight, about 50 mg / kg body weight, about 75 mg / kg body weight, about 100 mg / kg body weight, about 125 mg / kg body weight, about 150 mg / kg body weight, about 175 mg / kg body weight, about 200 mg / kg body weight, about 225 mg / kg body weight, about 250 mg / kg body weight, about 275 mg / kg body weight, or about 300 mg / kg body weight.

[0134] Compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided dosage of two, three or four times daily.

[0135] In a preferred embodiment of the invention, "subject", "individual", or "patient" is understood to be an individual organism, preferably a vertebrate, more preferably a mammal, even more preferably a primate and most preferably a human. The dose as defined herein is preferably suitable for administration to humans.

[0136] In a further preferred embodiment of the invention, the human is an adult, e.g. a person that is 18 years or older. In addition, it is herein understood that the average weight of an adult person is 62 kg, although the average weight is known to vary between countries. In another embodiment of the invention the average weight of an adult person is therefore between about 50 - 90 kg. It is herein understood that the effective dose as defined herein is not confined to subjects having an average weight. Preferably, the subject has a BMI (Body Mass Index) between 18.0 to 40.0 kg / m2, and more preferably a BMI between 18.0 to 30.0 kg / m2.

[0137] Alternatively, the subject to be treated is a child, e.g. a person that is 17 years or younger. In addition, the subject to be treated may be a person between birth and puberty or between puberty and adulthood. It is herein understood that puberty starts for females at the age of 10 -11 years and for males at the age of 11 - 12 year. Furthermore, the subject to be treated may be a neonate (first 28 days after birth), an infant (0-1 year), a toddler (1-3 years), a preschooler (3-5 years); a school-aged child (5-12 years) or an adolescent (13-18 years).

[0138] A compound for use as defined herein may be administered as a composition. The compositions comprising the compounds as described above, can be prepared as a medicinal or pharmaceutical preparation or in various other media, such as foods for humans or animals, including medical foods and dietary supplements. A "medical food" is a product that is intended for the specific dietary management of a disease or condition for which distinctive nutritional requirements exist. By way of example medical foods may include vitamin and mineral formulations fed through a feeding tube (referred to as enteral administration). A "dietary supplement" shall mean a product that is intended to supplement the human diet and is typically provided in the form of a pill, capsule, and tablet or like formulation. By way of example a dietary supplement may include one or more of the following ingredients: vitamins, minerals, herbs, botanicals; amino acids, dietary substances intended to supplement the diet by increasing total dietary intake, and concentrates, metabolites, constituents, extracts or combinations of any of the foregoing. Dietary supplements may also be incorporated into food, including, but not limited to, food bars, beverages, powders, cereals, cooked foods, food additives and candies; or other functional foods designed to promote bone health.

[0139] The subject compositions thus may be compounded with other physiologically acceptable materials that can be ingested including, but not limited to, foods. In addition or alternatively, the compositions for use as described herein may be administered orally in combination with (the separate) administration of food. A preferred composition comprises the compound as defined herein and a pharmaceutically acceptable excipient. The composition is preferably for use as defined herein.

[0140] The compositions may be administered alone or in combination with other pharmaceutical or cosmetic agents and can be combined with a physiologically acceptable carrier thereof. In particular, the compounds described herein can be formulated as pharmaceutical or cosmetic compositions by formulation with additives such as pharmaceutically or physiologically acceptable excipients carriers, and vehicles. Suitable pharmaceutically or physiologically acceptable excipients, carriers and vehicles include processing agents and drug delivery modifiers and enhancers, such as, for example, calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-P-cyclodextrin, polyvinylpyrrolidinone, low melting waxes, ion exchange resins, and the like, as well as combinations of any two or more thereof. Other suitable pharmaceutically acceptable excipients are described in "Remington's Pharmaceutical Sciences, " Mack Pub. Co. , New Jersey (1991), and "Remington: The Science and Practice of Pharmacy, " Lippincott Williams & Wilkins, Philadelphia, 20th edition (2003), 21stedition (2005) and 22ndedition (2012).

[0141] Pharmaceutical or cosmetic compositions containing the compounds for use according to the invention may be in any form suitable for the intended method of administration, including, for example, a solution, a suspension, or an emulsion. In a preferred embodiment, the compound is administered in a solid form or in a liquid form.

[0142] Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound may be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may also comprise additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.

[0143] Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water or saline. Such compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, cyclodextrins, and sweetening, flavoring, and perfuming agents.

[0144] Liquid carriers are typically used in preparing solutions, suspensions, and emulsions. In a preferred embodiment, liquid carriers / liquid dosage forms contemplated for use in the practice of the present invention include, for example, water, saline, pharmaceutically acceptable organic solvent(s), pharmaceutically acceptable oils or fats, and the like, as well as mixtures of two or more thereof. In a preferred embodiment, the compound for use as defined herein is admixed with an aqueous solution prior to administration. The aqueous solution should be suitable for administration and such aqueous solutions are well known in the art. It is further known in the art that the suitability of an aqueous solution for administration may be dependent on the route of administration.

[0145] In a preferred embodiment, the aqueous solution is an isotonic aqueous solution. The isotonic aqueous solution preferably is almost (or completely) isotonic to blood plasma. In an even more preferred embodiment, the isotonic aqueous solution is saline.

[0146] The liquid carrier may contain other suitable pharmaceutically acceptable additives such as solubilizers, emulsifiers, nutrients, buffers, preservatives, suspending agents, thickening agents, viscosity regulators, stabilizers, flavorants and the like. Preferred flavorants are sweeteners, such as monosaccharides and / or disaccharides. Suitable organic solvents include, for example, monohydric alcohols, such as ethanol, and polyhydric alcohols, such as glycols. Suitable oils include, for example, soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, and the like.

[0147] For parenteral administration, the carrier can also be an oily ester such as ethyl oleate, isopropyl myristate, and the like. Compositions for use in the present invention may also be in the form of microparticles, microcapsules, liposomal encapsulates, and the like, as well as combinations of any two or more thereof.

[0148] Time-release, sustained release or controlled release delivery systems may be used, such as a diffusion controlled matrix system or an erodible system, as described for example in: Lee, "Diffusion- Controlled Matrix Systems", pp. 155-198 and Ron and Langer, "Erodible Systems", pp. 199-224, in "Treatise on Controlled Drug Delivery", A. Kydonieus Ed. , Marcel Dekker, Inc. , New York 1992. The matrix may be, for example, a biodegradable material that can degrade spontaneously in situ and in vivo for, example, by hydrolysis or enzymatic cleavage, e.g. , by proteases. The delivery system may be, for example, a naturally occurring or synthetic polymer or copolymer, for example in the form of a hydrogel. Exemplary polymers with cleavable linkages include polyesters, polyorthoesters, polyanhydrides, polysaccharides, poly(phosphoesters), polyamides, polyurethanes, poly(imidocarbonates) and poly(phosphazenes).

[0149] The compounds of the present invention can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a compound as defined herein, stabilizers, preservatives, excipients, and the like. The preferred lipids are the phospholipids and phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N. Y., p. 33 et seq (1976).

[0150] A pharmaceutical or cosmetic composition can comprise a unit dose formulation, where the unit dose is a dose sufficient to have a therapeutic or suppressive effect of a disorder or condition as defined herein. The unit dose may be sufficient as a single dose to have a therapeutic or suppressive effect of a disorder or condition as defined herein. Alternatively, the unit dose may be a dose administered periodically in a course of treatment or suppression of a disorder or condition as defined herein. During the course of the treatment, the concentration of the subject compositions may be monitored to insure that the desired level of the compound of the invention is maintained.

[0151] In a preferred embodiment the invention pertains to a compound as defined herein for use in a method of treating or preventing a bone disorder by administration of an effective total daily dose, and wherein preferably the compound reaches a blood steady state level within 5 days. More preferably steady state levels are reached within 4 days, even more preferably within 3 days and most preferably steady state levels are reached within 2 days after the first administration.

[0152] Steady state is herein understood that the overall intake of a compound as defined above is (roughly) in dynamic equilibrium with its elimination. During steady state, the plasma levels of the compound preferably maintained within the effective therapeutic range. Put differently, the levels of the compound in the blood are maintained between the minimum therapeutically effective concentration and the maximum therapeutically effective concentration. Below the minimum concentration, the compound does not have sufficient therapeutic effect to be considered efficacious. Above the maximum concentration, side effects increase eventually leading to toxicity.

[0153] To maintain an effective therapeutic range during treatment, the average plasma concentrations (Cav) of the compound as defined herein is maintained between about 10 ng / ml to about 20000 ng / ml, or about 20 ng / ml to about 10000 ng / ml, or about 30 ng / ml to about 5000 ng / ml, or between about 30 ng / ml to about 4000 ng / ml, or between about 30 ng / ml to about 3000 ng / ml, or between about 30 ng / ml to about 2000 ng / ml, or about 30 ng / ml to about 1000 ng / ml, or between about 50 ng / ml to about 5000 ng / ml, or between about 100 ng / ml to about 5000 ng / ml, or between about 50 ng / ml to about 4000 ng / ml, or between about 50 ng / ml to about 3000 ng / ml, or between about 50 ng / ml to about 2000 ng / ml, or between about 50 ng / ml to about 1000 ng / ml. In a more preferred embodiment, the average plasma concentration of the compound is maintained between about 50 ng / ml - 500 ng / ml or 100 ng / ml - 500 ng / ml.

[0154] The average plasma concentrations may be determined using any conventional method known in the art. However in a preferred embodiment, the plasma concentrations are determined by extracting the compound as defined herein from human plasma by protein precipitation, followed by Liquid Chromatography - Tandem Mass Spectrometry (LC-MS / MS). The concentration of the compound may subsequently be determined using calibration standards.

[0155] The compound as defined herein may be metabolized and instead of, or in addition to the nonmetabolized compound, the effective therapeutic range of the metabolized compound may be maintained during treatment. In a preferred embodiment of the invention, the average plasma concentrations (Cav) of the metabolized compound is maintained between about 5 ng / ml to about 5000 ng / ml, or about 10 ng / ml to about 2000 ng / ml, or about 20 ng / ml to about 1000 ng / ml, or between about 20 ng / ml to about 800 ng / ml, or between about 20 ng / ml to about 600 ng / ml, or between about 20 ng / ml to about 400 ng / ml, or about 20 ng / ml to about 200 ng / ml, or between about 30 ng / ml to about 1000 ng / ml, or between about 50 ng / ml to about 1000 ng / ml, or between about 30 ng / ml to about 800 ng / ml, or between about 30 ng / ml to about 600 ng / ml, or between about 30 ng / ml to about 400 ng / ml, or between about 30 ng / ml to about 200 ng / ml. In a more preferred embodiment, the average plasma concentration of the compound is maintained between about 40 ng / ml - 500 ng / ml or 50 ng / ml - 200 ng / ml.

[0156] During or after administration of the compound as defined herein, the maximum plasma concentrations (Cmax) remain below about 20000 ng / ml or below 10000 ng / ml or below 5000 ng / ml or below about 4000 ng / ml or below about 3000 ng / ml or below about 2000 ng / ml or below about 1000 ng / ml. In the most preferred embodiment, the maximum plasma concentrations remain below about 500 ng / ml.

[0157] Similarly, the maximum plasma concentrations of the metabolized compound remain below about 5000 ng / ml, or 2000 ng / ml, or 1000 ng / ml, or below about 800 ng / ml or below about 600 ng / ml or below about 400 ng / ml. In the most preferred embodiment, the maximum plasma concentrations of the metabolized compound remain below about 250 ng / ml.

[0158] To maintain an effective range during treatment, the compound may be administered once a day, or once every two, three, four or five days. However preferably, the compound may be administered at least once a day. Hence in a preferred embodiment, the invention pertains to a compound as defined herein above, for use in a method of treating or preventing a bone disorder by administration of an effective total daily dose, wherein the effective dose is defined herein above. The total daily dose may be administered as a single daily dose. Alternatively, the compound is administered at least twice daily. Hence, the compound as defined herein may be administered once, twice, three, four or five times a day. As such, the total daily dose may be divided over the several doses (units) resulting in the administration of the total daily dose as defined herein. In a preferred embodiment, the compound is administered twice daily. It is further understood that the terms “twice daily”, “bid” and “bis in die” can be used interchangeable herein.

[0159] In a preferred embodiment, the total daily dose is divided over several doses per day. These separate doses may differ in amount. For example for each total daily dose, the first dose may have a larger amount of the compound than the second dose or vice versa. However preferably, the compound is administered in similar or equal doses. Therefore in a most preferred embodiment, the compound is administered twice daily in two similar or equal doses.

