Treatment of post-acute infection syndromes such as long covid and related symptoms

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 post-acute infection syndromes such as long COVID. It was found that the compounds have beneficial effects in reducing symptoms of post-acute infection syndromes, such as post-exertional malaise and persistent fatigue.

Description

[0001] Treatment of post-acute infection syndromes such as long COVID and related symptoms

[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 post-acute infection syndromes such as long COVID. It was found that the compounds have beneficial effects in reducing symptoms of post-acute infection syndromes, such as post-exertional malaise and persistent fatigue.

[0004] Background art

[0005] Chronic sequelae after acute infections, also known as post-acute infection syndromes (PAIS), can contribute to debilitating conditions which affect millions worldwide. For instance, after an acute SARS- CoV-2 infection, some patients suffer from post-acute sequelae of COVID-19 (PASC), referred to herein as long COVID. Oft reported symptoms of long COVID include limited exercise tolerance and post- exertional malaise, representing the worsening of symptoms after mental or physical exertion.

[0006] PAIS represents a great burden for which effective treatment is not always available. For instance, the underlying cause of long COVID is unknown, making the search for curative treatments challenging. Several hypotheses have been proposed, including viral persistence, autoimmunity, and microclots.

[0007] 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.

[0008] There is a need for methods of treatment of subjects suffering from PAIS such as long COVID. There is a need for small molecule therapies for treatment of subjects suffering from PAIS such as long COVID, and for small molecule therapies that can prevent and / or treat PAIS such as long COVID, for instance via multiple simultaneous mechanisms. Furthermore, there is a need for therapies with long- lasting effects on preventing and / or treating long COVID.

[0009] Summary of the invention

[0010] The inventors have surprisingly found that chromanyl derivatives as described herein can be used to treat post-acute infection syndromes (PAIS), post-viral disease, such as long COVID and related symptoms, as well as influenza. The compounds showed an attractive combination of three mechanisms of action, namely as reductive distress modulator (helping cellular metabolism), as oxidative distress modulator (protecting cells from ferroptotic cell death), and as a specific inhibitor of mPGES-1 (providing anti-inflammatory effects by reducing the production of the prostaglandin PGE2). Accordingly the invention provides a compound represented by general structure (la) or (lb): wherein,

[0011] - L is a linker comprising 1 10 optionally substituted backbone atoms selected from carbon, nitrogen and oxygen;

[0012] - 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;

[0013] - 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

[0014] - 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;

[0015] - R7is in each instance individually a Ci - Ce alkyl;

[0016] - X is an anion when R4is not absent and is absent when R4is absent; for use in a method of treating or preventing influenza, PAIS such as long COVID, or a symptom thereof.

[0017] In preferred embodiments each R7is methyl. Preferably X is a pharmaceutically acceptable anion. Linker L is preferably selected from

[0018] -(CH2)2-,

[0019] -(CH2)2NHC(O)CH2-,

[0020] -(CH2)3-,

[0021] -(CH2)2NHC(NH2)=,

[0022] -(CH2)2NHC(O)CH2NHC(NH2)=,

[0023] -(CH2)3NHC(NH2)=,

[0024] -(CH2)2NHC(Me)=, -(CH2)2NHC(O)CH2NHC(Me)=,

[0025] -(CH2)3NHC(Me)=,

[0026] -(CH2)2NR1’C(NH2)=,

[0027] -C(CO2H)(CH2)3-,

[0028] -C(CO2H)(CH2)3NHC(NH2)=,

[0029] -C(CO2H)CH2-

[0030] -C(CO2H)(CH2)2-,

[0031] -C(CO2H)(CH2)4-,

[0032] -(CH2)4-

[0033] -(CH2)5-,

[0034] -CHR2C(O)-,

[0035] -CHR2’CH2-

[0036] -CHR5CH2NR5C(Me)=,

[0037] -CHR2’(CH2)2-

[0038] -(CH2)2CHR1-,

[0039] -(CH2)2CHR1NHC(O)C(Me)-,

[0040] -CH2CHR1-,

[0041] -CH2CHR1NHC(Me)=,

[0042] -CHR5(CH2)2CHR5-,

[0043] -CHR2’CHR3’(CH2)2-, and

[0044] -CR5=CH-CH=CR5’-CH2-, wherein

[0045] R1together with R1forms a bridging moiety that is a further linker L;

[0046] R2together with R2forms a bridging moiety that is a further linker L;

[0047] 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.

[0048] Preferably, when a further linker L is present, 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, more preferably that cyclic structure is a 6 membered heterocycle. In preferred embodiments the compound is represented by structure (Vila), (Vllb), (Vile), (Vlld), (Vile), or (Vllf):

[0049] (Vila) (Vllb)

[0050]

[0051] (Vile) (VHf).

[0052] In some embodiments the method is for treatment of a symptom of PAIS such as long COVID, wherein the symptom is post-exertional malaise, fatigue, shortness of breath, difficulty concentrating, cognitive impairment, dysautonomia, palpitations, tachycardia, postural orthostatic tachycardia syndrome, dizziness, nausea, loss of taste, loss of smell, distorted smell, sleep difficulties, brain fog, seizures, persistent cough, diarrhea, chest pain, joint pain, muscle pain, exercise-induced muscle damage, hair loss, increased risk of stroke, increased risk of pulmonary embolism, increased risk of myocardial infarction, increased risk of diabetes, persistent fever, sore throat, difficulty sleeping, disrupted fertility, disrupted menstrual cycle, perimenopausal symptoms, gonadal function disorders, or ovarian insufficiency. Preferably the method is for treatment of post-exertional malaise. Preferably the treatment improves the outcome of Fatigue questionnaries preferably the NeuroQoL short form Fatigue questionnaire, or improves exercise capacity, or reduces mental fatigue, or reduces physical fatigue, or heightens the post-exertional malaise threshold, or shortens post-exertional malaise duration, or reduces post-exertional malaise severity.

[0053] In some embodiments the subject does not have a primary mitochondrial disease, preferably wherein the subject does not have an m.3243A>G mutation. In some embodiments the subject is a human of 6 to 18 years of age. Preferably the treatment comprises reduction of immune cell infiltration in skeletal muscle of the subject. Also provided is a method for treating or preventing influenza, PAIS such as long COVID, or a symptom thereof, the method comprising the step of administering a compound as defined above to a subject.

[0054] Description of the invention

[0055] The inventors have surprisingly found that chromanyl derivatives as described herein can be used to treat post-acute infection syndromes (PAIS) such as long COVID and related symptoms, as well as influenza. The compounds showed an attractive combination of three mechanisms of action, namely as reductive distress modulator (helping cellular metabolism), as oxidative distress modulator (protecting cells from ferroptotic cell death), and as a specific inhibitor of mPGES-1 (providing antiinflammatory effects by reducing the production of the prostaglandin PGE2). Compounds

[0056] The invention relates to compounds useful for practicing methods of treating PAIS such as long COVID and related symptoms, as well as influenza. The compounds for use according to the invention are represented by general structure (la) or (lb): wherein,

[0057] - L is a linker comprising 1 to 10 optionally substituted backbone atoms selected from carbon, nitrogen and oxygen;

[0058] - 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;

[0059] - 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

[0060] - 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;

[0061] - R7is in each instance individually a Ci - Ce alkyl moiety;

[0062] - X is an anion when R4is not absent and is absent when R4is absent.

[0063] 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.

[0064] 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.

[0065] 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).

[0066] (IVa) (IVb)

[0067] 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.

[0068] 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. 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.

[0069] 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.

[0070] 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-.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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).

[0085] 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.

[0086]

[0087] In the above linkers, R1' together with R1forms a bridging moiety that is a further linker L;

[0088] R2together with R2forms a bridging moiety that is a further linker L;

[0089] R3together with R3forms a bridging moiety that is a further linker L; and

[0090] R5together with R5forms a bridging moiety that is a further linker L.

[0091] 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.

[0092] 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.

[0093] 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-

[0094] 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.

[0095] 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.

[0096] 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 C* as marked above, 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 C* as marked above, and the second designator thereof defines the configuration of the additional stereocenter that may be present in the compound according to the invention.

[0097] 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.

[0098] 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

[0099] 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.

[0100] In a preferred embodiment, the compound is represented by general structure (la) or (lb), wherein:

[0101] - 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;

[0102] - 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

[0103] - 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.

[0104] 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.

[0105] In a preferred embodiment, the compound of general structure (la) or (lb) is represented by structure (Vila), (Vllb), (Vile), (Vlld), (Vile), or (Vllf):

[0106]

[0107] 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.

[0108] 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.

[0109] In a preferred embodiment, the compound of general structure (I) is represented by structure (Villa) or (VIII b) :

[0110] 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).

[0111] In a preferred embodiment, the compound of general structure (la) or (lb) is represented by structure

[0112] (IXa) (IXb)

[0113] 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).

[0114] 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:

[0115] (A) L = L1, R1-R2= L1, R3= H; (B) L = L1, R1= H, R2= H, R3= H;

[0116] (C) L = L2, R1= H, R2= H, R3= H;

[0117] (D) L = L3, R1= H, R2= H, R3= H;

[0118] (E) L = L4, R1= H, R2= H, R3= absent;

[0119] (F) L = L5, R1= H, R2= H, R3= absent;

[0120] (G) L = L6, R1= H, R2= H, R3= absent;

[0121] (H) L = L3, R1= H, R2= Me, R3= Me;

[0122] (I) L = L1, R1= H, R2= Me, R3= Me;

[0123] (J) L = L7, R1= H, R2= H, R3= absent;

[0124] (K) L = L8, R1= H, R2= H, R3= absent;

[0125] (L) L = L9, R1= H, R2= H, R3= absent;

[0126] (M) L = L10, R1-R1= L1, R2= H, R3= absent;

[0127] (N) L = L11, R1= H, R2= H, R3= H;

[0128] (O) L = L12, R1= H, R2= H, R3= absent;

[0129] (P) L = L13, R1= H, R2= H, R3= H;

[0130] (Q) L = L14, R1= H, R2= H, R3= H;

[0131] (R) L = L15, R1= H, R2= H, R3= H;

[0132] (S) L = L11, R1= H, R2= Me, R3= Me

[0133] (T) L = L16, R1= H, R2= H, R3= H;

[0134] (U) L = L17, R1= H, R2= H, R3= H;

[0135] (V) L = L16, R1= H, R2= Me, R3= Me;

[0136] (W) L = L18, R1= H, R2-R2= L3, R3= H;

[0137] (X) L = L19, R1= H, R2-R2= L3, R3= H;

[0138] (Y) L = L20, R1= H, R2= H, R5-R5’ = L3, R3= absent;

[0139] (Z) L = L21, R1= H, R2-R2= L1, R3= H;

[0140] (AA) L = L22, R1-R1= L1, R2= H, R3= H;

[0141] (AB) L = L23, R1-R1= L1, R2= H, R3= H;

[0142] (AC) L = L24, R1-R1= L3, R2= H, R3= H;

[0143] (AD) L = L25, R1-R1= L3, R2= H, R3= absent;

[0144] (AE) L = L26, R1= H, R2= H, R5-R5’ = L1, R3= H.