[0160] In a further preferred embodiment of the invention, the total daily dose of the compound as defined herein above is administered in at least two separate doses. The interval between the administration of the at least two separate doses is at least about 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours, preferably the interval between the at least two separate doses is at least about 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours and more preferably the interval between the at least two separate doses is at least about 8, 9, 10, 11 or 12 hours. The composition can be administered in an effective total daily dose as defined herein, either as a prophylaxis or treatment, to a patient in any of a number of methods. In particular, the method of administration can vary based on the individual subject, the condition or the stage of disease, and other factors evident to one skilled in the art.

[0161] The compounds for a use as defined herein may be administered enterally, orally, parenterally, sublingually, by inhalation (e. g. as mists or sprays), rectally, or topically in dosage unit formulations containing conventional nontoxic pharmaceutically or physiologically acceptable carriers, adjuvants, and vehicles as desired. For example, suitable modes of administration include oral, subcutaneous, transdermal, transmucosal, iontophoretic, intravenous, intraarterial, intramuscular, intraperitoneal, intranasal (e. g. via nasal mucosa), subdural, rectal, gastrointestinal, and the like, and directly to a specific or affected organ or tissue. For delivery to the central nervous system, spinal and epidural administration, or administration to cerebral ventricles, can be used. Topical administration may also involve the use of transdermal administration such as transdermal patches or iontophoresis devices. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection, or infusion techniques.

[0162] The compounds are mixed with pharmaceutically acceptable carriers, adjuvants, and vehicles appropriate for the desired route of administration. Oral administration is a preferred route of administration, and formulations suitable for oral administration are preferred formulations. Alternatively, the compounds may be administered by supplementation via gastric or percutaneous tubes.

[0163] Hence, in a preferred embodiment the invention pertains to a compound as defined herein above, for use in a method of treating or preventing a bone disorder by administration of an effective total daily dose, wherein compound is administered orally.

[0164] The oral route is the preferred means of administration and (at least for adults) preferably the dosage form used is a solid oral dosage form. The class of solid oral dosage forms consists primarily of tablets and capsules, although other forms are known in the art and can be equally suitable. When used as a solid oral dosage form, the compound as defined herein may e.g. be administered in the form of an immediate release tablet (or a capsule and the like) or a sustained release tablet (or a capsule and the like). Any suitable immediate release or sustained release solid dosage forms can be used in the context of the invention as will be evident for the skilled person.

[0165] The compounds described for use as described herein can be administered in solid form, in liquid form, in aerosol form, or in the form of tablets, pills, powder mixtures, capsules, granules, injectables, creams, solutions, suppositories, enemas, colonic irrigations, emulsions, dispersions, food premixes, and in other suitable forms. The compounds can also be administered in liposome formulations. The compounds can also be administered as prodrugs, where the prodrug undergoes transformation in the treated subject to a form which is therapeutically effective. Additional methods of administration are known in the art.

[0166] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in propylene glycol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

[0167] Suppositories for rectal administration of the drug can be prepared by mixing the drug with a suitable non-irritating excipient such as cocoa butter and polyethylene glycols that are solid at room temperature but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.

[0168] While the compounds for use as described herein can be administered as the sole active pharmaceutical (or cosmetic) agent, they can also be used in combination with one or more other agents used in the treatment or suppression of disorders.

[0169] When additional active agents are used in combination with the compounds of the present invention, the additional active agents may generally be employed in therapeutic amounts as indicated in the Physicians' Desk Reference (PDR) 53rd Edition (1999), or such therapeutically useful amounts as would be known to one of ordinary skill in the art. The compounds of the invention and the other therapeutically active agents can be administered at the recommended maximum clinical dosage or at lower doses. Dosage levels of the active compounds in the compositions of the invention may be varied so as to obtain a desired therapeutic response depending on the route of administration, severity of the disease and the response of the patient. When administered in combination with other therapeutic agents, the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.

[0170] Medical Use

[0171] The compounds and compositions as described above are for use in a method of treating or preventing a bone disorder. Preventing a bone disorder can encompass the avoidance of occurrence of a bone disorder, which can encompass the prevention of bone fractures, preferably through increase of bone health, or of bone mass, or of bone mineralization.

[0172] Bone disorders are commonly known. Examples of suitable bone disorders to be treated are bone fracture risk, osteoporosis, diabetic bone disease and particularly type-l diabetic bone disease, low osteogenic potential of marrow stromal cells (MSC), excessive glycolytic ATP production in MSC, low proliferation of MSC, low sternness of MSC, low bone formation capacity, and low mineralization capacity of MSC. Thus preferably the method is for decreasing bone fracture risk, for treating osteoporosis, for treating type-l diabetic bone disease, for increasing the osteogenic potential of marrow stromal cells (MSC), for decreasing glycolytic ATP production in MSC, for increasing proliferation of MSC, for increasing sternness of MSC, for increasing bone formation capacity, or for increasing the mineralization capacity of MSC.

[0173] Further examples of bone disorders are osteopetrosis (such as autosomal dominant 1), osteosclerosis, bone mineral density quantitative trait locus 1 , endosteal hyperostosis (autosomal dominant), endosteal hyperostosis (Worth type), Van Buchem disease, Van Buchem disease type 2, osteoporosis-pseudoglioma syndrome, LRP5-related primary osteoporosis, osteosclerosis- developmental delay-craniosynostosis syndrome, osteoporosis, and brittle bone disorder. Of these, it is particularly preferred to treat LRP5-related primary osteoporosis, osteosclerosis-developmental delay-craniosynostosis syndrome, osteoporosis, and / or brittle bone disorder using compounds and methods as described herein. A further preferred osteoporosis is Wnt1 -related osteoporosis.

[0174] The inventors found that the compounds had a specific effect on the epigenetic profile and activity of bone cells. Examples of bone cells are osteoblasts, osteocytes, and osteoclasts. Osteoblasts and osteocytes are derived from osteoprogenitor cells which derive from mesenchymal stem cells. Bone cells, particularly osteoprogenitor cells, contribute to good bone health, for instance through their osteogenic potential or through their ability to resorb bone that is in need of repair or remodeling. For instance, bone remodeling is a dynamic and coordinated process between bone formation by osteoblast and bone resorption by osteoclasts. A preferred cell type to modulate the activity of is a marrow stromal cell or an osteoblast. More preferably it is an osteoblast. Accordingly in preferred embodiments the method is for increasing the osteogenic potential of osteoblasts, for decreasing glycolytic ATP production in osteoblasts, for increasing proliferation of osteoblasts, for increasing bone formation capacity by osteoblasts, or for increasing the mineralization capacity of osteoblasts. Assays for determining these parameters are known in the art and are preferably as described in the examples.

[0175] Treatment with compounds as described herein improved various parameters in bone marrow stromal cells (BMSC), particularly in m.3243A>G human BMSC (hBMSC), e.g., OXPHOS, MSR, and mitophagy were improved. In those cells an upregulation of differentially expressed (DE) genes that are associated with enriched terms related to bone formation was observed, and key osteoblastogenesis genes, e.g. LRP5, RUNX2, TGFB3, and to a smaller extent GDF5, were upregulated. LRP5 and RUNX2 were particularly improved, with the most marked effect being found for LRP5. It was found that this correlated to improved osteogenic capacity of the hBMSC because an increased mineralization was observed for cells treated with the compounds.

[0176] Accordingly the compounds are particularly useful for use as described herein, wherein the bone disorder is associated with aberrant expression of LRP5 or RUNX2, preferably associated with insufficient expression of LRP5 or RUNX2. Similarly, in preferred embodiments the bone disorder is not associated with aberrant expression of GDF5, preferably it is not associated with insufficient expression of GDF5. In preferred embodiments the compound is for increasing expression of LRP5 or RUNX2. In more preferred embodiments the compound is for increasing expression of LRP5. Preferably, increasing expression of LRP5 or RUNX2 is increasing expression of functional LRP5 or functional RUNX2.

[0177] Low-density lipoprotein receptor-related protein 5 (LRP5) is a protein that in humans is encoded by the LRP5 gene. It is a key component of the LRP5 / LRP6 / Frizzled co-receptor group that is involved in canonical Wnt pathway. Mutations in LRP5 can lead to considerable changes in bone mass, or to other pathologies. Some bone conditions are associated with defective LRP5, such as bone mineral density quantitative trait locus 1 , endosteal hyperostosis (autosomal dominant), endosteal hyperostosis (worth type), Van Buchem disease, and Van Buchem disease type 2. In preferred embodiments the compound is not for treatment of these conditions.

[0178] Some bone conditions are known to not be associated with LRP5, such as osteopetrosis and osteosclerosis. It can also be known that further expression of LRP5 would not be beneficial, such as for osteoporosis-pseudoglioma syndrome. In preferred embodiments the compound is not for treatment of these conditions. Particularly, in preferred embodiments the subject does not suffer from LRP5-linked osteoporosis-pseudoglioma syndrome. Accordingly, in more preferred embodiments the compounds is not for use in treatment of osteoporosis-pseudoglioma syndrome, bone mineral density quantitative trait locus 1 , endosteal hyperostosis (autosomal dominant), endosteal hyperostosis (worth type), Van Buchem disease, and Van Buchem disease type 2, osteopetrosis, osteosclerosis, or osteoporosis- pseudoglioma syndrome. Accordingly in some embodiments the compound is for use in a method of treating or preventing a bone disorder, wherein the bone disorder is Lrp5-related primary osteoporosis, osteosclerosis-developmental delay-craniosynostosis syndrome, osteoporosis, or brittle bone disorder. In highly preferred embodiments the bone disorder is osteoporosis or brittle bone disorder. A preferred osteoporosis is not associated with osteoporosis-pseudoglioma syndrome.

[0179] Runt-related transcription factor 2 (RUNX2) also known as core-binding factor subunit alpha-1 (CBF-alpha-1) is a protein that in humans is encoded by the RUNX2 gene. RUNX2 is an important transcription factor associated with osteoblast differentiation. It has been suggested that RUNX2 plays a cell proliferation regulatory role in cell cycle entry and exit in osteoblasts, as well as endothelial cells. RUNX2 suppresses pre-osteoblast proliferation by affecting cell cycle progression in the G1 phase. In osteoblasts, the levels of RUNX2 is highest in G1 phase and is lowest in S, G2, and M. The comprehensive cell cycle regulatory mechanisms that RUNX2 may play are still unknown, although it is generally accepted that its varying activity and levels throughout the cell cycle contribute to cell cycle entry and exit, as well as cell cycle progression. These functions are important when discussing bone cancer, particularly osteosarcoma development. Accordingly in preferred embodiments the compound is not for treatment of bone cancer, more preferably not for treatment of osteosarcoma.

[0180] Mutations in RUNX2 are associated with the disease cleidocranial dysostosis, which is proposed to arises partly due to RUNX2 dosage insufficiencies. Because RUNX2 promotes exit from the cell cycle, insufficient amounts of RUNX2 are related to undesirably increased proliferation of osteoblasts observed in patients with cleidocranial disostosis. Accordingly in preferred embodiments the compound is for treatment of cleidocranial disostosis.

[0181] In some embodiments the compound for use is for increasing gene expression of one or more of COX1, COX2, ND4, ND4L, ND5, ND6, MT-RNR1, and MT-RNR5, preferably one or more of COX2, MT-RNR1, and MT-RNR2, more preferably of MT-RNR1. In some embodiments the compound for use is for decreasing gene expression of ND3. In some embodiments the compound for use is for increasing protein levels of one or more of NDUFS1 , NDUFB3, NDUFA13, NDUFB1 , NDUFA9, UQRC1 and UQCRFS1 , preferably of NDUFB1. In some embodiments the compound for use is for decreasing protein levels of one or more of ATP5MC3 and TCIRG1 , preferably of ATP5MC3. In preferred embodiments the subject to be treated suffers a mitochondrial disease, preferably a primary mitochondrial disease. Examples of mitochondrial diseases are Myoclonic epilepsy; Myoclonic Epilepsy with Ragged Red Fibers (MERRF); Leber's Hereditary Optic Neuropathy (LHON); neuropathy ataxia and retinitis pigmentosa (NARP); Mitochondrial Myopathy, Encephalopathy, Lactic acidosis, Stroke-like episodes (MELAS); Leigh syndrome; Leigh-like syndrome; Dominant Optic atrophy (DOA); Kearns-Sayre Syndrome (KSS); Maternally Inherited Diabetes and Deafness (MIDD); Alpers- Huttenlocher syndrome; Ataxia Neuropathy spectrum; Chronic Progressive External Ophthalmoplegia (CPEO); Pearson syndrome; Mitochondrial Neuro-Gastro-lntestinal Encephalopathy (MNGIE); Sengers syndrome; 3-methylglutaconic aciduria, sensorineural deafness, encephalopathy and neuro- radiological findings of Leigh-like syndrome (MEGDEL); myopathy; mitochondrial myopathy; cardiomyopathy; and encephalomyopathy, SURF1 (COX deficient Leigh syndrome due to complex IV surfeit protein deficiency) and isolated or combined OXPHOS deficiencies with so far unsolved genetic defect including disturbed pyruvate oxidation and ATP plus PCr production rates. Preferably, the mitochondrial disorder is associated with a m.3242A>G mutation of the mitochondrial tRNA(leu) gene. Accordingly in preferred embodiments the subject has a mitochondrial disease, more preferably a primary mitochondrial disease. In preferred embodiments the subject carries a mitochondrial DNA m.3243A>G mutation. In this light it is relevant that the inventors found that between healthy controls and subjects carrying a mitochondrial DNA m.3243A>G mutation there was no difference between groups in major regulators of bone homeostasis (such as p-25OHD, pCa2+, PTH, TSH, and liver and kidney function). Accordingly, in other preferred embodiments the subject does or does not carry a mitochondrial DNA m.3243A>G mutation.