[0145] (AF) L = L19, R1= H, R2-R2= L3, R3= Me;

[0146] (AG) L = L19, R1= H, R2-R2= L1, R3= H;

[0147] (AH) L = L21, R1= H, R2-R2= L1, R3= Me;

[0148] (Al) L = L27, R1= H, R2-R2’ = -CH2-, R3-R3’ = L1, preferably R4= H, preferably X = Cl;

[0149] (AJ) L = L28, R1= H, R2= H, R3= H, preferably R4= H, preferably X = Cl;

[0150] (AK) L = L1, R1-R2= L1, R3= -CH2CH2OH, R4= H, X = Cl;

[0151] (AL) L = L1, R1-R2= L1, R3= -CH2CH2OH, R4= absent, X = absent.

[0152] 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.

[0153] 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

[0154] 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

[0155] 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 C* as marked above, 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 Reconfiguration or a mixture thereof, most preferably compound AJ has the R,R-confuguration.

[0156] 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).

[0157] 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

[0158] 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

[0159] S, R-configuration (S,R-X).

[0160] 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.

[0161] 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

[0162] 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

[0163] S, R-configuration (S,R-X).

[0164] 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), even more preferably of structure (la).

[0165] 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), even more preferably of structure (la), 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), even more preferably of structure (la), wherein R4= H and X = Cl. 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,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), even more preferably of structure (la), 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), even more preferably of structure (la).

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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 100 mg.

[0171] An 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 11 mg / kg.

[0172] The total daily dose for children is preferably at most 250 mg, or more 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.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] In an 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.

[0177] 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). Also envisaged is treatment of young adults (18-25 years) or children (0-17 years). Also envisaged is treatment of young adults (18-25 years). In some preferred embodiments the subject is 6-18 years of age. 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 reduce symptoms as described herein.

[0178] 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.

[0179] 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).

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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, more preferably it is dissolved in water. 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.

[0184] 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. In another highly preferred embodiment the aqueous solution essentially consists of the compound for use dissolved in water.

[0185] 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.

[0186] 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.

[0187] 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). 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).

[0188] 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.

[0189] In a preferred embodiment the invention pertains to a compound as defined herein for use in a method of treating or preventing influenza, PAIS such as long COVID, or a symptom thereof 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.

[0190] 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.

[0191] 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. 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.

[0192] 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.

[0193] 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.

[0194] 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.

[0195] 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 influenza, PAIS such as long COVID, or a symptom thereof 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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. 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.

[0200] 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.

[0201] Hence, in a preferred embodiment the invention pertains to a compound as defined herein above, for use in a method of treating or preventing influenza, PAIS such as long COVID, or a symptom thereof by administration of an effective total daily dose, wherein compound is administered orally.

[0202] 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.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] Medical Use

[0209] The compounds and compositions as described above are for use in a method of treating or preventing influenza, PAIS such as long COVID, or a symptom thereof. Treating or preventing influenza, PAIS such as long COVID, or a symptom thereof can encompass the avoidance of occurrence of the symptom, and it can also encompass the mitigation of the frequency or severity of the symptom. In some embodiments the method is for treating PAIS such as long COVID. In some embodiments the method is for treating a symptom of PAIS such as long COVID. In some embodiments the method is for treating influenza. In some embodiments the method is for treating long COVID. In some embodiments the method is for treating a symptom of long COVID. Treatment of a symptom preferably comprises reduction of the frequency or of the severity of that symptom. Preferably the symptom is reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, which may mean that the symptom no longer occurs, or that the symptom can no longer be detected.

[0210] It was found that the compounds as described herein can be used to treat influenza, PAIS such as long COVID, and related symptoms. The compounds showed an attractive combination of three mechanisms of action, namely as reductive distress modulator (helping cellular metabolism), as oxidative distress modulator (protecting cells from ferroptotic cell death), and as a specific inhibitor of mPGES-1 (providing anti-inflammatory effects by reducing the production of the prostaglandin PGE2). The compounds have an unexpected effect on the immune cells of a subject suffering influenza, or PAIS such as long COVID.

[0211] Long COVID is a type of chronic sequelae after an acute infection. For an acute SARS-CoV-2 infection, these chronic sequelae are referred to as post-acute sequelae of COVID-19 (PASC), long COVID, or post-COVID. In some embodiments the compound is for treating chronic sequelae after an acute infection, particularly after an acute SARS-CoV-2 infection.

[0212] Symptoms of long COVID can be expediently treated. In preferred embodiments the method is for treatment of a symptom of long COVID, wherein the symptom is post-exertional malaise, fatigue, shortness of breath, difficulty concentrating, cognitive impairment, dysautonomia, palpitations, tachycardia, postural orthostatic tachycardia syndrome, dizziness, nausea, loss of taste, loss of smell, distorted smell, sleep difficulties, brain fog, seizures, persistent cough, diarrhea, chest pain, joint pain, muscle pain, exercise-induced muscle damage, hair loss, increased risk of stroke, increased risk of pulmonary embolism, increased risk of myocardial infarction, increased risk of diabetes, persistent fever, sore throat, difficulty sleeping, disrupted fertility, disrupted menstrual cycle, perimenopausal symptoms, gonadal function disorders, or ovarian insufficiency.

[0213] In some embodiments the symptom is post-exertional malaise, tachycardia, postural orthostatic tachycardia syndrome, exercise-induced muscle damage. In some embodiments the symptom is disrupted fertility, disrupted menstrual cycle, perimenopausal symptoms, gonadal function disorders, or ovarian insufficiency.

[0214] Of particular interest is the treatment of post-exertional malaise. Post-exertional malaise (PEM), sometimes referred to as post-exertional symptom exacerbation (PESE) or post-exertional neuroimmune exhaustion (PENE), is a worsening of symptoms that occurs after minimal exertion. It is known as a symptom of myalgic encephalomyelitis / chronic fatigue syndrome (ME / CFS) and fibromyalgia, and it is also a common symptom of long COVID. Post-exertional malaise is often severe enough to be disabling, and is triggered by ordinary activities that healthy people tolerate. Typically, it begins 12-48 hours after the activity that triggers it, and it can last for days. To date, there is no cure, and management of post-exertional malaise is symptom-based. It can include pacing of activities to avoid triggering post-exertional malaise.

[0215] In preferred embodiments, the method is for treatment of post-exertional malaise. The effect of treatment of post-exertional malaise can be monitored with the 2-day Cardiopulmonary Exercise Test (CPET). In preferred embodiments the treatment improves the outcome of Fatigue questionnaires, preferably the NeuroQoL short form Fatigue questionnaire, or improves exercise capacity, or reduces mental fatigue, or reduces physical fatigue, or heightens the post-exertional malaise threshold, or shortens post-exertional malaise duration, or reduces post-exertional malaise severity. More preferably the treatment improves the outcome of the NeuroQoL short form Fatigue questionnaire, or heightens the post-exertional malaise threshold, or shortens post-exertional malaise duration, or reduces post- exertional malaise severity. Even more preferably the treatment heightens the post-exertional malaise threshold, or shortens post-exertional malaise duration, or reduces post-exertional malaise severity. Most preferably the treatment heightens the post-exertional malaise threshold, and shortens post- exertional malaise duration, and reduces post-exertional malaise severity.

[0216] In some embodiments the method is for improving the outcome of the NeuroQoL-SF Fatigue questionnaire, preferably after 13 weeks of treatment. Neuro-QoL (Quality of Life in Neurological Disorders) is a set of self-report measures that assesses the health-related quality of life (HRQOL) of adults and children with neurological disorders. It is a measurement system that evaluates and monitors the physical, mental, and social effects experienced by adults and children living with neurological conditions. It is designed to be completed in approximately one minute.

[0217] In some embodiments the method is for improving quality of life as assessed with the EQ-5D- 5L questionnaire, preferably after 13 weeks of treatment. The EQ-5D-5L generally has 2 pages: the EQ-5D descriptive system and the EQ visual analogue scale (EQ VAS). The descriptive system comprises five dimensions: mobility, self-care, usual activities, pain / discomfort and anxiety / depression. Each dimension has 5 levels: no problems, slight problems, moderate problems, severe problems and extreme problems. The patient is asked to indicate their health state by ticking the box next to the most appropriate statement in each of the five dimensions. This decision results in a 1 -digit number that expresses the level selected for that dimension. The digits for the five dimensions can be combined into a 5-digit number that describes the patient’s health state. The EQ VAS records the patient’s self-rated health on a vertical visual analogue scale where the endpoints are labelled ‘The best health you can image’ and ‘The worst health you can image’. The VAS can be used as a quantitative measure of health outcome that reflects the patient’s own judgement.

[0218] In some embodiments the method is for reducing post-exertional malaise burden as assessed with the FUNCAP questionnaire, preferably after 13 weeks of treatment. FUNCAP is a questionnaire that has been developed to assess functional capacity in patients with diseases where post exertional malaise is present. FUNCAP55 is a longer version comprising 55 questions, developed for improved diagnostic and disability benefit / insurance FC assessments; and FUNCAP27 is a shorter version for clinical patient follow-up.

[0219] In some embodiments the method is for reducing post-exertional malaise burden as assessed with the PROMIS questionnaire, preferably after 13 weeks of treatment. The Patient-Reported Outcomes Measurement Information System (PROMIS) is a National Institutes of Health initiative to develop self-report measures to assess functioning and well-being in physical, mental and social domains of health. PROMIS-29 assesses pain intensity using 7 health domains (physical function, fatigue, pain interference, depressive symptoms, anxiety, ability to participate in social roles and P62041316WQ 32 activities, and sleep disturbance). The PROMIS-29 assesses each of the 7 domains with 4 questions with an additional pain intensity numeric rating scale (NRS).

[0220] In some embodiments the method is for improving quality of life as assessed with the DePaul Symptom Questionnaire, preferably after 13 weeks of treatment. The DePaul Symptom Questionnaire (DSQ) was developed to assess the symptomatology and case definition fulfillment of individuals with myalgic encephalomyelitis (ME) and chronic fatigue syndrome (CFS). The DSQ-2 questionnaire is a revised version, to improve its psychometric properties, increase its diagnostic reliability, and assess symptoms required by case definitions.