[0182] Treatments as described herein are attractive because they allow the modulation of epigenetic profiles without requiring the administration of complex biological substances. An example of medical use of biologicals for treating bone disorders is the use of teriparatide, sold under the brand name Forteo, which is a form of parathyroid hormone (PTH). It is an anabolic agent that promotes bone formation and therefore it is used in the treatment of some forms of osteoporosis. Related hereto, the protein sclerostin has been identified as binding to LRP5 / 6 receptors and inhibiting the Wnt signaling pathway, leading to decreased bone formation. Therapies wherein sclerostin is bound by antibodies, to inhibit its function and thus to promote bone formation, have been under development. The invention allows for the use of small molecules as described herein to achieve similar effects while avoiding the need for antibodies or hormones. In preferred embodiments the subject does not undergo concomitant therapy using hormones or antibodies for increasing bone formation, preferably the subject does not undergo concomitant therapy using parathyroid hormone or a form thereof. The compounds of the invention modulate the epigenetic profile of bone cells, and accordingly their effect can persist after the substances themselves have been cleared.

[0183] The above compounds for use can also be used in a method for treating or preventing a bone disorder, the method comprising the step of administering a compound as defined herein to a subject. The compound is preferably administered in an effective amount. The subject is preferably in need of treatment, or is known to be or suspected of being at risk of suffering a bone disorder.

[0184] General Definitions

[0185] In this document and in its claims, the verb "to comprise" and its conjugations is used in its nonlimiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, the verb “to consist” may be replaced by “to consist essentially of’ meaning that a combination or a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0186] The use of a substance as a medicament as described in this document can also be interpreted as the use of said substance in the manufacture of a medicament. Similarly, whenever a substance is used for treatment or as a medicament, it can also be used for the manufacture of a medicament for treatment. Products for use as a medicament described herein can be used in methods of treatments, wherein such methods of treatment comprise the administration of the product for use.

[0187] In the context of this invention, a decrease or increase of a parameter to be assessed means a change of at least 5% of the value corresponding to that parameter. More preferably, a decrease or increase of the value means a change of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, or 100%. In this latter case, it can be the case that there is no longer a detectable value associated with the parameter.

[0188] The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 5% of the value.

[0189] Each embodiment as identified herein may be combined together unless otherwise indicated. The invention has been described above with reference to a number of embodiments. A skilled person could envision trivial variations for some elements of the embodiments. These are included in the scope of protection as defined in the appended claims. All patent and literature references cited are hereby incorporated by reference in their entirety.

[0190] Description of the figures

[0191] Fig. 1A - Resting plasma lactate. As in panel 1 B - 1 D, n = 20 (10 m.3243A>G carriers and 10 controls matched by age, sex, and BMI). The statistical analysis performed was paired Wilcox test. *: p-value < 0.05.

[0192] Fig. 1B - Heteroplasmy of hBMSC analysed by digital PCR. n = 20 (10 m.3243A>G carriers and 10 controls matched by age, sex, and BMI).

[0193] Fig. 1C - Mitochondrial function analysis of hBMSC by respirometry using Seahorse analyzer. The scatter plot shows the basal mitochondrial and glycolytic ATP production.

[0194] Fig. 1D - Mitochondrial function analysis of hBMSC by respirometry using Seahorse analyzer. The scatter plot shows the basal mitochondrial respiration and glycolysis rates. Fig. 2A - Protein levels of glycolysis related proteins analyzed by mass spectrometry based proteomics. ENO2: Gamma-enolase; HK2: Hexokinase-2; LDHA: lactate dehydrogenaseA; LDHC: lactate dehydrogenase C. As in panel 2B and 2C, n = 20 (10 m.3243A>G carriers and 10 controls matched by age, sex, and BMI). The statistical analysis performed was paired Wilcox test. *: p-value < 0.05.

[0195] Fig. 2B - GDF-15 and FGF-21 expression levels in hBMSC analyzed by RNAsequencing. CFU-f: colony forming units; GDF-15: Growth Differentiation Factor 15-gene; FGF-21 : Fibroblast Growth Factor 21 - gene; hBMSC: human bone marrow mesenchymal stem cells.

[0196] Fig. 2C - CFU-f from m.3243A>G hBMSC at pO showed decreased proliferation.

[0197] Fig. 3A - Gene expression levels of bone-formation related genes analyzed by RNA sequencing. SMAD5: SMAD family member 5-gene; SOX11 : SRY-BOX 11 -gene; WWTR1 : WW domain-containing transcription regulator 1 -gene; YAP1 : YES-associated trancriptional regulator. As in panel 3B - 3D, n = 20 (10 m.3243A<G carriers and 10 controls matched by age, sex, and BMI). The statistical analysis performed was paired Wilcoxon test. * p-value < 0.05

[0198] Fig. 3B - Correlation between age and ALP activity

[0199] Fig. 3C - Correlation between age and Nile Red

[0200] Fig. 3D - Quantification of newly formed bone, following a heterotropic bone formation assay using hBMSC and hydroxyapatite / tricalcium phosphate as a scaffold that was implanted subcutaneously in an immunodeficient mice for 8 weeks.

[0201] Fig. 4A - Heteroplasmy analysis of mature OCs analysed by digital PCR. Peripheral blood mononuclear cells were isolated from peripheral blood and cultures with 25 ng / mL M-CFS until day 2, when 25 ng / mL RANKL was added. Cell culture media was collected at each media change for TRAcP activity. OCs were considered mature at day 9 when mitochondrial function analysis using Seahorse Extracellular analyzer was done, DNA was collected, and OCs were re-seeded onto bone slices for resorptive activity. M-CFS: Macrophage colony-stimulating factor; RANKL: Receptor activator of nuclear factor kappa-B ligand; OC: osteoclast; TRAcP: Tartrate-resistant acid phosphatase. As in panel 4B - 4I, n = 20 (10 m.3243A<G carriers and 10 controls matched by age, sex, and BMI). The statistical analysis performed was paired Wilcoxon test. * p-value < 0.05

[0202] Fig. 4B - Pearson correlation of heteroplasmy in mature OCs and hBMSCs from m.3243A>G carriers. Grey shadow shows 95% confidence interval.

[0203] Fig. 4C - Scatter plot showing the basal mitochondrial respiration and glycolysis of mature OC following mitochondrial function analysis of mature OCs by respirometry using Seahorse analyzer. ECAR: extracelluar acidification rate, OCAR: oxygen consumption rate

[0204] Fig. 4D - Scatter plot showing the basal mitochondrial and glycolytic ATP production following mitochondrial function analysis of mature OCs by respirometry using Seahorse analyzer.

[0205] Fig. 4E - Proportion of mitochondrial ATP and glycolytic ATP following mitochondrial function analysis of mature OCs by respirometry using Seahorse analyzer.

[0206] Fig. 4F- Resorption activity of mature OCs quantified as percentage of eroded surface by bone surface.

[0207] Fig. 4G - Extracellular TRAcP activity of mature OCs on bone slices.

[0208] Fig. 4H - Extracellular TRAcP activity of cells under RANKL stimulation for differentiation

[0209] Fig. 41 - Number of nuclei per mature OCs quantified by brightfield imaging. Fig. 5A - Bone density determined as BV / TV = bone volume / total volume. Trabecular bone microarchitecture is determined by uCT. As in panel 5B - 5G, n = 14 (7 carriers and 7 controls matched by age and sex), except MAR where n = 8 (4 carriers and 4 controls matched by age and sex). The statistical analysis performed was paired Wilcox test. * p-value < 0.05.

[0210] Fig. 5B - Spearman correlation analysis between BV / TV (%) and trabecular parameter Tb.Th (trabecular thickness)

[0211] Fig. 5C- Spearman correlation analysis between BV / TV (%) and trabecular parameter Tb.N (trabecular number).

[0212] Fig. 5D - Cortical bone microarchitecture determined by pCT. Ct.Th = cortical thickness.

[0213] Fig. 5E - Extension of the eroded surface (ES) related to the total bone surface (BS).

[0214] Fig. 5F - Extension of the osteoid surface (OS) related to the total bone surface (BS).

[0215] Fig. 5G - Quantification of bone formation analysed as Mineral Apposition Rare (MAR) following tetracycling labelling to bone formation by green fluorescence.

[0216] Fig. 6A - Scatter plot showing the basal mitochondrial respiration and glycolysis following mitochondrial function analysis of hBMSC by respirometry using Seahorse analyzer. As for panel 6B - 6G, n = 8, and the statistical analysis performed was paired Wilcox test. * p-value < 0.05.

[0217] Fig. 6B - Scatter plot showing the basal mitochondrial and glycolytic ATP production following mitochondrial function analysis of hBMSC by respirometry using Seahorse analyzer.

[0218] Fig. 6C - Proportion of mitocondrial ATP and glycolytic ATP production.

[0219] Fig. 6D - Total ATP production following mitochondrial function analysis of hBMSC by respirometry using Seahorse analyzer.

[0220] Fig. 6E - Gene expression analysis of COX7A isoforms measured by RNA sequencing. COX7A: COX7A2-like protein-gene.

[0221] Fig. 6F - Gene expression analysis of PDK isoforms measured by RNA sequencing. PDK: pyrovate dehydrogenase kinase isoenzyme-gene.

[0222] Fig. 6G - Protein levels of TCA cycle related proteins measured by mass spectrometry. TCA: tricaboxylic acid.

[0223] Fig. 7A - Transcriptional downregulation of MSR genes. hBMSCs were cultured for 3 days with KH183 (5 uM) or with DMSO as control. As for panel 7B - 7B, n = 16 (8 m.3243A>G carriers and 8 controls matched by age, sex, and BMI). The statistical analysis performed was paired Wilcox test. * p-value < 0.05.

[0224] Fig. 7B - Heat map showing the false discovery rate (goseq) for the enrichment of the RNA-seq signatures associated with bone formation.

[0225] Fig. 7C - Gene expression levels of bone formation genes. GDF5: growth differentiation factor 5; LRP5: low density lipoprotein receptor related protein 5; RUNX2: Runt related transcription factor 2

[0226] Fig. 7D - Mineralization capacity of hBMSC stained with Alizarin Red after 12-days in osteoblast induction media.

[0227] Examples

[0228] Examples Example 1 - Introduction

[0229] Bone is a dynamic tissue that is remodelled throughout life to maintain bone mass equilibrium and mechanical competencies. The continuous process of bone degradation by bone-resorbing osteoclasts followed by the formation of bone matrix by bone-forming osteoblasts, a process known as bone remodeling, requires high amounts of energy in the form of adenosine triphosphate (ATP), which is generated via mitochondrial oxidative phosphorylation (OXPHOS) and cytoplasmatic glycolysis. The significance of sufficient ATP supply for bone remodelling has been studied in progenitor cells and fully differentiated osteoblasts and osteoclasts. The main energy source of bone is glucose, and accordingly, glucose uptake is particularly high in skeletal sites with substantial bone remodelling, as demonstrated by radio-tracer studies in adults. Osteoprogenitors cells prefer glycolysis for proliferation and selfrenewal, which is in agreement with somatic stem cells from other tissues. However, data on osteoblasts and osteoclasts is less uniform.

[0230] Most studies show OXPHOS activation during osteogenic differentiation while glycolysis remains unchanged. Furthermore, the importance of OXPHOS to skeletal integrity is supported by the finding of low bone mass, bone deformities and fragility fractures in mice with impaired ATP supply caused by OXPHOS deficiency in complex I, III, IV, and V. Besides ATP production, mitochondria are multifunctional organelles, and thus, impaired OXPHOS function affects other mitochondrial functions than ATP production, e.g. substrates production for epigenetic reactions such as tricarboxylic acid (TCA) cycle intermediates, regeneration of NAD+ for other reactions like malate dehydrogenase (MDH2), regulation of mitochondrial stress responses (MSR).

[0231] Osteoprogenitors showed metabolic flexibility as demonstrated by shifting from OXPHOS to glycolysis after pharmacological inhibition of complex III and vice versa after lactate dehydrogenase (LDH) inhibition, where increased OXPHOS activity exerted a bone anabolic effect. This data shows the potential for a novel target for preventing and treating bone fragility in individuals with defects in mitochondrial function or conditions characterized by inadequate bone formation, such as osteoporosis, including glucocorticoid-induced osteoporosis.