[0221] In some embodiments the method is for improving orthostatic intolerance, preferably as assessed with the NASA 10-minute Lean Test, preferably after 13 weeks of treatment. The NASA 10- minute Lean Test is a variant of a test used by NASA researchers to test for orthostatic intolerance; it reduces muscular influences on venous return, a major cause of variability in orthostatic testing. The test involves measurement of blood pressure and heart rate while resting supine and every minute for 10 min while standing with shoulder-blades on the wall for a relaxed stance.

[0222] In some embodiments the method is for improving the outcome of muscle function and balance tests, preferably the Five Times Sit-to-Stand test (5xSTS). The Five Times Sit-to-Stand Test measures the time taken to stand five times from a sitting position as fast as possible. The test provides a method to quantify functional lower extremity strength and / or identify movement strategies a patient uses to complete transitional movements. In other embodiments the method is for reducing heart rate variability. In other embodiments the method is for increasing the number of steps per day of a subject, preferably as measured with an accelerometer.

[0223] The inventors found that the compounds had a specific effect on immune cells. Particularly, the compounds mitigate reduced mitochondrial function in peripheral blood monocytes (PBMCs) of post- COVID patients. In preferred embodiments, the treatment is for promoting mitochondrial function in peripheral blood monocytes of subjects suffering from long COVID. It was found that long COVID patients showed increased immune cell infiltration in their skeletal muscle. In preferred embodiments the treatment comprises reduction of immune cell infiltration in skeletal muscle of the subject. In some embodiments the method is for improving mitochondrial respiration measured in immune cells.

[0224] The treatment promotes skeletal muscle health. In preferred embodiments the method is for promoting skeletal muscle health. More preferably the method is for reducing the number of small and / or unconnected mitochondria. Preferably the method is for promoting mitochondrial connection in skeletal muscle. Preferably the method is for promoting the presence of intact sarcomere structure. Preferably the method is for promoting crista presence in skeletal muscle mitochondria.

[0225] Effects of the treatment are preferably as determined after at least 4 weeks, preferably after 8 weeks, more preferably after 12 or even 13 weeks of treatment, optionally even longer. Assays for determining these parameters are known in the art.

[0226] In preferred embodiments the subject to be treated does not suffer 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 subject does not have a mitochondrial disorder that is associated with a m.3242A>G mutation of the mitochondrial tRNA(leu) gene. In some embodiments the subject carries a mitochondrial DNA m.3243A>G mutation. In preferred embodiments the subject does not carry a mitochondrial DNA m.3243A>G mutation.

[0227] Treatments as described herein are attractive because they allow the treatment of conditions resulting from viral infection without requiring the administration of complex biological substances. 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. The compounds of the invention modulate cells of the immune system and accordingly their effect can persist after the substances themselves have been cleared.

[0228] In some embodiments the compound is for treatment of viral infection or PAIS, associated post- viral disease, or a symptom thereof, wherein the viral infection is preferably respiratory viral infection, more preferably influenza or coronaviral infection such as SARS-CoV-2 infection. In some embodiments the compound is for treatment of PAIS associated with viral infection, wherein the viral infection is preferably respiratory viral infection, more preferably coronaviral infection such as SARS-CoV-2 infection. When the compound is for treatment of PAIS associated with viral infection, the subject to be treated is preferably no longer infected with the associated virus. In some embodiments the compound is for the treatment of influenza. The treatment is preferably for reducing lung pathology related to viral infection or associated PAIS, more preferably for reducing the inflammatory phenotype of immune cells involved in lung pathology related to viral infection or associated PAIS. In some embodiments the treatment is for preventing, mitigating, or limiting long-term tissue damage, preferably lung tissue damage, related to viral infection or associated PAIS, more preferably related to influenza or PAIS, most preferably related to long COVID.

[0229] Influenza is caused by infection with influenza A virus (IAV), and a common embodiment thereof is seasonal flu; preferred treatment of influenza is treatment of seasonal flu. Treatment of influenza preferably comprises reduction of disease severity. Preferably treatment of influenza comprises improving oxygen saturation. Oxygen saturation is preferably increased to at least about 92%, preferably 92.5%, more preferably 93%, still more preferably 93.5%, more preferably 94%, even more preferably 94.5%, most preferably at least 95%. In preferred embodiments the treatment of influenza comprises the reduction of the inflammatory profile, preferably of lung monocytes. In preferred embodiments the treatment of influenza comprises improving lung function. In preferred embodiments the treatment of influenza comprises reducing inflammatory cytokines such as CXCL1 or IFNy or CCL5, preferably CXCL1 and CCL5, more preferably CXCL1 and IFNy and CCL5. Preferably, reduction of the inflammatory profile comprises increasing the frequency of Ly6Cl0monocytes. Preferably, reduction of the inflammatory profile comprises decreasing pro-inflammatory monocytes, more preferably decreasing Ly6Chimonocytes. Preferably, reduction of the inflammatory profile comprises reducing neutrophils. Preferably, reduction of the inflammatory profile comprises reduced cytokine levels, more preferably reduced CXCL1 and / or CCL5 levels. Preferably, reduction of the inflammatory profile comprises decreased IFN-y levels, preferably in the lungs. Preferably, reduction of the inflammatory profile comprises reduced IL-6 levels, preferably in serum.

[0230] In highly preferred embodiments treatment of influenza comprises reduction of the inflammatory profile, which comprises increasing the frequency of Ly6Cl0monocytes, and decreasing pro- inflammatory monocytes, more preferably decreasing Ly6Chimonocytes. In highly preferred embodiments treatment of influenza comprises reduction of the inflammatory profile, which comprises increasing the frequency of Ly6Cl0monocytes, and decreasing pro-inflammatory monocytes, more preferably decreasing Ly6Chimonocytes, and reducing neutrophils. Influenza can drive M2 macrophages towards a phenotype marked by high PGE2 production, fueling maladaptive inflammatory responses. Preferably the compound is for preventing, ameliorating, or reducing maladaptive inflammatory responses associated with influenza.

[0231] In highly preferred embodiments reduction ofthe inflammatory profile comprises reduced CXCL1 and CCL5 levels, and decreased IFN-y levels, preferably in the lungs, and reduced IL-6 levels, preferably in serum.

[0232] In some embodiments the compound is for treatment of long COVID, wherein the compound is for treatment of symptoms persisting for at least three months following initial SARS-CoV-2 infection, wherein the symptoms are preferably at least one of fatigue, shortness of breath, cognitive impairment, chest pain, neurological complications, cardiovascular complications, and respiratory complications, more preferably all those symptoms.

[0233] In some embodiments the compound is for treatment of viral infection, or a symptom thereof, wherein the viral infection is preferably respiratory viral infection, more preferably coronaviral infection such as SARS-CoV-2 infection, wherein the treatment is for increasing the frequency of monocytes. Preferably, increasing the frequency of monocytes includes increasing the Ly6Chisubset. Preferably, increasing the frequency of monocytes includes increasing the Ly6Cl0subset. More preferably, increasing the frequency of monocytes includes increasing the Ly6Chisubset and the Ly6Cl0subset. In preferred embodiments this is for increasing neutrophils. In preferred embodiments, the treatment is for reducing CD8+T cell frequencies. In preferred embodiments, the treatment is for reducing CD4+T cell frequencies. More preferably it is for reducing CD4+and CD8+T cell frequencies. Preferably the treatment is for a subject that is not deficient in Ly6Chimonocytes. Preferably the treatment is for a subject that is not deficient in Ly6Cl0monocytes. Preferably the treatment is for a subject that is not deficient in CD4+ T cells. Preferably the treatment is for a subject that is not deficient in CD8+ T cells.

[0234] When the compound is for treating respiratory viral infection as described above, preferably for treating SARS-CoV-2 infection, the compound is preferably for reducing cytokines. In preferred embodiments the cytokine is CXCL1 , preferably in lung tissue. In preferred embodiments the cytokine is IFN-y, preferably in lung tissue. In preferred embodiments the cytokine is IFN-a, preferably in serum. More preferably the compound is for reducing CXCL1 , preferably in lung tissue, and IFN-y, preferably in lung tissue. Most preferably the compound is for reducing CXCL1 , preferably in lung tissue, and IFN- y, preferably in lung tissue, and IFN-a, preferably in serum. Accordingly the treatment is preferably for altering lung immune cell dynamics, wherein the alteration comprises shifting toward increased innate cell populations, optionally while reducing cytokine production, preferably as described above.

[0235] When the compound is for treating long COVID, it is preferably for reducing lung inflammation, more preferably chronic lung inflammation. When the compound is for treating long COVID, it is preferably for restoring mean lung density to healthy levels. When the compound is for treating long COVID, it is preferably for mitigating pulmonary and systemic inflammation. When the compound is for treating long COVID, it is preferably for reducing vascular changes. When the compound is for treating long COVID, it is preferably fortreating without changing lung immune cell frequencies. Preferably when the compound is fortreating long COVID, it is for maintaining minimal interstitial lymphoid cell infiltration, or for preventing progression of perivascular and interstitial lymphoid cell infiltration.

[0236] Serum and lung tissue from subjects suffering from long COVID can show increased cytokine production compared to healthy subjects. In preferred embodiments, when the compound is for treating long COVID, it is for reducing cytokine levels, preferably towards a healthy phenotype, preferably in serum and / or lung tissue, more preferably in both. In preferred embodiments, the compound is for reducing CXCL1 , preferably in lung tissue. In preferred embodiments, the compound is for reducing CXCL10, preferably in lung tissue. In preferred embodiments, the compound is for reducing CCL5, preferably in serum. Accordingly, the compound is preferably for reducing cytokine levels in serum and lung tissue, wherein preferred cytokines are CXCL10, CXCL1 , and CCL5.

[0237] Bone marrow cells from subjects suffering from long COVID can show increased cytokine production compared to healthy subjects. In preferred embodiments, when the compound is for treating long COVID, it is for reducing a hyperresponsive immune cell phenotype towards a healthy phenotype, more preferably it is for reducing at least one of TNF-a, IFN-a, CCL2, IL-6, and IFN-p, preferably for reducing at least TNF-a and IFN-a, more preferably for reducing all of TNF-a, IFN-a, CCL2, IL-6, and IFN-p. Accordingly, the compound is preferably for reducing hyperresponsiveness of bone marrow progenitors.

[0238] The compounds were found to alter metabolic and chromatin-associated pathways in lungs of subjects suffering from long COVID. In preferred embodiments, when the compound is for treating long COVID, it is for downregulating pathways related to OCR chemokine receptor binding or related to structural constituents of chromatin, preferably for downregulating both pathways. More preferably it is for downregulation of CCR chemokine receptor binding genes such as Ccl5, Cell 7, Cell 9, Ccl21 a, Ccl22, and Ccl25, preferably at least one of Ccl5, Cell 7, and Cell 9, more preferably all three of those, even more preferably all six of Ccl5, Cell 7, Cell 9, Ccl21 a, Ccl22, and Ccl25. In other embodiments it is for downregulation of CCR chemokine receptor binding genes such as Ccl5, Ccl21 a, Nes, Ccl22, Cnih4, Cell 9, Ccl25, and Cell 7, preferably at least one of Ccl5 and Ccl21 a, more preferably both Ccl5 and Ccl21 a, even more preferably all eight of Ccl5, Ccl21 a, Nes, Ccl22, Cnih4, Cell 9, Ccl25, and Ccl17.

[0239] Preferably the compound is for reducing chemokine-driven recruitment of lymphocytes and / or dendritic cells, preferably both. The compound is preferably for dampening chronic immune infiltration. Preferably the compound is for reducing chemokine-driven recruitment of lymphoid cells, wherein lymphoid cells can include T cells, B cells, and NK cells. Preferably the compound is for reducing chemokine-driven recruitment of myeloid cells, which can include monocytes, basophils, eosinophils, and dendritic cells. More preferably Preferably the compound is for reducing chemokine-driven recruitment of both lymphoid cells and myeloid cells. The compound is preferably for reducing tissue inflammation in lungs of long COVID patients.

[0240] In preferred embodiments, when the compound is for treating long COVID, it is for upregulating lipid droplets pathways, more preferably for promoting lipid homeostasis and / or lipid catabolic processes, preferably both. In such cases the compound is preferably for downregulating genes linked to histone gene expression. Other preferred uses of the compound are for downregulation of pathways related to alcoholism, for downregulation of pathways related to systemic lupus erythematosus, and for upregulation of pathways related to fructose and mannose metabolism, more preferably for increasing expression of phosphomannomutase. When the compound is for downregulating CCL5, it is preferably for treating, reducing, ameliorating, or preventing hypoxia, chronic inflammation, and vascular activation.

[0241] Because it was found that the compound acts as a host-directed therapeutic with distinct effects in acute viral infection and PAIS, the compound is preferably for recalibrating maladaptive trained immunity and / or immunometabolism, more preferably both. Preferably the compound is for reducing hyperresponsiveness of bone marrow progenitors related to long COVID. Preferably the compound is for attenuating trained innate immunity associated with long COVID. Preferably the compound is for promoting a repair-oriented immune response.

[0242] The above compounds for use can also be used in a method for treating or preventing influenza, PAIS such as long COVID, or a symptom thereof, 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. Further details can be as described above.

[0243] General Definitions

[0244] 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".

[0245] 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.

[0246] 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.

[0247] 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.

[0248] 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.

[0249] Description of the Drawings

[0250] Fig. 1A - Compound 176 reduces influenza A virus (IAV) disease severity in mice. Male C57BL / 6J mice (13 weeks old) were intranasally infected with 2,500 PFU of H1 N1 / Auckland / 09. At 2 days post-infection (dpi), mice were treated once daily by oral gavage with compound 176 (10 mg / kg) or vehicle (0.9% saline) until euthanasia at 7 dpi. Each point represents an individual mouse. Data was pooled from two independent experiments and presented as mean ± SEM. *P < 0.05, **P < 0.01. Oxygen saturation shown.

[0251] Fig. 1 B - As 1 A, but frequencies of immune cell subsets in enzymatically digested lungs shown.

[0252] Fig. 1 C - As 1A, cytokine concentrations (pg / mL) in lung homogenates.

[0253] Fig. 1 D - As 1A, cytokine concentrations (pg / mL) in serum.

[0254] Fig. 2A - Compound 176 modulates the immune response to SARS-CoV-2 infection in vivo. Male C57BL / 6J mice (13 weeks old) were intranasally infected with 104PFU of mouse-adapted SARS-CoV- 2. At 1 day post-infection (dpi), mice received compound 176 (10 mg / kg) or vehicle (0.9% saline) via oral gavage once daily until euthanasia at 4 dpi. Each point represents an individual mouse. Data are pooled from two independent experiments and presented as mean ± SEM. *P < 0.05, **P < 0.01. Frequencies of immune cell subsets in enzymatically digested lungs are shown.

[0255] Fig. 2B - As 2A, but cytokine concentrations (pg / mL) in lung homogenates shown.

[0256] Fig. 2C - As 2A, but cytokine concentrations (pg / mL) in serum shown.

[0257] Fig. 3 - Evidence of systemic inflammation in PASC mouse model. Female obese mice (>12 weeks on high-fat diet; 16 weeks of age) were infected with mouse-adapted SARS-CoV-2, allowed to recover, and culled at 28 dpi. Cytokine production by ex vivo stimulated bone marrow progenitor cells is shown. Bone marrow cells isolated from mice in the PASC model were stimulated ex vivo for 24 hours with R848 (TLR7 / 8 agonist), LPS (TLR4 agonist), PAM3Cys (TLR2 agonist), or RPMI as a control. Cytokine concentrations (pg / mL) in culture supernatants were measured using a multiplex cytokine assay.

[0258] Fig. 4A - Murine model of post-acute sequelae of SARS-CoV-2 (PASC) with compound 176 treatment. Female C57BL / 6J mice (4 weeks old) were fed a high-fat diet for 12 weeks prior to intranasal infection with 104PFU of mouse-adapted SARS-CoV-2 or PBS (mock). At 14 days post-infection, infected mice were treated with compound 176 (10 mg / kg) or vehicle (0.9% saline) daily by oral gavage for 12 days until euthanasia at 26 dpi. Data are pooled from two independent experiments and presented as mean ± SEM. *P < 0.05, **P < 0.01 . Body weight shown relative to the first day of treatment.

[0259] Fig. 4B - As 4A, but showing lung histopathology scores, including total score, vascular changes (leukocyte margination, transmural migration, perivascular infiltration), and lung parenchyma changes (bronchitis, interstitial and alveolar inflammation, pneumocyte hypertrophy, and pleuritis). Each point represents an individual mouse.

[0260] Fig. 5 - Compound 176 reduces systemic inflammation associated with trained phenotype in PASC bone marrow progenitors. Bone marrow cells isolated from mice in the PASC model were stimulated ex vivo for 24 hours with R848 (TLR7 / 8 agonist), LPS (TLR4 agonist), PAM3Cys (TLR2 agonist), or RPMI as a control. Cytokine concentrations (pg / mL) in culture supernatants were measured using a multiplex cytokine assay. Dotted line indicates limit of detection of the assay.

[0261] Fig. 6A - Ridgeplots from Gene Set Enrichment Analysis (GSEA) of bulk lung RNA sequencing in PASC mice treated with compound 176 or vehicle. Gene Ontology Biological Process (BP) shown.

[0262] Fig. 6B - As 6A, but Gene Ontology Molecular Function (MF) shown.

[0263] Fig. 6C - As 6A, but Gene Ontology Cellular Component (CC) shown.

[0264] Examples

[0265] Example 1 . Summary of clinical trial

[0266] Patients with long COVID (or post-COVID, pC) report persistent fatigue and post-exertional malaise (PEM) as major complaints, for which there is no treatment. After an acute COVID-19 infection, a subset of patients suffer from these long-term consequences. Long COVID is a multi-systemic disease and common symptoms are persistent fatigue and post-exertional malaise (PEM). PEM is the worsening of symptoms after mental or physical exertion, and is considered to be the most disabling symptom of the disease. In pC patients, we found abnormalities in the structure and function of mitochondria. Studies have shown that both the architecture and function of the mitochondria are affected.

[0267] The clinical symptoms and the abnormalities in the architecture and function (such as complex I deficiency) of long COVID were found to be consistent with symptoms and muscle abnormalities in patients with genetic mitochondrial diseases such as MELAS spectrum (m.3243A>G) spectrum diseases. Intervention studies aiming to improve metabolism are needed to increase the induction threshold for PEM and to shorten the PEM duration. In this study, the efficacy of a mitochondrial drug candidate (shown below) is tested in post-COVID patients with persistent fatigue, muscle weakness, and PEM symptoms. The tested compound has beneficial effects for patients with mitochondrial disease, and improves quality of life (phase 1 , phase 2a and phase 2b program completed) while showing no serious side effects to date and having a good benefit / risk profile.

[0268] Objectives and study design: The study is a randomized, double-blind, placebo-controlled trial investigating whether the tested compound is effective in reducing persistent fatigue, muscle weakness and PEM in 80 adults (18-65 years) with post-COVID. Patients have all been in good health before post-COVID (WHO performance 0), and experienced post-exertional malaise (PEM) at the time of inclusion (DSQ). Exclusion criteria include a history of post-COVID-like complaints, serious cardiovascular disease, autoimmune disease, treatment with anticoagulants, pregnancy or breastfeeding.

[0269] Primary outcome: a reduction of post-COVID fatigue measured with the NeuroQoL-SF Fatigue questionnaire at week 13.

[0270] Secondary outcomes: (1) a reduction in fatigue during a repeated handgrip strength test of the dominant hand; (2) an increase in the number of steps per day measured with an accelerometer, in combination with a diary, and PEM app (available via Solve ME); (3) reduction in outcome measures of the DePaul Symptom Questionnaire (DSQ2); (4) an improvement in quality of life measured with the EQ-5D-5L; (5) a reduction in disease burden measured with “patient global impression of severity (PGIS)”, FUNCAP27 / 55 and promis-29 (including improvement in social, societal and work participation).

[0271] Results: the tested compound is an effective, safe and well-tolerated treatment method to reduce PEM-related complaints in post-COVID patients.

[0272] Example 2. Set-up of clinical trial

[0273] Clinical research questions and patient engagement

[0274] This post-COVID study is an intervention study aimed at reducing post-COVID symptoms and improving quality of life. The aim of applying the tested compound to post-COVID patients is to improve patients’ quality of life by reducing persistent fatigue and muscle weakness, raising the PEM threshold (i.e., reducing frequency and severity), and shortening PEM duration. As a major cause of reduced quality of life is persistent fatigue and the exacerbation of symptoms with mental and physical exhaustion after exertion (post-exertional malaise), this clinical trial aims to alleviate significant symptoms.

[0275] The choice for a placebo-controlled design is relevant to account for the placebo effect in the analysis, allowing an objective assessment of the intervention's effectiveness. The placebo group is relevant as there are numerous study visits that could provoke PEM and therefore induce a worse outcome on the questionnaires, which could be unrelated to the tested compound. Positive results can immediately scale up to a Phase 3 study, and will extend the research to children and severely affected patients.

[0276] The design of the post-COVID study considers patient burden. The study team has extensive knowledge of the severely debilitating symptoms of PEM and general fatigue. The study design minimizes patient burden by reducing the number of required study visits and setting primary and secondary outcome measures as low-threshold as possible. COVID conscious location is taken into account. Hotel accommodations are arranged for pre- and post-assessments if needed.