[0232] Clinical data linking mitochondrial function and specifically OXPHOS to skeletal integrity are limited. Increased bone fragility has been reported in an epidemiological study of subjects with inherited mitochondrial disorders (Gandhi, S.S., et al. 2017) and in case reports of young patients with mitochondrial diseases showing bone loss and fractures (Catheline, S.E., et al. 2023). Mitochondrial proteins are encoded by nuclear DNA (nDNA) and mitochondrial DNA (mDNA), that encodes 13 OXPHOS proteins and the mitochondrial translation machinery. The most common pathogenic mDNA variant m.3243A>G in the MT-TL1 gene that leads to defective translation of mDNA encoded proteins resulting in impairment of OXPHOS. We have previously reported thinner cortical bone and lower bone mineral density (BMD) in adult men and women carrying m.3243A>G pathogenic variant (Langdahl, J.H., et al., (2017).

[0233] Collectively, pre-clinically and observational studies suggest that impaired OXPHOS activity compromises bone cell differentiation and function, potentially impairing skeletal integrity. However, the role of energy metabolism in bone and its clinical significance remains understudied. Here, we used osteoprogenitors from m.3243A>G carriers to investigate the mechanisms of bone regulation by mitochondria in humans. We show how a favourable glycolytic bioenergetic program in osteoprogenitors reduces their osteogenic potential by regulating their transcriptional program. Furthermore, pharmacological enhancement of OXPHOS, restored the pro-osteogenic transcriptional program of osteoprogenitors. Overall, these findings extend the knowledge of the effect of OXPHOS on hBMSCs and osteoblasts and propose OXPHOS as a potential therapeutic target for improving bone formation and supporting skeletal health.

[0234] Example 2 - Materials and Methods

[0235] Participants and sample collection

[0236] Participants were subjects >18 years know to carry the pathogenic mitochondrial genetic variant, m.3243A>G and healthy matched controls matched for sex and age. Criteria of exclusion: Renal (creatinine > 90 pmol / l) or liver dysfunction (AST > 3 times the upper limit), medical treatment influencing bone metabolism (oral corticosteroid <12 weeks, anti-osteoporosis treatment, sex steroids, anticonvulsant), pregnancy, excessive consumption of alcohol (>14 unit / week), anticoagulants or preexisting coagulopathy, allergy to lidocaine, morphine or diazepam.

[0237] This is a cross-sectional, case-control study including two parts: (A) collection and study of bone biopsies and (B) collection of cells and analysis of cellular studies. For part (A), tetracycline labelled bone biopsies were collected in 2016 from seven individuals carrying the m.3243A>G pathogenic variant and seven age- and sex-matched healthy individuals as controls. For part (B), ten individuals carrying the m.3243A>G participated in the cellular studies, five of them were part of part A study, with ten age-, sex-, and BMI-matched healthy individuals as controls. Bone marrow aspiration, blood samples and scans were assessed in 2021 .

[0238] Isolation of bone marrow mesenchymal stem cells from bone marrow aspirates

[0239] Bone marrow samples were obtained from the iliac crest by aspiration of 10-15 mL after infiltration of the area with local anaesthetic (10 mg / mL lidocaine) (Amgros). The samples were mixed 1 :1 with Minimum Essential Media (MEM) with heparin (100 U / mL) (Amgros). Low-density mononuclear cells were isolated through centrifugation with a Lymphoprep density gradient (density = 1 .077 ± 0.001 g / cm2) (ILS). hBMSC were selected through the process of plastic adherence. Cells were cultured in MEM containing 10% FBS, 1 % Penicillin / Streptomycin (P / S) (Gibco # 15140130), 1 mM Sodium Pyruvate (Gibco # 1 1360039), 2 mM Glutamax (Gibco # 35050-038), 1 % nonessential amino acids (Gibco # 11140050), and 50 pg / mL uridine (Sigma-Aldrich # U3003) incubated at 5% CO2 at 37°C. The media was changed for the first time after 7 days and every second day after that until 70% confluence.

[0240] Culture of MSC

[0241] MSCs were sub-cultured in MEM containing 10% FBS, 1 % Penicillin / Streptomycin (P / S) (Gibco # 15140130), 1 mM Sodium Pyruvate (Gibco # 11360039), 2 mM Glutamax (Gibco # 35050-038), 1 % nonessential amino acids (Gibco # 11 140050), and 50 pg / mL uridine (Sigma-Aldrich # U3003) in standard cell culture conditions (37°C, 85% humidity, and 5% CO2). MSCs were routinely tested for Mycoplasma and cultured in Mycoplasma-negative conditions. The medium was changed every other day. Cultured cells (passage 0) were cryopreserved and sub-cultured (passage 1) for further studies.

[0242] Mitochondrial DNA m.3243A>G heteroplasmy analysis

[0243] To quantify the fractional abundance of mitochondrial DNA with m.3243A>G, droplet digital PCR (ddPCR) was conducted. Each 22 pl ddPCR reaction consisted of 11 pl 2x ddPCR SuperMix for probes (no dUTP) (Bio-Rad, Hercules, California, USA), 909 nM of each primer, 568 nM of each probe, and 5 pl template DNA. Reactions were prepared in semi-skirted 96 well PCR plates (Bio-Rad, Hercules, California), and droplets were generated using an Automated Droplet Generator (Bio-Rad, Hercules, California). Following droplet generation, PCR plates were sealed with pierceable foil (Bio-Rad, Hercules, California) at 180°C for 5 seconds, and PCR was performed on a CFX96 Touch Deep Well Real-Time PCR System (Bio-Rad, Hercules, California). After optimization of the assay, PCR conditions were 95°C for 10 minutes, 50 cycles of 94°C for 30 seconds and 56°C for 1 minute, and 98°C for 10 minutes with a ramp rate of 1 °C / s. The plate was kept on hold at 4°C for a minimum of 30 minutes or at 12°C for a minimum of 4 hours, after which the plate was incubated for 10 minutes at room temperature and read on QX200™ Droplet Reader (Bio-Rad, Hercules, California).

[0244] Every ddPCR run included a positive template control run in duplicates, a negative template control run in five wells, and a minimum of two no-template control (NTC) for a clean workflow quality control check. Only wells with a minimum of 10,000 accepted droplets were analyzed, and thresholds were manually set at 1 ,200 in channel 1 (FAM) and 2,200 in channel 2 (HEX). Thresholds were defined during optimization using positive and negative controls with gating based on fluorescence amplitude in 1 D and 2D plots. Data was analyzed with QuantaSoft Analysis Pro 1.0 software (Bio-Rad, Hercules, California). A sample was scored positive for m.3243A>G if the concentration was above the false positive rate. The fractional abundance was calculated based on the concentration of both m.3243A>G and wild-type mitochondrial DNA for each sample.

[0245] Bioenergetic analysis of MSC

[0246] Mitochondrial bioenergetics was performed using Seahorse XFe96 (Seahorse, Agilent, USA) extracellular flux analyzer to measure the Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) simultaneously. hBMSC were seeded at 6,000 cells per well S-MEM media (MEM + 10% FBS + 1 % P / S + 50 pg / mL uridine) in a Seahorse 96-well cell culture plate (Agilent). After 24 hours, the cells were treated with either a vehicle (DMSO) or KH183 (5 pM) in cell culture media (MEM + 10% FBS + 1 % P / S) for 72 hours. An hour before the measurement, the cell culture medium was replaced with the assay medium and incubated in a non-CO2 environment at 37°C. The assay medium was prepared by supplementing Seahorse XF DMEM medium (Agilent, #103575-100) with 10mM glucose (Sigma-Aldrich, G8769), 1 mM Sodium Pyruvate (Gibco, 11360-039), and 2 mM Glutamine (Sigma-Aldrich, G7513).

[0247] For the analysis of mitochondrial respiration, we used the Mito Stress Test assay (Agilent, #103015-100). Two groups of sequential drug injections were used: (1) uncoupler FCCP (Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; 1 pM final / well), and Rotenone / Antimycin A (0.5 pM final / well); (2) ATP-synthetase inhibitor Oligomycin (1 pM final / well), and Rotenone / Antimycin A (0.5 pM final / well). For the ATP production analysis, we used the second group of injections with Oligomycin and Rotenone / Antimycin A. The parameters obtained were calculated using the average of the three cycles measured according to the manufacturer’s recommendations. For the glycolysis analysis, we used the Glycolytic Rate assay (Agilent ##103344-1) by injecting sequentially Rotenone / Antimycin A (0.5 pM final / well) and 2-deoxyglucose (50 mM final / well).

[0248] After OCR and ECAR measurements were completed, we added Hoechst 33342 to each well as nuclear staining (20 pM / final well, Invitrogen, H3570) for 15 minutes in the dark at 37°C. The cells were imaged by Cytationl (Biotek, Agilent) using Cell Imaging software (Agilent) to count the number of cells in each well. The OCR and ECAR values were normalized to the Hoechst-stained cell counts, with the normalization unit set to 1 ,000 cells. The normalized OCR and ECAR data were exported from Wave Software (Agilent) to Seahorse Analytics for the calculations according to the manufacturer’s instructions.

[0249] Glucose uptake

[0250] The glucose uptake was determined by seeding hBMSCs in a 96-well black / clear bottom plate (Perkin Elmer) at a density of 6000 cells / well. After 24 hours, the cells were starved for 6 hours using low glucose (1 g / L) DMEM without FBS. Next, cells were incubated with glucose-free media without FBS with 10 pg / mL fluorescent 2-deoxyglucose (2-NBDG) (Caiman) for 2 hours. Finally, the cells were stained with Hoechst (1 pg / mL) for 10 min. The fluorescence was measured using the Cytation 1 Cell Imaging Multi-Mode Reader (BioTek Instruments, Agilent) using the DAPI channel for Hoechst 33342 (377 nm / 477 nm) and the GFP channel for 2-NBDG (Excitation 500 / 24 nm, Emission 542 / 27 nm). The mean fluorescent intensity of 2-NBDG was normalized to the number of Hoechst-stained cells.

[0251] Mitochondrial Mass Determination with MitoTracker™ Green

[0252] Mitochondrial mass was determined with MitoTracker™ Green FM (Invitrogen, USA, M7514). We seeded 4,000 cells in a 96-well black / clear bottom plate (Perkin Elmer). After 24 hours, cells were washed with PBS and incubated with 100 nM MitoTracker Green and 20 pM Hoechst in HBSS (Hanks' Balanced Salt Solution) for 30 minutes at 37°C. The fluorescence was measured using the Cytation 1 Cell Imaging Multi-Mode Reader (BioTek Instruments, Agilent) using the DAPI channel for Hoechst 33342 (377 nm / 447 nm) and the GFP channel for MitoTracker Green (469 nm, 525 nm). The mean fluorescent intensity of MitoTracker Green was normalized to the number of Hoechst-stained cells.

[0253] RNA isolation hBMSC were seeded in 6-well plates at a density of 100,000 / well in S-MEM media (MEM + 10% FBS + 1 % P / S + 50 pg / mL uridine). After 24 hours, the cells were treated with either a vehicle (DMSO) or KH183 (5 pM) in cell culture media (MEM + 10% FBS + 1 % P / S) for 72 hours. Cells were washed once with PBS and added 250 pL of TRIzol (Invitrogen #15596-018). The cells were then detached using a cell scraper (VWR, #734-2602) and, collected in a pre-chilled microtube and stored at -80°C for later use for RNA sequencing. RNA isolation was performed using TRIzol, including DNase treatment with a DNA-free Kit (Ambion, AM1906) according to the manufacturer’s instructions. Total RNA concentrations were determined with a Synergy H1 plate reader equipped with Take3 Plate (BioTek, Agilent). Quality control analysis was performed with an automated 4200 TapeStation System (Agilent, G2991 AA), and RNA integrity number (RIN) values were determined by RNA ScreenTape Assay for the 4200 TapeStation System according to the manufacturer’s instructions. RIN values were above 9.3 and 8.3 for all samples of the cell and zebrafish models, respectively.