[0277] Paediatric population

[0278] In adults, the tested compound reduces persistent fatigue and muscle weakness in patients with mitochondrial disease (m.3243A>G MELAS spectrum disorders), and improves quality of life (phase 1 , phase 2a and phase 2b program completed), and >2.5 years of safety data is available. It was considered by the inventors that similar mechanisms can play a role across age groups. Children with post-COVID, like adults, suffer from symptoms that negatively impact their daily activities, including social interactions and school attendance. As similar mechanisms are thought to play a role across age groups, increasing the understanding of post-COVID in adults will also be valuable for children. The study includes young adults (18-25 years), and findings are extrapolated to paediatric populations. PBPK modelling has been performed to determine the adult-equivalent dose for children. In the age- group of children ranged 6-18 years PK analysis has confirmed the adequate dose-range in this population (Smeitink J., et al., 2022).

[0279] Recruitment

[0280] Patients were recruited via the RIVM portal and from a biobank, the Post-COVID Network Netherlands, and from previous inclusion in existing cohorts. This enriches the existing biobank and ensures well- defined patient inclusions.

[0281] Relevance

[0282] The underlying cause of post-COVID is unknown, making the search for curative treatments challenging. Several hypotheses have been proposed, including viral persistence, autoimmunity, and microclots. The inventors considered that long COVID patients may have reduced mitochondrial function in skeletal muscle. Mitochondria produce energy via their final biochemical pathway, the oxidative phosphorylation system. This system consists of five multi-protein enzyme complexes (complex l-V). A deficient activity of one or more of these complexes lead to severe multisystem disorders termed mitochondrial disease.

[0283] In this study, post-exertional malaise is induced in 25 post-COVID patients. Muscle biopsies are taken from the vastus lateralis muscle before and one day after PEM induction through a maximal cycling test. Mitochondrial function is lower at both time points, one week before and one day after the cycling test, compared to healthy controls. It was shown that one or more of the OXPHOS enzyme complex activities are decreased in post-COVID patients. Patients with mitochondrial diseases exhibit a similarly diverse array of symptoms as post-COVID patients, including fatigue and muscle weakness. The pathophysiology of mitochondrial diseases is based on a deficient activity of one or more OXPHOS enzyme complexes, but is not related to viral infection like long COVID. Nonetheless the inventors considered that long COVID may include increased production of harmful compounds (oxygen radicals) causing lipid peroxidation induced cell death (ferroptosis), disrupted cellular metabolism (abnormal redox balance), and the in mitochondrial disease observed increased production of inflammatory mediators (PGE2).

[0284] Problem definition and objective

[0285] Post-COVID is a severe post-acute infectious disease without an established pathogenesis. This study establishes a 'proof of concept' clinical trial using a biomedical-driven therapy under controlled conditions. This study addresses the following question: does treatment with the tested compound lead to a reduction in post-COVID symptoms, especially persistent fatigue and post exertional malaise symptoms caused by mitochondrial dysfunction? The trial also provides information about the different mechanisms affecting the mitochondria, and about a mitochondrial function test to screen for responders and non-responders.

[0286] Approach

[0287] A schematic overview of the study is presented in the table below. This is a double-blind, placebo- controlled clinical trial. The treatment arm in this study will receive 100 mg of the tested compound twice daily for 13 weeks (Gr. I in the table below). The placebo arm will follow the same schedule, the tested compound and the placebo are provided by Khondrion. The effect of the treatment will be evaluated at weeks 4, 8, and 13 by measuring the outcomes and collecting participant experiences, including safety assessments. All participants will be seen by a post-COVID physician before the study begins (week - 4 to -2). A two-week period between screening and the start of the treatment has been chosen to allow participants to recover from the screening and baseline measurements before start of the treatment. Study visits, will take place in Amsterdam.

[0288] Table 1 . Trial design. Gr=group, Wk=week.

[0289] Study Population: patients with post-COVID aged 18 to 65 years, diagnosed by a panel of medical post- COVID physicians, will be included. Patients have been in good health before post-COVID (WHO performance 0) and experience persistent fatigue and PEM (DSQ-PEM). Patients are not bedbound (moderate-severe, according to CCC criteria). Inclusion and exclusion criteria are similar to earlier clinical trials with the tested compound (Smeitink, J., et al., 2022).

[0290] Intervention: the tested compound 100 mg, administered orally twice daily as a powder dissolved in water, for a period of 12 weeks, with the last assessment at week 13.

[0291] Randomization: a stratified randomization method (1 :1) will be used. Duration of infection, seriousness of complaints, age categories and gender will serve as controlled covariates.

[0292] Primary outcome: reduction of post-COVID fatigue measured by the NeuroQoL-SF Fatigue questionnaire at week 13.

[0293] Secondary outcomes:

[0294] • reduction in fatigue with a handgrip strength test of the dominant hand,

[0295] • increase in the number of steps per day measured with an accelerometer,

[0296] • reduction in DSQ-2 questionnaire outcomes,

[0297] • improvement in quality of life measured by the EQ-5D-5L, and

[0298] • reduction in disease burden measured by patient global impression of severity, FUNCAP27 / 55, and PROMIS-29.

[0299] Exploratory outcomes:

[0300] • NASA LEAN test,

[0301] • mitochondrial respiration measured in immune cells,

[0302] • heart rate variability and sympathetic nervous system activity,

[0303] • “Five Times Sit-to-Stand test (5xSTS)”,

[0304] • social, societal, and work participation, and

[0305] • safety and tolerability.

[0306] The compound thus treats long COVID and its symptoms.

[0307] It was found that subjects suffering post-exertional malaise had skeletal muscle disarray and multiple broken myofibrils were observed, together with very small and unconnected mitochondria. In some subjects a complete loss of cristae and mitochondrial connectiveness was observed. Treatment with the tested compound promoted a return to a healthy state, which is healthy skeletal muscle featuring mitochondria that are well-connected, with densely packed cristae and intact sarcomere structure.

[0308] Safety: although no related serious adverse events have been reported during over two years of treatment, the safety of the tested compound is monitored throughout the study. Rationale for endpoints: the primary endpoint chosen is a fatigue questionnaire. Secondary endpoints focus on both short-term and long-term improvements in fatigue, muscle weakness, daily activity, POTS, post-COVID-related symptoms, and quality of life.

[0309] Safety monitoring: the study will be monitored by a Data Safety Monitoring Board (DSMB). Scheduled monitoring points are at weeks 4 and 8. The DSMB consists of independent experts.

[0310] Power calculation: with a standard deviation of the NeuroQoL-SF Fatigue of 5.7, a one-sided alpha (0.05), and a power of 80%, 34 participants per group are required. Considering an expected dropout rate of 10%, as observed in the LARGO trial, the number of participants per group will be 40.

[0311] Statistical methods: statistical significance is calculated with a t-test or Mann-Whitney U test on the specific outcome time. Longitudinal statistical significance is calculated using generalized linear mixed models. Post-hoc analyses is conducted with "Empirical Mean Differences" with correction for multiple testing.

[0312] References

[0313] Guo L, Appelman B, Mooij-Kalverda K, Houtkooper RH, van Weeghel M, Vaz FM, et al. EBioMedicine [Internet]. 2023 Aug 1 [cited 2023 Oct 5];94.

[0314] Jiang X, Renkema H, Pennings B, Pecheritsyna S, Schoeman JC, Hankemeier T, et al. Sci Rep [Internet], 2021 Dec 1 [cited 2024 Mar 6];11 (1).

[0315] Smeitink J, van Maanen R, de Boer L, Ruiterkamp G, Renkema H. BMC Neurol [Internet]. 2022 Dec 1 [cited 2024 Mar 6];22(1).

[0316] Example 3. Further investigation of treatment of post-viral disease

[0317] 3. 1 summary

[0318] There is a need for improved treatment of disease in influenza A virus (IAV) and SARS-CoV-2 infections, as well as for improved treatment of persistent symptoms in post-acute sequelae of viral infections such as SARS-CoV-2 infection (PASO, or “long COVID”). Therapies targeting mitochondrial pathways in these conditions remain limited. Compound 176, shown in Example 1 , is a small molecule that modulates mitochondrial redox balance via antioxidant activity and enhanced peroxiredoxin activity, and mPGES-1 inhibition by reducing ROS-driven inflammation. It has shown promise in treating mitochondrial disease in Phase Ila clinical trial and the Phase 2b program. We here explore its potential in acute viral infection and post-acute infection syndromes (PAIS). We evaluated compound 176 in mouse models of acute influenza A virus (IAV), SARS-CoV-2 infection, and PASC. In lAV-infected mice, compound 176 reduced disease severity, improving oxygen saturation and shifting lung monocytes toward a less inflammatory profile. In SARS-CoV-2 infection, compound 176 altered lung immune cell composition and lowered cytokine levels but did not impact weight loss or lung pathology. In our PASC model, characterized by persistent lung pathology and systemic inflammation, compound 176 showed a trend towards reduced lung pathology and attenuated cytokine hyperresponsiveness in progenitor cells, suggesting a reduction of chronic inflammation. To further explore the mechanisms in PASC lungs, bulk RNA sequencing was performed, revealing 179 differentially expressed genes following compound 176 treatment. Gene set enrichment analysis identified downregulation of CCR chemokine receptor binding and core histone genes, alongside upregulation of pathways related to lipid metabolism, lipid droplet biology, and fructose / mannose metabolism (CCR, or CC chemokine receptors, are also known as beta chemokine receptors). Together, these findings indicate that compound 176 modulates immune pathways during acute viral infection and influences inflammation, metabolism, and potentially epigenetic processes in PASC, supporting its further investigation as a host-directed therapeutic strategy for viral infections.

[0319] 3.2 introduction

[0320] Acute infection with influenza A virus (IAV) and SARS-CoV-2 can cause severe respiratory disease, leading to substantial morbidity and mortality worldwide. Despite widespread vaccination, the rapid emergence of novel viral strains and the inevitable delay in vaccine availability during the early stages of pandemics highlight the pressing need for effective post-infection treatments. Moreover, a subset of patients develop post-acute sequelae of SARS-CoV-2 infection (PASC, or “long COVID”), which can be defined as symptoms persisting for at least three months following initial SARS-CoV-2 infection, with no alternative explanation. PASC is characterized by more than 200 reported symptoms, including fatigue, shortness of breath, cognitive impairment (“brain fog”), chest pain, and diverse neurological, cardiovascular, and respiratory complications. Consequently, these persistent inflammatory responses highlight the need for therapies that not only mitigate acute disease but also limit long-term tissue damage.