[0254] Proteomics sample preparation hBMSC were seeded in T25 flasks at a density of 250,000 in MEM media (MEM + 10% FBS + 1 % P / S + 1 mM Sodium Pyruvate + 2 mM Glutamax + 1 % nonessential amino acids + 50 pg / mL uridine). After 24 hours, the cells were treated with either a vehicle (DMSO) or KH183 (5 pM) in cell culture media (MEM + 10% FBS + 1 % P / S) for 72 hours. Then, the cells were washed once with PBS, 1 mL of trypsin was added to the flasks and incubated for 3-5 min at 37°C. Samples were centrifuged at 200 g for 5 minutes, the supernatant was removed. The cell pellet was resuspended in 1 mL PBS and centrifuged at 16,000 g at 4°C. The supernatant was removed, the cell pellet was resuspended in 1 mL PBS. The centrifugation step was repeated. The supernatant was discarded, and the pellet was stored at -80°C. Cell pellets were lysed in a HEPES lysis buffer (4% SDS (Invitrogen), 50 mM HEPES (Sigma Aldrich), and protease inhibitor (Roche, Complete mini), pH 7.4). Samples were further lysed by sonication in a water bath (Branson 2800 Ultrasonic Cleaner) for 10 seconds with 30 seconds of ice incubation between cycles (total of 3 cycles). Sonicated lysates were centrifuged at 12,000 g for 10 minutes at 4°C. Protein concentrations of the supernatants were determined with the BCA protein assay (Thermo Scientific) according to the manufacturer’s instructions.

[0255] Tandem Mass Tag Labeling

[0256] MSC samples were tagged with TMT plex™ and TMTpro™16plex Isobaric Label Reagent Set (Thermo Scientific, #90111 and #A44520, respectively). From each sample, 100 pg total protein was in-solution trypsin digested and TMT labelled according to the manufacturer’s instructions. Following the TMT labelling, all samples within each study were pooled. After a strong cation exchange (SCX) purification (Phenomenex, #8B-S010-EAK), peptides were loaded to an Immobiline™ DryStrip Gel (GE Healthcare, #11534985) isoelectric focusing (IEF) separation was performed. The IEF gels were then cut into 10 equal pieces, purified in PepCIean C18 spin columns (Thermo Scientific, #11824141) according to the manufacturer’s instructions, and then vacuum centrifuged until dryness and stored at -20°C until nanoLC-MS / MS analyses.

[0257] NanoLC-MS / MS and Proteomics Database Search

[0258] Nano-liquid chromatography tandem-mass spectrometry (nanoLC-MS / MS) was performed on an EASY nanoLC-1200 coupled to a Q-Exactive™ HF-X Quadrupole-Orbitrap™ Mass Spectrometer (Thermo Scientific). Briefly, MS was operated in positive mode using pre- and analytical columns: acclaim PepMap 100, 75 pm x 2 cm (Thermo Scientific) and EASY-Spray PepMap RSLC C18, 2pm, 100 A, 75 pM x 25 cm (Thermo Scientific), respectively, to trap and separate peptides in a 170-minute gradient with 5-40 % acetonitrile, 0.1 % formic acid. Higher-energy collisional dissociation (HCD) was used for peptide fragmentation, and the normalized collision energy (NCE) was 35. Full scan (MS1) and fragment scan resolutions were set at 60,000 and 45,000, respectively. Automatic gain control (AGC) for MS1 and MS2 were set at 1 *106with a scan range between 372-1 ,500 m / z and at 1 *105with a fixed first mass of 110 m / z, respectively. Up to 12 of the most intense peaks of the full scan were fragmented with data-dependent analysis (DDA). Unassigned and +1 charge states were excluded from fragmentation, and the dynamic exclusion duration was 15 seconds. Each fraction was LC-MS / MS analyzed twice, where peptides identified from >9 scans in the first analysis were excluded from fragmentation in the second analysis.

[0259] Proteomics Database Search

[0260] All LC-MS / MS results were merged and submitted for the database search for identification and quantification of proteins using Proteome Discoverer 3.0 (Thermo Scientific). 20,401 reviewed Homo sapiens Uniprot sequences were used as reference proteome (Swiss-Prot; downloaded on 02.12.2022) using the Sequest algorithm. Precursor mass tolerance was 10 ppm, and fragment mass tolerance was 20 mmu. The maximum number of allowed missed cleavages was two. Oxidation of methionine was set as dynamic modification and static modifications were carbamidomethylation of cysteines and TMT- plex-labels on lysine and peptide N-terminus. The co-isolation threshold was set at 35%, and the identification false discovery rate was 0.01 at both peptide and protein levels. Proteins with more than 2 quantitative peptide scans and at least one unique peptide were considered as quantified and were included in the further proteomics data analysis. Criteria for the differentially expressed proteins were set at p<0.05 and | FC|> 1 .2 for protein abundance.

[0261] Colony forming units -fibroblast (CFU-f) assay

[0262] For assessment of CFUs, hBMSC (pO) were plated at a density of 0.5 and 1 million cells in T25 flasks. Media was renewed on days 7 and 10. After 14 days, colonies displaying more than 50 cells were counted using Crystal Violet staining (Sigma-Aldrich).

[0263] Cell proliferation

[0264] BMSCs at passage 3 were plated in 96-well plates at 1 ,000 cells / well in MEM medium supplemented with 10% FBS and 1 % P / S. The cell medium was changed every other day. Each sample was measured in eight wells of technical replicates. Cell number was evaluated on days 1 , 3, 5, 7, 9, 13 and 14. On each assay day, the cells were washed with PBS and stained with Hoechst 33342 (1 pg / mL) (Thermo Fisher) for 15 min at 37°C. The Hoechst staining was analysed using Cytation 1 Cell Imaging Multi-Mode Reader (BioTek Instruments, Agilent) with ) using the DAPI channel (377 nm / 477 nm). Four pictures covering the entire surface of each well were imaged. The cell proliferation rate was calculated by normalizing each cell count by the cell count at 24 hours post-seeding.

[0265] Metabolomics sample preparation hBMSC were seeded in 6-well plates at 100,000 cells / well density in S-MEM media (MEM + 10% FBS + 1 % P / S + 50 pg / mL uridine). After 24 hours, the cells were treated with either a vehicle (DMSO) or KH183 (5 pM) in cell culture media (MEM + 10% FBS + 1 % P / S) for 72 hours. Cells were washed once with warm PBS and incubated for 90 min at 37°C with MEM with 2.5 mM of [U-13C]glucose. After incubation, the medium was collected, and the cells were washed with cold PBS (4°C) to stop metabolic reactions. The cells were lysed, and metabolites were extracted with 70% cold ethanol. Cells were centrifuged at 20,000g for 20 min at 4°C to separate soluble and insoluble components. Pellets were dissolved in KOH (1 M) at room temperature and analyzed for protein content by the BCA assay (ThermoFisher) according to manufacturer’s instructions.

[0266] Metabolic mapping using gas chromatography coupled to mass spectrometry (GC-MS)13C-enrichment of metabolites was determined using GC-MS. Briefly, extracts were reconstituted in water, acidified, and metabolites were extracted into an organic phase with 96% ethanol / benzene and derivatized using N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide. The relative abundance of ions based on their mass-to-charge (m / z) values was determined. Natural13C-abundance was corrected and calculated. Data are presented as a percentage of labelling of the isotopologue M + X, where M corresponds to the molecular weight of the unlabelled molecule and X is the number of13C-enriched carbon atoms in the molecule.

[0267] Cell Viability

[0268] Cellular viability was determined by adding the CellTiter-Blue solution (Promega, Madison, USA) for 1 hour according to the manufacturer’s instructions. The absorbance was measured at a wavelength of 570 nm in a FLUOmega plate reader.

[0269] Osteoblasts differentiation

[0270] BMSCs (passage 1) were plated at a density of 20,000 cells / cm2in MEM supplemented with 10% FBS and 1 % P / S. Osteoblastogenesis was induced on the following day by media supplemented with 10 nM b-glycerophosphate (Sigma-Aldrich), 10 nM dexamethasone (Sigma-Aldrich), 50 ug / mL vitamin C (Sigma-Aldrich) and 50 ug / mL vitamin D (Sigma-Aldrich). The medium was changed every other day for the duration of the differentiation.

[0271] Alkaline phosphatase (ALP) activity assay

[0272] ALP activity was analysed after 7 days of osteoblasts differentiation. The cell number (viable cells) was determined as described before. Subsequently, the cells were rinsed with TBS (20 mM Trizma base (Sigma-Aldrich), 150 mM NaCI (Thermo Fisher), pH = 7.5) and fixed in 3.7% formaldehyde-90% ethanol (Sigma-Aldrich) for 30 seconds at room temperature. A reaction mixture containing 50 mM NaHCO3, 1 mM MgCI2 (Sigma-Aldrich), and 1 mg / ml of p-nitrophenyl phosphate (Sigma-Aldrich) was added into each well and incubated at 37 °C for 20 minutes. The reaction was stopped by adding 50 pL of 3 M NaOH. Absorbance was measured at 405 nm in a FLUOmega plate reader. ALP enzymatic activity was normalized to cellular viability. Alizarin Red Staining hBMSCs were seeded in 4-well plates at 40,000 cells / cm2. The day after, cells were induced to differentiate into osteoblasts using osteoblastic induction medium. At day 14 of osteoblast differentiation, mineralized matrix formation was measured using Alizarin Red staining (AR-S). Briefly, cells were fixed with 70% ice-cold ethanol for 1 hour at -20°C before adding AR-S (40 mM; Sigma- Aldrich) for 10 minutes at room temperature (RT). The level of calcium deposition was quantified by elution of AR-S. The absorbance of the eluted dye was assessed at 570 nm in a FLUOmega plate reader.

[0273] In vivo heterotropic bone formation assay

[0274] The in vivo heterotropic bone formation assay is used to evaluate the in vivo bone formation capacity of hBMSC by implanting them subcutaneously in immunodeficient mice. Briefly, implantation we seeded 500,000 cells onto scaffold granules in 200 pL of cell culture medium (MEM + 10% FBS + 1 % P / S) in cut-off syringes incubated them overnight at 37°C and 5% CO2. The scaffold granules were Hydroxyapatite / tricalcium phosphate (HA / TCP) granules (Zimmer Scandinavia, Horsholm, Denmark). Each mouse received two implants from the same participant. The mice were 8-week-old females NOD / SCID (NOD / LtSz-Prkdcscid).

[0275] Adipocyte differentiation hBMSCs in p1 were plated at 30.000 cells / cm2density. Adipogenesis was induced on the following day by DMEM supplemented with 10% FBS, 1 % P / S, 100 nM dexamethasone (Sigma-Aldrich), 0.5 mM 3- isobutyl-1 -methylxanthine (IBMX) (Sigma-Aldrich), 1 pM BRL (Rosiglitazone) (Sigma-Aldrich), 2 pg / mL insulin (Sigma-Aldrich). The medium was changed every other day for the duration of the differentiation.

[0276] Lipid droplets quantification

[0277] Lipid droplets quantification was used to evaluate adipocyte differentiation, which was determined with Nile Red (Sigma-Aldrich). We seeded 13,000 cells per well in a 96-well black / clear bottom plate (Perkin Elmer). After 24 hours, cells were induced to differentiate into adipocytes using adipogenic induction medium. At day 7, cells were washed with PBS and incubated with Nile Red (5 pg / mL) for 15 minutes in the dark at 37 °C. The fluorescence was quantified using FLUOmega plate reader at 485 nm excitation and 572 nm emission. The Nile Red fluorescent was normalized to cellular viability per well.

[0278] Osteoclasts differentiation

[0279] Peripheral Blood Mononuclear Cells (PBMCs) were isolated from 50 mL of EDTA blood by differential centrifugation using Ficoll-Paque (Cytiva) as a density gradient. The PBMC were washed twice with PBS (Thermo Fisher) before counting the cells with trypan blue (Thermo Fisher) using an automated cell counter - Countess (Invitrogen). Cells were seeded at a density of 50x106in T75 or 16.7x106in T25 culture flasks and differentiated to mature OC over 9 days in alpha-MEM (Thermo Fisher) with 10% FBS (Thermo Fisher) and 1 % P / S (100 U / mL penicillin (GIBCO), and 100 ug / mL streptomycin (GIBCO)) (Invitrogen). Forthe first 2 days, the cells were induced with 25 ng / mL M-CSF (R&D Systems, Abingdon, UK), after they were induced with 25 ng / mL M-CSF and 25 ng / mL RANKL (R&D Systems, Abingdon, UK). After 9 days of maturation, 12 systematic and evenly distributed pictures were taken to quantify the number of nuclei / OC and the number of OCs with more than 2 nuclei using a ckx41 microscope with an SC30 camera (Olympus Corporation, Tokyo, Japan).

[0280] Bone resorption assay

[0281] Matured osteoclasts (day 9 of induction) were detached by accutase treatment (PSS, Pasching, Austria) and were seeded onto bone slices (50,000 cells / bone slice) (www.boneslices.com). The cells were cultivated for 72 hours in alpha-MEM with 10% FBS, 1 % P / S, 25 ng / mL M-CSF, and 25 ng / mL RANKL. The media was stored at -20 °C for measurements of TRAcP activity. The bone slices were washed with water to remove the remaining cells and stained with toluidine blue (Thermo Fisher). Bone resorption was quantified by assessing the percentage of eroded surface per bone surface and the number of two types of resorption patterns: pits and trenches. A pit was defined as an excavation, circular in appearance, with well-defined edges, and where the ratio between the length and the width of the excavation did not exceed two. A trench was defined as an elongated and continuous excavation with well-defined edges and at least two times longer than its width. The analysis was performed blinded and randomized.

[0282] TRAcP activity analysis

[0283] TRAcP activity was analyzed in frozen cell culture media collected during the differentiation and resorption. In brief, 10 pL of cell culture media was analyzed in duplicate by incubation with TRAcP reaction buffer (1 M acetate (Sigma-Aldrich), 0.5% Triton X-100 (Sigma-Aldrich), 1 M NaCI (Sigma Aldrich), 10 mM EDTA (VWR), pH 5.5), 50 mM L-Ascorbic acid (Sigma-Aldrich), 0.2 M disodium tartrate (Sigma-Aldrich), and 82 mM 4-nitrophenylphosphate (Sigma-Aldrich), for 15 minutes at 37C in the dark. The reaction was stopped by adding 100 pL of 0.3 M sodium hydroxide (VWR). The absorbance was measured at 400 nm using a microplate reader (Synergy HT, Biotek).

[0284] Bioenergetic analysis of mature osteoclasts

[0285] The mature osteoclasts (day 8 of induction) were detached by accutase (Biowest) and seeded in seahorse cell culture plates (40,000 cells / well) (Agilent). The cells were incubated in alpha-MEM with 10% FBS, 1 % P / S, 25 ng / mL M-CSF, and 25 ng / mL RANKL for 24 hours. The cells were washed twice with Seahorse media (non-buffered DMEM (Agilent # 103575-100) supplemented with 1 mM glucose (Agilent), 1 mM sodium pyruvate (Agilent), and 2 mM glutamine (Agilent). Next, 180 pL of Seahorse media was added to each well, and the plate was incubated in a non-CO2 incubator for 60 min.

[0286] For the analysis of mitochondrial respiration, we used the Mito Stress Test assay (Agilent, #103015-100). Briefly, two groups of sequential drug injections were used: (1) uncoupler FCCP (Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; 2-2 pM final / well), and Rotenone / Antimycin A (0.5 pM final / well); (2) ATP-synthetase inhibitor Oligomycin (1 .5 pM final / well), and Rotenone / Antimycin A (0.5 pM final / well). For the ATP production analysis, we used the second group of injections with Oligomycin and Rotenone / Antimycin A. The parameters obtained were calculated using the average of the three cycles measured according to the manufacturer’s recommendations. For the glycolysis analysis, we used the Glycolytic Rate assay (Agilent #103344-100) by injecting sequentially Rotenone / Antimycin A (0.5 pM final / well) and 2-deoxyglucose (50 mM final / well).

[0287] Bone biopsies

[0288] Transiliac crest bone biopsies were collected using a modified Bordier procedure after each study subject underwent double labelling with tetracycline administrated orally. Prior to the bone biopsy, the participants ingested tetracycline hydrochloride 250 mg three times daily on day 1 -3 and day 13-15. On day 20, a 7 mm diameter core was obtained across the iliac crest. The specimens were subsequently fixed and stored in 70% ethanol / 30% water at 4° C until dehydrated and embedded non-decalcified in methyl-methacrylate. A Jung Model K microtome was used to cut 7-pm-thick sections for detailed bone and adipocyte histomorphometric analyses.

[0289] Micro-CT scanning

[0290] The whole transiliac bone biopsy specimens were scanned using a pCT scanner (pCT 50, Scanco Medical AG, Bruttisellen, Switzerland) with an isotropic voxel size of 6 pm (X-ray tube: 155 pA, 90 kVp, integration time 1500 ms) to quantify the 3D microarchitectural properties of the cancellous bone. All specimens were scanned in the same orientation. The 3D-image data sets, filtered with a Gaussian filter (sigma = 0.8, support = 1) and segmented with an optimal threshold of 180, consisted of approximately 600 consecutive 16-bit grey scale images. These images were used to quantify (after segmentation with a fixed optimal threshold) bone volume fraction (BV / TV, %), trabecular thickness (Tb.Th, pm), trabecular spacing (Tb.Sp, pm), trabecular number (Tb.N, mm'1), structure model index (SMI) , connectivity density (mm'3), trabecular bone density (TBD, pg / cm3), cortical thickness (Ct.Th, mm) and cortical porosity (Ct.Po, %). Bone histomorphometry

[0291] The transiliac bone biopsy specimens were fixed in 70 % Ethanol and undecalcified embedded methylmethacrylate (MMA), before pCT scanned and sectioned for the bone histomorphometry on a SM2500 heavy duty microtome (Leica). Central seven-pm-thick sections were either Masson’s trichrome stained for static histomorphometry or unstained for dynamic histomorphometry of tetracycline (mineralization) label and imaged on a VS200 slide scanner (Olympus). The slide scans were performed using a combination of bright-field and polarized light microscopy on the Masson’s stained sections, and fluorescence microscopy of the tetracycline double-labels on the unstained sections, and manually analysed using the VS200 desktop software (Olympus). The static histomorphometry measures included osteoid surface per bone surface (OS / BS, %) and eroded surfaces per bone surface (ES / BS, %), where the eroded surfaces were divided according to whether they had neighbouring osteoid surfaces [(ES with neighbouring OS / BS, %) and (ES without neighbouring OS / BS, %)]. The dynamic histomorphometry measures included the single and double labelled perimeter (sL.S / Pm and dL.S / Pm, pm) derived into the MS / BS (%) = M.Pm / B.Pm = (1 / 2 x sL.Pm + dL.S / Pm) / B.Pm, and mean interlabel width (II. W, pm) derived into the mineral apposition rate (MAR, pm / day) = II.W x ll.time (days) x p / 4.

[0292] Adipocyte histomorphometry

[0293] The bone marrow adiposity was investigated in scans of Masson’s trichrome stained sections, using point- and box-grids. The point-grid measures allow us to estimate the adiposity area per marrow area (Ad.Ar / M.Ar, %), while the box-grid measures allow us to estimate the adipocyte density as the adipocyte profile number per marrow area (Ad.Pf.N / M.Ar), as well as the mean adipocyte profile diameter (Ad.Pf.Dm).

[0294] DXA scan

[0295] Areal bone mineral density (aBMD) was measured at the lumbar spine (L1 -L4), total hip and the femoral neck using DXA (Hologic Discovery, Waltham, Massachusetts, USA). Z-scores and T-scores were calculated using the reference range provided by the manufacturer and the Third National Health and Nutrition Examination Survey reference.

[0296] Bioinformatics and Enrichment Analysis

[0297] RNA sequencing data analysis was performed in R. Briefly, Differential gene expression was analyzed using DESeq2 with a factor design (design = -Timepoint + Donor), which regresses donor variation to determine differentiation-associated genes, and a linear variable (design: -Resorption) was used for each individual time point to determine resorption-associated genes. Differentially expressed genes were selected based on an adjusted p < 10-4. Gene Ontology (GO) and Reactome pathway92 analyses were performed using GOseqseq.93

[0298] Mitochondrial proteins and their encoded genes were selected using Human MitoCarta3.0 in R. ID conversion and alignment for human proteins, genes and transcripts was done based on HGNC Biomart (https: / / biomart.genenames.org / martform). Enrichment analysis of the differentially expressed proteins (proteins p-value <0.05 and | log2FC| >0.26) was performed on the Enrichr using the gene name for each protein. The enriched terms are shown as a negative Iog10 of p-values from the Fisher exact test.

[0299] Statistics

[0300] All data is presented as median ± standard deviation (SD), unless otherwise stated. The statistical significance was determined by a paired Wilcox non-parametric test. A p-value of p<0.05 was considered significant (* p < 0.05, “ p > 0.01 and *** p >0.001). The n values correspond to the number of individuals analysed in each experiment, the number of technical replicates is specified in each section of the materials and methods. The indicated statistical tests and graphs were performed using R version 4.1.1. Data was processed using dplyr (v.) and tidyr (v.). The visualization of the data was done with R packages ggplot2 (v.), ggthemes.

[0301] Example 2 - Results hBMSC from m.3243A>G carriers present a transcriptional, proteomic, and metabolic reprogramming from OXPHOS to glycolytic bioenergetics profile

[0302] To study the effect of impaired OXPHOS on bone, we did a comprehensive assessment of the impact of m.3243A>G on bone progenitor and bone cell function, in vivo bone formation, and clinical measures, including DXA. We recruited 10 adult subjects carrying the m.3243A>G; tRNALeu(UUR)- including symptomatic and asymptomatic individuals - and 10 healthy controls matched on age, sex and body mass index (BMI). There was no difference between groups in major regulators of bone homeostasis such as p-25OHD, pCa2+, PTH, TSH, and liver and kidney function. Resting plasma lactate levels were higher in m.3243A>G carriers (Fold-change (FC)=1.58, p< 0.01) (Fig. 1A). To study the effect of impaired mDNA translation of mDNA-encoded OXPHOS proteins in bone cells, we isolated hBMSCs from bone marrow aspirates. Carriers of m.3243A>G have a variable level of heteroplasmy within cells and tissues. Heteroplasmy in hBMSCs was analyzed by digital PCR and ranged from 12-82% in m.3243A>G carriers, while the levels were below 0.1 % in matched controls (Fig. 1 B).

[0303] To characterize the effect of the pathogenic variant m.3243A>G in hBMSC we performed a comprehensive analysis using RNA sequencing, discovery proteomics and bioenergetic metabolic analysis. We performed a transcriptional, proteomic, and metabolic reprogramming of hBMSCs from m.3243A>G carriers. RNA sequencing of cultured hBMSCs showed 1941 differentially expressed (DE) genes that are enriched mainly in mitochondrial functions, including mitochondrial metabolism, morphology, and biogenesis. The transcriptomic profile of hBMSCs separated m.3243A>G carriers and healthy individuals in two groups with only minor overlap. To evaluate whether this transcriptional regulation is reflected in the protein levels, we performed large-discovery proteomics in hBMSC. The proteome showed altered levels of metabolic proteins, specifically mitochondrial metabolism, such as electron transport chain (ETC), citric acid (TCA) cycle, and branched-chain amino acid catabolism (BCAA) (Table 1).

[0304] Table 1

[0305] Enrichment analysis of differentially expressed proteins of hBMSC from m.3243A>G compared to control using EnrichR with Reactome as database.

[0306] Overall, the transcriptome and proteome show differential regulation of mitochondrial metabolism in m.3243A>G hBMSC. One of the key roles of mitochondria is the production of cellular energy in the form of adenosine triphosphate (ATP), which plays a role in highly active tissues such as bone, e.g., for hBMSC differentiation to osteoblast. We found a metabolic reprogramming of hBMSCs from m.3243A>G carriers to a favourable glycolytic profile. Specifically, the bioenergetic profile of m.3243A>G hBMSC demonstrated increased glycolytic ATP production (FC = 1.48, p<0.01) and decreased mitochondrial ATP production (FC = 0.8) (Fig. 1 C). Importantly, the net ATP production rate was unchanged, with a tendency to be increased in m.3243A>G hBMSC (Fig. 1 C). This shift in ATP production rates is the consequence of a metabolic reprogramming characterized by increased glycolytic rates (FC = 1.56, p>0.01) (Fig. 1 D).

[0307] Increasing heteroplasmy levels of m.3243A>G is generally associated with an earlier debut of symptoms and a more severe clinical phenotype. Accordingly, the levels of heteroplasmy correlated with the metabolic reprogramming, specifically negatively with metabolic reprogramming index (r = - 0.72), and mitochondrial ATP production (r = -0.84) and positively with glycolytic ATP production (r = 0.84). Furthermore, 98% of the DE genes in m.3243A>G correlate with the percentage of heteroplasmy. On the contrary, heteroplasmy levels showed little correlation with the proteomic profiles (6.3%), indicating that posttranscriptional regulation is independent of the heteroplasmy level in hBMSCs.

[0308] OXPHOS and TCA activities are tightly coupled as oxidation of NADH and FADH2 by OXPHOS complexes I and II are required for the TCA cycle (Da, W. et al., 2021). To evaluate the effects of decreased OXPHOS activity observed in MSC from m.3243A>G carriers on TCA cycle, we performed dynamic metabolic mapping using uniformly labelled glucose ([U-13C]glucose) to measure13C-labeled cellular metabolites derived from glucose, which is the major energy substrate source for MSC and OB. [U-13C]glucose is metabolized to [U-13C]pyruvate that can be converted into: (1)13C-labelled lactate, (2)13C-labelled alanine or (3)13C-labelled citrate in the mitochondria via acetyl-CoA upon entry in the TCA cycle. hBMSC showed a low13C enrichment in measured metabolites, indicating a slow glucose metabolism with a tendency to increase13C-labeled lactate in line with the increased glycolytic rates observed in the respirometry assays. Furthermore, we observed increased levels of rate-limiting glycolytic enzymes such as hexokinase 2 (HK2) and gamma enolase (ENO2), and lactate dehydrogenase isoforms (LDHA and LDHB) without changes in their gene expression, indicating a posttranslational regulation of glycolysis (Fig. 2A). Pyruvate enters the mitochondria and is metabolized to acetyl-CoA that enters the TCA cycle. The relative abundance of intermediate TCA metabolites showed a tendency to be reduced in the first turn of the cycle, while13C-labeled malate was increased.