[0321] Mitochondrial dysfunction was identified as a possibly central feature of IAV, SARS-CoV-2, and PASC pathogenesis. Viral infections were thought to disrupt mitochondrial dynamics, impair energy production by shifting metabolism from oxidative phosphorylation (OxPhos) to glycolysis, and promote excessive reactive oxygen species (ROS) generation, amplifying inflammatory responses and contributing to tissue injury. In patients with PASC, abnormalities have been observed in OxPhos, mitochondrial membrane potential, and altered expression of mitochondria-related genes in PBMCs and skeletal muscle biopsies, consistent with systemic mitochondrial dysfunction. These mitochondrial defects resemble those seen in myalgic encephalomyelitis / chronic fatigue syndrome (ME / CFS), a well- characterized mitochondria-associated disorder.

[0322] These insights have spurred growing interest in the inventors for targeting host metabolic pathways to modulate inflammation and improve clinical outcomes. However, known mitochondria-specific therapeutics for acute respiratory infections and PASC remain limited. For example, a Phase II clinical trial found that treatment with CoQ10, a natural component of the electron transport chain (ETC), did not significantly reduce the frequency or severity of PASC symptoms compared to placebo (Hansen, K.S., et al., High-dose coenzyme Q10 therapy versus placebo in patients with post CO VID-19 condition: a randomized, phase 2, crossover trial. The Lancet Regional Health - Europe, 2023. 24). In contrast, another trial suggested that MitoQ, a derivative of CoQ10, reduced the likelihood of SARS-CoV-2 infection and shortened the duration of viral symptoms (Chen, K., N.J. Jackson, and T. Kelesidis, Mitoquinone mesylate as post-exposure prophylaxis against SARS-CoV-2 infection in humans: an exploratory single center pragmatic open label non-randomized pilot clinical trial with matched controls. eBioMedicine, 2024. 102). Other mitochondria-related therapeutics, such as metformin, which partially inhibits complex I of the ETC, have shown moderate promise, with data indicating a roughly 41 % reduction in the risk of developing PASC when administered in outpatient settings (Bramante, C.T., et al., Outpatient treatment of COVID-19 and incidence of post-COVID-19 condition over 10 months (COVID-OUT): a multicentre, randomised, quadruple-blind, parallel-group, phase 3 trial. The Lancet Infectious Diseases, 2023. 23(10): p. 1119-1129).

[0323] Compound 176 is a small molecule that modulates mitochondrial redox pathways. It activates the thioredoxin system / peroxiredoxin enzyme machinery and attenuates induced lipid peroxidation, thereby preventing ferroptosis. Furthermore, it selectively inhibits microsomal prostaglandin synthase E-1- mediated PGE2 biosynthesis and attenuates inflammation. mPGES-1 is a key enzyme in prostaglandin E2 (PGE2) production, which can drive excessive inflammation during respiratory viral infection, while peroxiredoxins help detoxify ROS and maintain mitochondrial and immune homeostasis. Compound 176 has primarily been developed for primary and secondary mitochondrial disorders, demonstrating efficacy in preclinical studies and promising results in Phase Phase II clinical trials. Its potential in acute viral disease and PASC has not yet been explored. Here, we confirm the therapeutic potential of compound 176 via in vivo models of acute IAV and SARS-CoV-2 infection, as well as PASC.

[0324] 3.3 materials & methods

[0325] 3.3.1 Viruses - The A / H1 N1 / Auckland / 1 / 2009 (H1 N1) isolate, hereafter referred to as influenza A virus (IAV), was expanded in embryonated chicken eggs following published protocols (Brauer, R. and P. Chen, Influenza Virus Propagation in Embryonated Chicken Eggs. Vol. 97. 2015: 1940-087X. e52421). In vivo experimentation made use of a mouse-adapted SARS-CoV-2 variant belonging to the B.1.351 lineage (hCoV-19 / Australia / QLD1520 / 2020; GISAID Accession EPI_ISL_968081), first developed by Foo et al., European Respiratory Journal, 2023. 61 (3): p. 2201306.

[0326] 3.3.2 Cell Culture - Vero E6 (ATCC CRL-1586) and MDCK (ATCC NBL-2) cells were cultured in DMEM (Gibco) supplemented with 10% FCS and 1 % penicillin-streptomycin. Vero E6 cells were used for SARS-CoV-2 plaque assays, while MDCK cells were used for IAV plaque assays with a semi-solid overlay. All cells were maintained at 37 °C with 5% CO2and passaged at 80-100% confluency.

[0327] 3.3.3 Mouse Acute Infection Models - Male C57BL / 6J mice (13 weeks old) were obtained from Ozgene ARC and housed in individually ventilated cages on a 12-h I ig ht / dark cycle with food and water available ad libitum. For IAV infection, mice were anesthetized with 4% isoflurane and given 2,500 PFU intranasally. For SARS-CoV-2, mice were anesthetized with methoxyflu rane and intranasally inoculated with 104PFU of a mouse-adapted strain. Treatment with compound 176 (10 mg / kg) or vehicle (0.9% NaCI) was administered once daily by oral gavage, beginning at 2 dpi for IAV or 1 dpi for SARS-CoV-2 and continued until study termination. At the endpoint of IAV experiments, oxygen saturation was measured using a MouseOx Plus pulse oximeter (STARR Life Sciences) equipped with a collar sensor.

[0328] 3.3.4 Post-Acute Sequelae of SARS-CoV-2 Mouse Model - Female C57BL / 6J mice (4-weeks old) were obtained from Ozgene ARC and housed in individually ventilated cages under a 12-h light / dark cycle with ad libitum access to food and water. Animals were maintained on a high-fat diet (Specialty Feeds, Australia) for 12 weeks prior to infection. Mice were anesthetized with methoxyflu rane and intranasally inoculated with 104PFU of mouse-adapted SARS-CoV-2. Beginning 2 weeks post-infection, mice received compound 176 (10 mg / kg) by oral gavage once daily until the experimental endpoint (26dpi).

[0329] 3.3.5 Blood collection and analysis - At the study endpoint, mice were euthanized with a pentobarbital overdose, and blood was collected by cardiac puncture. Samples were kept at 4 °C overnight, and serum was isolated by centrifugation at 10,000 x g for 10 minutes.

[0330] 3.3.6 Lung Processing - The right superior, middle, and post-caval lung lobes were collected into DMEM (Gibco) and mechanically homogenized using a Qiagen Tissuelyser II. The left lung lobe was fixed in 10% neutral-buffered formalin for downstream histopathology analysis. The inferior lobe, reserved for single-cell suspension analysis, was enzymatically digested in DMEM containing 0.25 mg / mL Liberase™ (Roche) and 0.15 mg / mL DNase I (Thermo Fisher Scientific). Tissue was finely minced with scissors and incubated at 37 °C for 20 minutes. The digested material was passed through a 70 pm cell strainer, pelleted at 400 x g for 5 minutes, and resuspended in 1 mL RBC lysis buffer (Invitrogen) at 25 °C for 1 minute. Following dilution with 25 mL PBS and a second centrifugation, cells were finally resuspended in 100 pL PBS containing EDTA (Sigma-Aldrich) for downstream flow cytometry staining.

[0331] 3.3.7 Bone Marrow Collection - Bone marrow was collected for the PASC mouse model for flow cytometry staining and ex vivo stimulation. Bones were cleaned in RPMI 1640 (Gibco) using a scalpel and forceps. A single-cell suspension was prepared by flushing the marrow with PBS (Gibco) through a 25-gauge needle and a 70 pm cell strainer (Corning). Cells were pelleted by centrifugation at 300 x g for 5 minutes, resuspended in 2 mL of freezing medium (90% FBS and 10% DMSO), and aliquoted into 1 mL cryovials. Samples were frozen in a Mr. Frosty™ freezing container (Thermo Fisher Scientific) at -80 °C.

[0332] 3.3.8 Histology - The left lung lobe was fixed in 10% neutral-buffered formalin for a minimum of 24 hours, then transferred to 70% ethanol for processing at the Core Histology Facility, Translational Research Institute. Samples were embedded in paraffin and sectioned at 5 pm thickness using a Hyrax M25 rotary microtome (Leica Biosystems). Sections were deparaffinized and rehydrated through graded xylene and ethanol washes prior to hematoxylin and eosin (H&E) staining and mounted with Depex (Sigma-Aldrich). A veterinary pathologist, blinded to the experimental groups, evaluated the sections following previously described criteria (Hulme, K.D., et al., eLife, 2021. 10: p. e61803). Assessment included vascular changes, bronchitis, interstitial inflammation, alveolar inflammation, pneumocyte hypertrophy, and pleuritis. The term “lung parenchyma changes” refers to the combined scores for bronchitis, interstitial inflammation, alveolar inflammation, pneumocyte hypertrophy, and pleuritis.

[0333] 3.3.9 Bone Marrow Stimulation - Prior to stimulation, cells from each vial were thawed in a 37 °C water bath and transferred into 5 mL of thawing medium consisting of RPMI 1640 (Gibco) and supplemented with 10% FBS and 1 % Pen-Strep (Gibco). The cell suspension was diluted with 25 mL PBS (Gibco), centrifuged at 400 x g for 5 minutes, and resuspended in thawing medium. Viable cells were counted using a 1 :10 dilution of Trypan Blue on a Neubauer hemocytometer (Sigma-Aldrich). Bone marrow were aliquoted into a 96 well plate (2.5 x 105cells / well) in 100mL of fresh RPMI 1640 medium. Samples were incubated for one hour at 37°C 5% CO2 prior to stimulation with 400ng / mL Resiquimod (R848, Invivogen, USA), 25ng / mL LPS (Invivogen, USA), 10ug / mL Pam3Cys (Invivogen, USA) or RPMI-1640 (control) for 24 hours at 37°C 5% CO2. Stimulants were added with an additional 10OuL RPMI medium. Supernatants were harvested to fresh plates for storage at -80°C prior to analysis by multiplex cytokine detection assay.

[0334] 3.3.10 Flow Cytometry Staining - Single-cell suspensions (5 x 105cells for lung) were pelleted at 300 x g for 5 minutes in 96-well round-bottom plates (Corning) and incubated for 20 minutes with Fixable Viability Stain 700 (1 :15,000; 564997, BD Biosciences). Cells were then blocked with 50 pL Fc Block Rat Anti-mouse (CD16 / 32, BD Biosciences) prior to staining with the antibody panels listed in Table 3.3.10. Single-color compensation controls were prepared using either remaining cells or the VersaComp Antibody Capture Kit (Beckman Coulter). Both controls and samples were washed twice with 200 pL PBS, fixed in 100 pL BD Cytofix Fixation Buffer (BD Biosciences), washed again, and transferred to 5 mL FACS tubes (Corning) for acquisition on a BD LSRFortessa X20 at the T ranslational Research Institute. Flow cytometry data were processed and analyzed in FlowJo v10.8 (Windows), with suitable gating strategies.