[0309] This OXPHOS-deficiency metabolic reprogramming has previously been described in other cells and tissues that showed a lack of metabolic flexibility, which is required for the cells to adapt to microenvironmental changes, e.g. alterations in nutrient supply. These cells have developed various adaptative responses, such as unfolded protein response (UPR) and integrated stress response (ISR) that act coordinately to counteract the effects of mitochondrial dysfunction by activating autophagy. Here, we summarize them under the term mitochondrial stress response (MSR). MSR is dysregulated in MSC from m.3243A>G as we foundterms related to stress responses to be enriched in DE genes. Most of the genes involved in these pathways are upregulated, indicating an upregulation of the MSR. ISR activates the secretion of circulating cytokines like growth / differentiation factor 15 (GDF-15) and fibroblast growth factor 21 (FGF-21), which are considered mitochondrial disease biomarkers. MSC from m.3243A>G carriers showed an increased expression of GDF-15 (logFC = 0.36), while FGF-21 expression was very low in these cells (Fig. 2B). MSR activates a transcriptional program resulting in autophagy, cell cycle arrest, decreased proliferation, apoptosis or cellular senescence. The transcriptional program of hBMSC from m.3243A>G showed enriched terms in these pathways, indicating an early aging phenotype. Culturing m.3243A>G hBMSCs showed fewer colony-forming units-fibroblasts (CFU-f) and altered proliferation rates under nutrient-restricted conditions, indicating decreased metabolic flexibility, a common consequence of MSR (Fig. 2C).

[0310] Collectively, the data show that hBMSC from m.3243A>G carriers present with impaired OXPHOS and an adaptative metabolic reprogramming from OXPHOS to a more glycolytic bioenergetic profile, preserving the total cellular ATP production. Together with impaired OXPHOS, hBMSC from m.3243A>G carrier showed activation of MSR resembling an early aging cellular phenotype. Metabolic reprogramming to glycolysis decreases m.3243A>G hBMSCs osteogenic capacity

[0311] Previous in vitro and in vivo animal studies have shown that mitochondrial function is important for bone e.g. mitochondrial ATP production, TCA cycle intermediate metabolites, MSR activation, all of which are affected in m.3243A>G cells. Human BMSCs are osteoprogenitor cells with a transcriptional profile predisposed to osteoblast differentiation. Hollenberg et al. 2020 showed that inhibition of glycolysis with direction to OXPHOS increased hBMCSs osteogenic potential with increase in the osteogenic marker RUNX2, and in vivo increased the bone mineral density in mice. We subsequently investigated if the metabolic reprogramming of m.3243A>G from OXPHOS to a glycolytic bioenergetic profile decreased the ability of hBMSCs to differentiate to osteoblasts and affected bone formation capacity. Here, the hBMSC transcriptome of m.3243A>G carriers showed alteration in the gene expression of bone-related pathways; specifically, we observed that transcription factors involved in bone formation i.e. WWTR1, SMAD5, SOX11 and YAP1 are significantly downregulated (Fig. 3B). Furthermore, m.3243A>G hBMSC showed an altered stem cell lineage determination compared to controls. The m.3243A>G hBMSC, but not control hBMSCs, exhibited decreased osteogenic capacity correlating with age measured by alkaline phosphatase (ALP), a bone marker reflecting osteogenic activity (Fig. 3B). On the contrary, adipocyte differentiation capacity assessed using lipid staining by Nile Red (NR) was not correlated with age in m.3243A>G hBMSC but directly correlated with age in controls (Fig. 3C). This difference in lineage determination pattern favorable to adipogenesis instead of osteogenesis has been observed in the elderly population (> 85 years old). To further evaluate the hBMSC bone formation capacity in vivo, we performed a heterotopic assay with subcutaneous implantation of a mixture of MSC and hydroxyapatite - as a scaffold - in immunodeficient mice for 8 weeks. The hematoxylin-eosin staining of the implants revealed a decreased bone formation capacity of m.3243A>G MSC compared to the matched controls (Fig. 3D).

[0312] Collectively, these data show that hBMSC from m.3243A>G carriers have decreased in vivo bone formation capacity despite unchanged total cellular energy production.

[0313] Osteoclasts show increased TRAcP activity without changes in mitochondrial function

[0314] Bone remodelling is a dynamic and coordinated process between bone formation by osteoblast and bone resorption by osteoclasts. Derived from the hematopoietic progenitors in bone marrow, bone degrading osteoclasts possess a high abundance of mitochondria and in mice, global mitochondrial dysfunction increases osteoclast activity and bone loss. While osteoclast differentiation depends on glycolysis and OXPHOS, the bioenergetic pathway reprograms to glycolysis as the main source of energy for human mature osteoclasts for resorption activity in vitro.

[0315] To further evaluate the effects of impaired OXPHOS caused by the m.3243A>G variant in the process of bone resorption, we studied osteoclasts derived from peripheral blood mononuclear cells from m.3243A>G carriers and their matched controls. In contrast to hBMSCs, matured osteoclasts (day 9 of differentiation) showed lower levels of heteroplasmy. The heteroplasmy of osteoclasts correlates positively with the heteroplasmy of hBMSCs (R = 0.82) (Fig. 1 A, 4A, and 4B). The variant m.3243A>G is selected against with every cellular division, thus replicative cells like monocytes show decreased level of heteroplasmy with age as we and others have previously reported. Accordingly, these lower heteroplasmy levels were not associated with changes in bioenergetics profile of mature OCs from m.3243A>G carriers compared to their matched controls (Fig. 4C-E). In addition, matured OCs reseeded on bone slices showed a tendency to increase bone resorption activity (Fig. 4F). At the same time, we observed an alteration in the resorption patterns with respect to pits and trenches. For the OCs from m.3243A>G carriers, no correlation was found between pits (round cavities made by immobile osteoclasts) and trenches elongated resorption made by osteoclasts moving across the surface) as it was observed with the OCs from the controls. Osteoclasts that primarily form pits are associated with increased eroded surface compared to trenches. During bone resorption, osteoclasts secrete tartrateresistant acid phosphatase (TRAcP) enzyme to aid in the degradation of bone tissue (Halleen, J.M., 2003). The activity of secreted TRAcP during resorption was significantly increased in m.3243A>G carriers (FC 1.41) compared to controls (Fig. 4G). Moreover, the activity of secreted TRAcP during differentiation (from day 2 to day 9) was also significantly increased in osteoclasts from m.3243A>G carriers (Fig. 4H). This, together with a tendency to an increased number of nuclei per OC (Fig. 4I), indicates a better differentiation process of OC. None of these parameters correlated with the heteroplasmy levels or the bioenergetic profile, indicating that other parameters, potentially without any relation to mitochondrial function, drive these changes. Overall, OC from m.3243A>G carriers showed no alterations in OXPHOS activity or cellular metabolism.

[0316] The bone biopsies from m.3243A>G indicate a delay in bone formation

[0317] The effects of the mitochondrial dysfunction on bone tissue were also evaluated in transiliac bone biopsies from m.3243A>G carriers (n=7) and healthy individuals matched on age and gender (n = 7)(see Materials and Methods). Micro-computer-tomography (pCT) of the bone biopsies showed a 50% decreased trabecular bone volume (BV / TV) associated with decreased trabecular thickness (Fig. 5A- C). No difference was observed in the cortical bone parameters (Fig. 5D). Bone histomorphometry of the trabecular bone surfaces showed no difference in bone surfaces between carriers and controls, analyzed as eroded surface (ES) per bone surface (ES / BS) reflecting the initial resorption and reversalresorption phase bone, osteoid surface (OS / BS), and mineralizing surface (MS / BS), both reflecting bone formation phase (Fig. 5E-F). However, the extend of ES without neighbouring OS was increased in carriers, indicating a slight delay in the initiation of bone formation on ES. Furthermore, the mineral apposition rate (MAR) could only be measured in four of the m.3243A>G carriers due to the lack of double tetracycline labelling in the other three, showing a decreased MAR that reflects a reduced rate of mineralization once the process has started (Fig. 5G). On the contrary, adipocyte histomorphometry showed an increased mean adipocyte diameter (Ad.Pf.Dm), with unchanged adipocyte area (Ad.Ar / Ma.Ar) and density (Ad.Pf.N / Ma.Ar). To further evaluate the effect of this microarchitecture changes on the macroarchitecture of the bone, we performed DXA scans in all 20 participants of the study. Because mitochondrial disease may affect muscle mass and body weight which are directly associated with bone mass, controls were closely matched on BML BMD values were numerically but not statistically significantly lower both at the lumbar spine, femoral neck, and total hip- We previously reported lower BMD in a substantially larger m.3243A>G cohort [1] which would be in line with lower BMD in cases in the present study. Importantly, the former study [1] did not include good matching on body weight, therefore, the overall effect of m.3243A>G on BMD remains undetermined.

[0318] Overall, these results indicate that the defective bone formation observed in cultured MSC from m.3243A>G carriers is also reflected in the bone tissue of these individuals by lower bone density in the trabecular bone, which is the more metabolically active bone compartment.

[0319] Treatment with KH183 promoted a bioenergetically more efficient OXPHOS in m.3243A>G carriers hBMSC

[0320] To further evaluate the effects of OXPHOS on bone formation, we treated hBMSC with KH183 (a compound of general structure (lb) wherein as per compound X the following apply: L = L19; R1= H; R2- R2’ = L3; R3= H, in the S,R-configuration, which is the active compound of Sonlicromanol, which is the same compound yet of formula (la)), a drug developed for treating primary mitochondrial diseases. After 3 days of treatment with KH183, we observed a restoration of the transcriptional, proteomic, and metabolic reprogramming of m.3243A>G carriers hBMSCs with increased mitochondrial respiration and decreased glycolysis. RNA sequencing analysis revealed 6846 DE genes enriched mainly in metabolic pathways and mitochondrial functions. Enriched terms in mitochondrial functions include morphological changes, mitochondrial ATP production, mitochondrial-induced apoptosis and mitophagy showed an overall downregulation of their gene expression. This transcriptional program was associated with a metabolic switch of increased mitochondrial ATP production and decreased glycolytic ATP production (Fig. 6A-C). No change in total ATP production was observed (Fig. 6D). Treatment with KH183 restored the energy metabolism profile of the hBMSC m.3243A>G with a 50% mitochondrial and 50% glycolytic ATP production and a negative correlation between them. This KH183-induced metabolic profile is similar to the BM-MSC from the controls (Fig. 1 C).

[0321] Treatment with KH183 promoted a bioenergetically more efficient OXPHOS by altering the composition of the OXPHOS complexes. COX7A isoforms promote the functional reorganization of two distinct MRC structures: C-MRC, which is more efficient OXPHOS bioenergetics, and S-MRC, which is more glycolytic bioenergetics (Alston, C.L., et al., 2017). Gene expression of COX7A isoforms changes upon KH183 treatment, increasing COX7A1 and decreasing COX7A2, indicating an abundance of C- MRC structures promoting a more OXPHOS metabolism in m.3243A>G carries hBMSC (Fig. 6E). Furthermore, the third COX7A isoform, COX7A2L - known as “SC-associated factor 1 ” (SCAFI) - promotes a more glycolytic metabolic program. In line with the metabolic reprogramming of KH183 treatment, COX7A2L gene expression is decreased upon KH183 treatment in m.3243A>G carriers MSC (Fig. 6E). The COX7A-isoform-dependent MRC organization is tightly regulated by the pyruvate dehydrogenase complex (PDH) activity that provides acetyl-CoA to enter the TCA cycle, providing substrates for OXPHOS. PHD activity is regulated by phosphorylation inhibition by pyruvate dehydrogenase kinases (PDKs). The most expressed PDK isoforms in the bone marrow are PDK1 and PDK3, which are downregulated after treatment with KH183, thereby promoting PDH activity (Fig. 6F).

[0322] Overall, this indicates that KH183 affects changes in the bioenergetics transcriptional program of m.3243A>G carriers MSC, leading to an increased abundance of C-MRC structures that are more OXPHOS efficient and, thus, restoring the metabolic program of these cells. The percentage of heteroplasmy correlates with 99% (6794 out of 6846) of the DE genes in m.3243A>G hBMSC, while heteroplasmy levels showed little correlation with the proteomic profiles (6.9%,), indicating that the protein response to KH183 treatment is independent of heteroplasmy levels.

[0323] To evaluate the effects of KH183 treatment on glucose metabolism, we incubated the cells with [U-13C]glucose and analyzed13C-labelled metabolites.13C-labelled lactate decreased after KH183 treatment in line with the decreased glycolysis observed, while13C-labelled alanine was increased (data not shown).13C-labelled succinate was significantly increased after treatment, and the other relabelled TCA metabolites tended to increase levels, indicating an increase in first-turn TCA cycle intermediates (Fig. 6G). Furthermore, SUCLA2 and SUCLG2 protein levels were also increased after KH183 treatment, overall indicating a regulation of the succinate, a TCA cycle metabolite that directly connects it with OXPHOS via the complex II (succinate dehydrogenase, SDH) which converts succinate into fumarate.

[0324] To further evaluate the effects of the metabolic reprogramming of KH183 treatment, we analyzed the gene expression of the 13 mDNA encoded OXPHOS proteins. We observed downregulation of 1 and upregulation of 6 mDNA genes encoding complex I and IV, and upregulation of the two ribosomal RNAs (rRNA) encoding in the mDNA. More specifically ND3 was downregulated and COX1, COX2, ND4, ND4L, ND5, ND6, MT-RNR1 and MT-RNR5 were upregulated. The genes COX2, MT-RNR1 and MT-RNR2 showed the strongest increase in expression.. On the contrary, the 38% nDNA OXPHOS genes showed a downregulation (44 genes), while only 9 OXPHOS proteins showed changes in the protein level. Among these, NDUFS1 , NDUFB3, NDUFA13, NDUFB1 , NDUFA9, UQRC1 and UQCRFS1 , were the proteins that showed increased expression, whereas ATP5MC3 and TCIRG1 were found to be downregulated. The strongest differences in protein expression were measured for NDUFB1 and ATP5MC3. The mitochondrial transcriptional profile of m.3243A>G was upregulated compared to matched control, a potential compensatory mechanism for the OXPHOS deficiency present in these cells (Fig. 1). This downregulation of OXPHOS gene expression indicates that treatment with KH183 not only improves mitochondrial ATP production, but also decreases the mitochondrial responses to OXPHOS deficiency. After treatment with KH183, we observed an overall downregulation of the mitochondrial pathways in m.3243 A>G hBMSC that were upregulated, leading to a transcriptional general downregulation of MSR genes together with a downregulation of mitochondrial disease biomarker GDF-15 (LogFC = - 0.58) while FGF-21 was very low expressed in these cells (Fig. 7A). Downregulation of MSR led to a downregulation of DE genes involved in autophagy, cell cycle arrest, decreased proliferation, apoptosis, and cellular senescence. This improved mitochondrial function associated with the downregulation of genes involved in cellular stress responses, indicating a healthier cellular status.

[0325] More efficient OXPHOS in m.3243A>G carriers hBMSC associates with a bone-forming transcriptional program

[0326] Previous studies have shown that several mitochondrial functions are important for osteoblastogenesis, which treatment of KH183 improved in m.3243A>G hBMSC, e.g., OXPHOS, MSR, mitophagy (Fig. 6 and Fig. 7). Thus, we hypothesized that treatment with KH183 can improve the osteogenic capacity of m.3243A>G hBMSC. In line with our hypothesis, we observed an upregulation of DE genes that are associated with enriched terms related to bone formation (Fig. 7B). Key osteoblastogenesis genes, e.g. LRP5, RUNX2, TGFB3, were upregulated (Fig. 7C). Unexpectedly, key osteoblastogenesis gene GDF5 was not upregulated. To further evaluate whether KH183 can improve the osteogenic capacity of m.3243A>G hBMSC, we analyzed the mineralization capacity of m.3243A>G hBMSC after treatment with KH183 and observed an increased mineralization in KH183 treated cells (Fig. 7D). Taken together, treatment with KH183 improved bone formation capacity.

[0327] Example 4 - Brief summary of some research findings

[0328] Mitochondria have emerged as regulators of stem cell function and fate. However, the cellular and molecular mechanisms underlying these changes in bone and how they affect patients with mitochondrial diseases remain unknown. Therefore, the metabolic program of bone-marrow mesenchymal stem cells (MSC) and their fate in mitochondrial disease patients was investigated. In total, 10 m.3243A>G carriers and 10 sex- age- and BMI-matched healthy controls were recruited to collect peripheral blood and mononuclear cells, bone-marrow mesenchymal stem cells (MSC) and iliac crest bone biopsies. The m.3243A>G carriers were found to have higher plasma lactate (Fold-change (FC) = 1 .58, p< 0.01). There was no difference between groups in parameters that may influence bone (p-25OHD, p-Ca2+, p-PTH, p-TSH, liver and kidney function). Cultured MSCs from m.3243A>G carriers presented a heteroplasmy of 12-82%, which was associated with lower mitochondrial respiration (FC=0.45, p<0.01) and mitochondrial ATP production(FC=0.74, p<0.01), together with a compensatory increase in glycolysis rate (FC=1.49, p<0.01) and glycolytic ATP production(FC=1 .27, p=0.02), leading to unchanged total ATP production. This metabolic reprogramming is associated with fewer colonyforming units, lower cell proliferation rates, and decreased in vivo heterotopic bone formation capacity (50% lower, p=0.03). Interestingly, heteroplasmy was lower in osteoclasts (16-30%), explaining similar metabolic programming, proliferation and differentiation rate of osteoclasts from carriers and controls. In line with the cellular results, bone formation was decreased in bone biopsies from carriers (p=0.05), leading to a 50% lower bone volume (p=0.01) driven by thinner trabeculae (r=0.85, p=0.05). Because compromised oxidative phosphorylation can change the metabolic programming of MSCs that impairs bone formation, we investigated if restoration of the mitochondrial function can improve bone formation. Treatment of MSCs from m.3243A>G carriers with the redox modulating molecule KH183 restored the metabolic program of these cells. This indicates that the metabolic reprograming in MSCs with mitochondrial dysfunction is reversible, and targeting mitochondrial dysfunction to enhance bone formation may benefit patients with mitochondrial disease. It was shown that m.3243A>G impairs bone formation by reprogramming bone-marrow MSC.

[0329] References

[0330] Alston, C.L., Rocha, M.C., Lax, N.Z., Turnbull, D.M., and Taylor, R.W. (2017). The genetics and pathology of mitochondrial disease. J. Pathol. 241, 236-250. 10.1002 / path.4809. Catheline, S.E., Kaiser, E., Eliseev, R.A. (2023). Mitochondrial Genetics and Function as Determinants of Bone Phenotype and Aging. Curr Osteoporos Rep. 2023 Oct;21 (5):540-551. doi: 10.1007 / s11914- 023-00816-4. Epub 2023 Aug 5.

[0331] Da, W., Tao, L., and Zhu, Y. (2021). The Role of Osteoclast Energy Metabolism in the Occurrence and Development of Osteoporosis. Front. Endocrinol. 12, 675385. 10.3389 / fendo.2021 .675385.

[0332] Gandhi, S.S., Muraresku, C., McCormick, E.M., Falk, M.J., and McCormack, S.E. (2017). Risk factors for poor bone health in primary mitochondrial disease. Journal of Inherited Metabolic Disease 40, 673- 683. 10.1007 / s10545-017-0046-2.

[0333] Hollenberg, A.M., Smith, C.O., Shum, L.C., Awad, H., and Eliseev, R.A. (2020). Lactate Dehydrogenase Inhibition With Oxamate Exerts Bone Anabolic Effect. J. Bone Miner. Res. 35, 2432-2443. 10.1002 / jbmr.4142.

[0334] Haleen, J.M. (2003). Tartrate-resistant acid phosphatase 5B is a specific and sensitive marker of bone resorption. Anticancer Res. 2003 Mar-Apr;23(2A):1027-9.

[0335] Langdahl, J.H., Frederiksen, A.L., Hansen, S.J., Andersen, P.H., Yderstraede, K.B., Duno, M., Vissing, J., and Frost, M. (2017). Mitochondrial Point Mutation m.3243A>G Associates With Lower Bone Mineral Density, Thinner Cortices, and Reduced Bone Strength: A Case-Control Study. Journal of Bone and Mineral Research 32, 2041-2048. 10.1002 / jbmr.3193.

Claims

Claims1 . A compound represented by general structure (la) or (lb):wherein,- L is a linker comprising 110 optionally substituted backbone atoms selected from carbon, nitrogen and oxygen;- R1and R2are each independently selected from H, Ci - Ce alkyl, or Ci - Ce alkenyl, or R1and R2together form a bridging moiety that is a further linker L, or R1is joined with a backbone atom of the linker L to form a bridging moiety that is a further linker L which forms a cyclic structure and / or R2is joined with a backbone atom of the linker L to form a bridging moiety that is a further linker L which forms a cyclic structure;- R3is selected from H, Ci - Ce alkyl, or Ci - Ce alkenyl, wherein the alkyl or alkenyl may be substituted with one or more halogen atoms, hydroxyl moieties, or (halo)alkoxy moieties, or R3is absent when the nitrogen atom to which it is connected is connected to L via a double bond; or R3is joined with a backbone atom of the linker L to form a bridging moiety that is a further linker L which forms a cyclic structure; and- R4is selected from H or Ci - Ce alkyl, wherein the alkyl may be substituted with one or more halogen atoms, hydroxyl moieties, or (halo)alkoxy moieties; or R4is absent;- R7is in each instance individually a Ci - Ce alkyl;- X is an anion when R4is not absent and is absent when R4is absent; for use in a method of treating or preventing a bone disorder.

2. The compound for use according to claim 1 , wherein each R7is methyl.

3. The compound for use according to claim 1 or 2, wherein X is a pharmaceutically acceptable anion.

4. The compound for use according to any one of claims 1 -3, wherein linker L is selected from -(CH2)2--(CH2)2NHC(O)CH2-, -(CH2)3- -(CH2)2NHC(NH2)=, -(CH2)2NHC(O)CH2NHC(NH2)=, -(CH2)3NHC(NH2)=, -(CH2)2NHC(Me)=, -(CH2)2NHC(O)CH2NHC(Me)=, -(CH2)3NHC(Me)=, -(CH2)2NR1C(NH2)=, -C(CO2H)(CH2)3- -C(CO2H)(CH2)3NHC(NH2)=, -C(CO2H)CH2- -C(CO2H)(CH2)2-, -C(CO2H)(CH2)4-, -(CH2)4- -(CH2)5-, -CHR2C(O)-, -CHR2’CH2- -CHR5CH2NR5C(Me)=, -CHR2(CH2)2- -(CH2)2CHR1-, -(CH2)2CHR1NHC(O)C(Me)-, -CH2CHR1-, -CH2CHR1NHC(Me)=, -CHR5(CH2)2CHR5-, -CHR2’CHR3’(CH2)2-, and -CR5=CH-CH=CR5’-CH2-, whereinR1together with R1forms a bridging moiety that is a further linker L;R2together with R2forms a bridging moiety that is a further linker L; R3together with R3forms a bridging moiety that is a further linker L; andR5together with R5forms a bridging moiety that is a further linker L.

5. The compound for use according to any one of claims 1 -4, wherein the further linker L is -CH2- or -(CH2)2- or -(CH2)3- or -(CH2)4-.

6. The compound for use according to any one of claims 1 -5, wherein linker L together with to at least one of R1or R2forms a bridging moiety that is a further linker L which forms a cyclic structure, wherein that cyclic structure is a 4-10 membered heterocycle.

7. The compound for use according to claim 6, wherein that cyclic structure is a 6 membered heterocycle.

8. The compound for use according to any one of claims 1-7, wherein the compound is represented by structure (Vila), (VII b) , (Vile), (Vlld), (Vile), or (VHf):The compound for use according to any one of claims 1 -8, wherein the bone disorder is associated with aberrant expression of LRP5 or RUNX2.

10. The compound for use according to any one of claims 1-9, wherein the bone disorder is not associated with aberrant expression of GDF5.

11. The compound for use according to any one of claims 1-10, wherein the method is for decreasing bone fracture risk, for treating osteoporosis, for treating Type-I diabetic bone disease, for increasing the osteogenic potential of marrow stromal cells (MSC), for decreasing glycolytic ATP production in MSC, for increasing proliferation of MSC, for increasing sternnessof MSC, for increasing bone formation capacity, or for increasing the mineralization capacity of MSC.

12. The compound for use according to any one of claims 1 -11 , wherein the subject has a mitochondrial disease.

13. The compound for use according to any one of claims 1 -12, wherein the subject does not suffer from LRP5-linked osteoporosis-pseudoglioma syndrome.

14. The compound for use according to any one of claims 1-13, wherein the subject does not undergo concomitant therapy using hormones or antibodies for increasing bone formation.

15. A method for treating or preventing a bone disorder, the method comprising the step of administering a compound as defined in claim 1 to a subject.

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