[0335] Table 3.3. 10 - Lung digest antibody panel

[0336] 3.3.1 1 Multiplex cytokine assay - A panel of 13 cytokines, including IFN-y, CXCL1 , TNF-a, CCL2, IL- 12p70, CCL5, IL-10, CXCL10, GM-CSF, IL-10, IFN-p, IFN-a, and IL-6, was measured in lung homogenates, serum, and supernatants from ex vivo bone marrow stimulations using the LEGENDplex Mouse Anti-Virus Response Panel (BioLegend) according to the manufacturer’s instructions. Samples were acquired on a BD Accuri C6 Plus flow cytometer (BD Biosciences).

[0337] 3.3.12 Quantification of IAV Titers - IAV plaque assays were performed on confluent MDCK cells using a semi-solid overlay. Cells were cultured in DMEM (Gibco) supplemented with 10% fetal calf serum (FCS) and 1 % penicillin-streptomycin, and seeded at 7 x 105cells per well in 12-well plates. The following day, monolayers were washed once with serum-free DMEM and inoculated with 10-fold serial dilutions of virus stocks or experimental samples. Plates were incubated at 37 °C with 5% CO2 for 1 hour, with gentle rocking every 15 minutes. After virus adsorption, wells were overlaid with serum-free MEM containing 0.1 % low EEO agarose (Sigma-Aldrich) and 1 pg / mL TPCK-treated trypsin (Worthington Biochemical). Plates were incubated for 3 days, after which the overlay was removed and cells were stained with 0.1 % crystal violet in 10% neutral-buffered formalin for 1 hour to visualize plaques.

[0338] 3.3.13 Quantification of SARS-CoV-2 Titers - SARS-CoV-2 plaque assays were conducted on confluent Vero E6 cells using a semi-solid overlay. Cells were cultured in DMEM (Gibco) supplemented with 10% fetal calf serum (FCS) and 1 % penicillin-streptomycin, and seeded at 2 x 105cells per well in 12-well plates. After two days, monolayers were inoculated with 10-fold serial dilutions of virus stocks or experimental samples and incubated at 37 °C with 5% CO2 for 1 hour, with gentle rocking every 15 minutes. Following virus adsorption, wells were overlaid with MEM containing 2% FCS and 0.1 % low EEO agarose (Sigma-Aldrich). Plates were incubated for 2 days for mouse-adapted SARS-CoV-2 or 3 days for Delta SARS-CoV-2. The overlay was then removed, and cells were stained with 0.1 % crystal violet in 10% neutral-buffered formalin for 1 hour to visualize plaques.

[0339] 3.3.14 RNA extraction - Total RNA was extracted using the RNeasy kit (QIAGEN) for RNA-sequencing samples, as per manufacturer’s protocols. RNA was treated with Turbo DNase (Thermo Fisher) to remove genomic DNA.

[0340] 3.3.15 Statistical analyses - Data analysis was performed using GraphPad Prism. Outliers were identified and removed using the ROUT method (Q = 1 %). Data normality was assessed with the Shapiro-Wilk test. Comparisons between two groups were conducted using Welch’s t test for normally distributed data or Mann-Whitney U tests for non-parametric data. Comparisons among three groups were performed using one-way ANOVA or Welch’s ANOVA if variance assumptions were not met (Bartlett’s test), or the Kruskal-Wallis test with appropriate post-hoc analyses, depending on data distribution. Weight loss data were analyzed by two-way ANOVA with Geisser-Greenhouse correction, followed by Tukey’s post-hoc test.

[0341] 3.3.16 RNA library preparation and sequencing - RNA-seq libraries were prepared from 80-100 ng of total RNA using the Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus kit (Illumina, 20040529) and RNA UD Indexes Ligation (Illumina, 20091657) following the manufacturer’s protocol. rRNA was depleted, RNA was fragmented, and first-strand cDNA was synthesized using random primers. Second-strand synthesis incorporated dUTP to preserve strand orientation. cDNA ends were adenylated, adapters ligated, and libraries amplified for 13 PCR cycles with unique indexes. Libraries were quantified on a LabChip GX Touch (Revvity) and pooled equimolarly. Sequencing was performed using the Illumina NovaSeq X Series sequencer (NovaSeq Control Software v1 .2.2.48004 and Real Time Analysis v4.6.7). The library pool was diluted and denatured according to the standard NovaSeq X protocol and sequenced to generate single-end 151 bp reads across 1 lane of a flow cell using a NovaSeq X Series 10B Reagent Kit (300 cycles) (Illumina, 20085594). Fastq files were generated with bcl2fastq2 (v2.20.0.422) and adapters trimmed. Library prep was conducted at the University of Queensland Sequencing Facility, with sequencing in collaboration with the Australian Genome Research Facility. 3.3.17 RNA-sequencinq analysis - Adapter sequences and low-quality bases were trimmed using Trim Galore (v0.6.10) with a quality cutoff of 20 (default). Post-trimming quality was verified with FastQC. Transcript quantification was performed using Salmon (v1 .10.0) with the -validateMappings, -seqBias, and -gcBias options enabled, and library type set to “A”. Salmon indices were generated from mouse transcriptome (GENCODE M37) without decoy sequences. For downstream analyses, transcript counts were imported and summarized to gene-level expression in R (v4.3.1). Genes with <20 counts were filtered out of the analysis. Differential expression was assessed using DESeq2 (v1.42.0). Surrogate variable analysis was applied to control for potential batch effects. Differentially expressed genes were identified as padj<0.05 and Iog2foldchange >1 . Analyses were restricted to protein-coding genes annotated in org.Mm.eg.db. Functional enrichment analyses included Gene Ontology (GO) term enrichment, GO Gene Set Enrichment Analysis (GSEA), and Kyoto Encyclopedia of Genes and Genomes (KEGG) GSEA to identify pathways and biological processes associated with differential gene expression. All enrichment analysis used default settings, with pvalueCutoff and qvalueCutoff set to 0.05.

[0342] 3.4 Results

[0343] 3.4.1 - Compound 176 mitigates disease severity in mice infected with IAV - Compound 176 was evaluated in vivo using an established mouse model of severe IAV infection. Mice were infected with IAV and treated daily starting 2dpi via oral gavage with compound 176 (10 mg / kg), a dose previously reported to be well tolerated, or vehicle (0.9% saline) until the experimental endpoint. Treatment initiation after infection was chosen to better reflect a clinical post-exposure setting. The primary disease severity readouts were weight loss, the conventional endpoint in influenza models, and blood oxygen saturation, which directly reflects lung function. Compound 176 treatment did not significantly alter IAV- induced weight loss, but it reduced disease burden as measured by improved oxygen saturation (Fig . 1 A). No significant differences were detected in total lung histopathology scores or viral titers. Immune profiling revealed that compound 176-treated lungs displayed an increased frequency of Ly6Cl0monocytes and a relative decrease in pro-inflammatory Ly6Chimonocytes, along with a trend toward reduced neutrophils (p = 0.068; Fig. 1 B). Cytokine analysis showed reduced CXCL1 and CCL5 levels, with trends toward decreased IFN-y in the lungs (p = 0.094) and lower serum IL-6 (p = 0.099; Fig. 1 C and Fig. 1 D). Overall, compound 176 reduced disease burden as reflected by improved oxygen saturation and was associated with a shift toward a less inflammatory immune profile during IAV infection.

[0344] 3.4.2 Compound 176 modulates immune response in severe SARS-CoV-2 infection - We also assessed the impact of compound 176 in SARS-CoV-2 infection. Mice were infected with mouse-adapted SARS- CoV-2 and treated daily with compound 176 (10 mg / kg) or vehicle (0.9% saline) by oral gavage until 4 dpi. In this model, disease severity was primarily measured by weight loss. Compound 176 treatment did not affect weight loss, lung histopathology scores, or viral titers. Immune profiling revealed that compound 176 increased the frequency of monocytes, including both Ly6Chiand Ly6Cl0subsets, with a trend toward increased neutrophils (p = 0.052). Conversely, CD4+and CD8+T cell frequencies were reduced in treated mice (Fig. 2A). Cytokine analysis showed reduced CXCL1 and IFN-y levels in the lungs, along with decreased serum IFN-a (Fig. 2B and Fig. 2C). Altogether, compound 176 altered lung immune cell dynamics, shifting toward increased innate cell populations while reducing cytokine production.

[0345] 3.4.3 Compound 176 treatment shows a trend of reduced lung pathology and systemic inflammation in the PASC model - A PASC mouse model was previously established (generated by Sinclair et al.). Female obese mice were selected for infection, as both female sex and obesity are major risk factors for PASC, while young healthy male mice were used as a control. Female mice maintained on a high- fat chow diet for at least 12 weeks were infected with mouse-adapted SARS-CoV-2 and subseguently recovered, with no evidence of ongoing viral replication. At one-month post-infection, these mice exhibited persistent features consistent with PASC, including chronic lung inflammation (histology scoring), altered mean lung density (Ct imaging). In contrast, healthy male mice infected with mouse- adapted SARS-CoV-2 and allowed to recover for one month did not develop persistent lung inflammation, suggesting that long-term seguelae consistent with PASC were specific to the obese female cohort.

[0346] These persistent changes in the female PASC mice were accompanied by systemic inflammatory responses (changes in serum proteomics) and proinflammatory response of bone marrow cells to ex vivo stimulation (Fig. 3). This is consistent with innate immune training; a process whereby innate immune cells acguire a memory-like state through metabolic and epigenetic reprogramming. Innate immune training has been implicated in chronic inflammation and persistent symptoms in post-viral syndromes.

[0347] Given compound 176’s reported ability to reduce ROS-mediated inflammation in other disease contexts, we investigated whether compound 176 could mitigate pulmonary and systemic inflammation in PASC. Female obese mice were infected (or mock-infected) with mouse-adapted SARS-CoV-2, as per laboratory PASC model. At two weeks post-infection, mice began receiving daily compound 176 treatment (10 mg / kg) or vehicle (0.9% saline) by oral gavage until 26 dpi. Compound 176 did not alter body weight loss relative to vehicle controls but improved relative weight gain compared to baseline by 12 days post-treatment (Fig. 4A). No SARS-CoV-2 viral titres or evidence of SARS-CoV-2 viral RNA was detected in the lungs of PASC (treated and untreated) compared to mock-infected controls. As expected, PASC mice exhibited significant lung pathology compared to mock controls (Fig. 4B). There was a trend towards reduced vascular changes in compound 176-treated mice compared to vehicle- treated. There were no observed changes in lung immune cell frequencies between treated and untreated. Histopathological assessment by a blinded pathologist indicated that most vehicle-treated mice had mild perivascular and interstitial lymphoid cell infiltration, whereas compound 176-treated mice exhibited predominantly minimal interstitial lymphoid cell infiltration. In the lungs, compound 176 treatment showed a trend toward reduced cytokine levels compared to vehicle-treated PASC controls. CXCL1 was lower in compound 176-treated mice (p = 0.073), although CXCL1 was not elevated in PASC relative to uninfected controls. CXCL10 tended to increase with PASC (uninfected vs. vehicle; p = 0.087) and showed a trend toward reduction following compound 176 treatment (p = 0.095), suggesting partial restoration toward baseline levels.

[0348] We next assessed whether compound 176 could reduce systemic inflammation associated with PASC. Compound 176 treatment showed a trend of reduced cytokine levels in serum compared to vehicle- treated PASC mice. Compound 176 treatment significantly reduced CCL5 compared to vehicle-treated mice. IL-10, a cytokine typically associated with anti-inflammatory and immunosuppressive effects, was significantly decreased in vehicle-treated PASC mice versus uninfected controls and showed an increase following compound 176 treatment.

[0349] We next assessed whether compound 176 could reduce systemic inflammation by examining bone marrow progenitor cells. Bone marrow cells from PASC mice showed increased cytokine production following stimulation compared to mock-infected controls (Fig. 5). Treatment with compound 176 partially reduced this hyperresponsive phenotype relative to vehicle-treated PASC mice. Specifically, reductions were observed in TNF-a (Pam3Cys stimulation) and IFN-a (LPS stimulation), with trends toward decreased CCL2 (LPS; p=0.065), IL-6 (Pam3Cys; p=0.108), and IFN-p (Pam3Cys; p=0.065).

[0350] Overall, compound 176 showed a trend toward reducing lung pathology and significantly reduced hyperresponsiveness of bone marrow progenitors, suggesting it may modulate systemic inflammation in PASC.

[0351] 3.4.4 Compound 176 alters metabolic and chromatin-associated pathways in PASC lungs - To further assess the effects of compound 176 in the lungs, bulk RNA-sequencing was performed on lung homogenates from PASC mice. Initial comparisons between mock and PASC lungs were limited by sample quality and number; therefore, subsequent analyses focused on PASC mice treated with compound 176 versus vehicle controls. Surrogate variable analysis corrected for batch effects, and replicates clustered consistently. Differential expression analysis (DESeq2) identified 179 proteincoding DEGs (padj < 0.05, |log2FC| > 1). Gene ontology (GO) over-representation analysis did not yield significant terms, prompting gene set enrichment analysis (GSEA).

[0352] Within molecular functions (MF), compound 176-treated lungs displayed downregulation of ‘CCR chemokine receptor binding’ and ‘structural constituent of chromatin’. The latter was driven by coordinated changes in multiple core histone genes, suggesting potential epigenetic alterations consistent with a trained immunity phenotype. Specifically, downregulation of CCR chemokine receptor binding genes (Ccl5, Cell 7, Cell 9, Ccl21 a, Ccl22, Ccl25; see table 3.3.4) points to reduced chemokine- driven recruitment of lymphocytes and dendritic cells, which may dampen chronic immune infiltration and tissue inflammation in PASC lungs. Ccl5 reached statistical significance (padj < 0.05). Table 3.3.4 - CCR Chemokine receptor binding genes from bulk RNA-seq of treated vs. vehicle PASC

[0353] Given compound 176’s reported role in mitochondrial regulation, we also examined cellular components (CC). Lipid droplets were the only upregulated CC pathway, aligning with biological process (BP) enrichments in lipid homeostasis and lipid catabolic processes (Fig. 6), while the nucleosome (linked to histone gene expression) was downregulated.

[0354] KEGG GSEA identified several enriched pathways, including downregulation of ‘alcoholism’ and ‘systemic lupus erythematosus’, and upregulation of ‘fructose and mannose metabolism’. The downregulated pathways were associated with multiple H2A and H3B histone family genes, while the upregulated pathway was linked to increased expression of phosphomannomutase (EC 5.4.2.8). These represent the differentially expressed genes (DEGs) driving the enrichment. Together, these findings indicate that compound 176 impacts metabolic, inflammatory, and chromatin-related pathways in the lungs, highlighting its role in modulating cellular metabolism and epigenetic regulation during PASC.

[0355] 3.5 Discussion

[0356] In this study, we investigated compound 176, a clinical-stage small molecule with activity as an oxidative stress modulator, a redox modulator via the thioredoxin / peroxiredoxin system, and as an inhibitor of the prostaglandin E2 (PGE2) synthase pathway (mPGES-1). In acute viral infections, compound 176 mitigated disease severity in IAV and altered immune responses during SARS-CoV-2 infection. Consistent with our working hypothesis that compound 176 would reduce disease severity, it improved oxygen saturation in lAV-infected mice. In SARS-CoV-2, compound 176 altered lung immune cell composition. Viral titers remained unchanged in both infections, consistent with compound 176 acting through host immune modulation rather than direct antiviral activity. Compound 176 reduced inflammatory cytokines (CXCL1 and IFNy for SARS-CoV-2, and the same plus CCL5 for IAV), indicating it dampens excessive immune activation linked to disease severity.

[0357] Lung immune profiling revealed virus-specific effects. In IAV, compound 176 increased frequencies of Ly6Cl0monocytes, associated with inflammation resolution, and reduced neutrophils which are often associated with less severe IAV. In SARS-CoV-2, frequencies of neutrophils and monocytes were increased while CD4+and CD8+T cells were decreased. This profile partially resembles patterns reported in severe COVID-19 patients, with elevated innate immune cells, but differs in that cytokine levels were not elevated. These findings provide proof of concept that compound 176 has beneficial effects in IAV and immunomodulatory effects in SARS-CoV-2.

[0358] Severe acute respiratory infections are associated with elevated ROS which leads to mitochondrial damage, and alterations to oxidative phosphorylation. Compound 176 has been shown in other disease contexts to limit ROS-induced damage and preserve mitochondrial function. Beyond redox effects, compound 176 also inhibits prostaglandin E2 (PGE2) signaling, which has been implicated in severe influenza through suppression of alveolar macrophage effector function. Notably, IAV infection can drive M2 macrophages towards a phenotype marked by high PGE2 production, fueling maladaptive inflammatory responses.

[0359] In the PASC model, compound 176 reduced the ex vivo hyperresponsiveness of bone marrow progenitors. This indicates compound 176 can attenuate trained innate immunity, a process driven by epigenetic and metabolic reprogramming of innate progenitors that has been implicated in PASC (Gu, J., et al., Frontiers in Immunology, 2023. Vol. 14). Human studies report persistent alterations in CD34+HSPCs for up to a year post-infection with SARS-CoV-2, consistent with our observation that PASC bone marrow progenitors showed heightened cytokine responses to TLR agonists. Compound 176 dampened this response, indicating that redox modulation can interfere with progenitor training. While ROS are not classical drivers of trained immunity, they can influence epigenetic enzymes (e.g., HDACs) and cellular metabolism. Similarly, transient increases in PGE2 have been observed in p-glucan training models, although its direct role in establishing trained immunity remains unclear (Kang, Y.Y., et al., Adv Sci (Weinh), 2024. 11 (14): p. e2308978).

[0360] Trained immunity is closely linked to myelopoiesis, with downstream myeloid cells derived from HSPCs retaining the trained phenotype. In PASC, post-COVID-19 monocytes exhibit epigenetic changes and produce elevated IL-6, GM-CSF, and TNF-a in response to R848 stimulation (Cheong, J.-G., et al., Cell, 2023. 186(18): p. 3882-3902. e24; You, M„ et al., Nat Cell Biol, 2021. 23(6): p. 620-630). Future studies can therefore extend analyses to differentiated populations, including BM-derived monocytes and macrophages.

[0361] Building on the progenitor-level effects and to further investigate the trend of reduced lung pathology observed in compound 176-treated PASC lungs, we performed RNA-seq on lung homogenates. RNA- seq revealed compound 176-induced changes in chromatin-associated genes (H2A and H2B), CCR chemokine receptor binding, lipid droplet biology, and fructose / mannose metabolism. Epigenetic and lipid metabolic reprogramming are central to trained immunity, and dysregulated lipid metabolism has been linked to PASC. Although lipid droplets have not been directly studied in PASC, they support SARS-CoV-2 replication and accumulate during infection. Altered lipid droplets have also been reported in myalgic encephalomyelitis / chronic fatigue syndrome (ME / CFS), a condition with overlapping symptoms with PASC (Jahanbani, F., et al., PLoS One, 2022. 17(8): p. e0272703). Upregulation of genes associated with lipid droplets and lipid homeostasis in compound 176-treated lungs can therefore reflect altered immune metabolism that influences inflammation resolution.

[0362] Compound 176 treatment upregulated phosphomannomutase (PMM), a key enzyme in fructose / mannose metabolism involved in N-glycosylation. PMM activity can influence mitochondrial structure, glucose metabolism, and protein glycosylation, suggesting potential effects on immune cell function (Radenkovic, S., et al., Cell Reports, 2024. 43(3)). Beyond metabolic pathways, compound 176 also impacted immune signaling, as evidenced by the downregulation of CCR chemokine receptor binding genes, which regulate recruitment of innate immune cells to the lung. Notably, CCL5, one of the downregulated genes, is highly elevated in the plasma of PASC patients and is associated with hypoxia, chronic inflammation, and vascular activation. Although lung immune cell frequencies were unchanged in our study, these transcriptional changes may reflect subtle anti-inflammatory effects or altered immune cell function, contributing to the trend of reduced vascular changes observed in compound 176- treated PASC lungs. Future studies can combine transmission electron microscopy (TEM) to assess mitochondrial structure and lipid droplet abundance with epigenomic analyses to further clarify how compound 176 impacts PASC biology.

[0363] Patients with PASC have been suggested to be more susceptible to secondary infections (Murray, H.C., et al., 2020-2023. International Journal of Infectious Diseases, 2024. 145: p. 107078). Moreover, augmented immune functions associated with trained immunity have been implicated in pathological tissue damage and chronic inflammatory diseases such as atherosclerosis, raising the possibility that persistent symptoms in PASC may reflect maladaptive innate immune training. Thus, trained immunity may represent a context-dependent process: possibly protective in acute heterologous infection, but maladaptive in the setting of chronic inflammation and tissue injury.

[0364] Overall, this study evaluated compound 176 in the context of viral infections. We identified compound 176 as a host-directed therapeutic with distinct effects in acute viral infection and PAIS. Compound 176 mitigated severe IAV by improving oxygenation and reducing inflammatory cytokines, while in SARS- CoV-2 it altered immune cell composition. In PASC, compound 176 dampened bone marrow progenitor hyperresponsiveness and modulated chemokine, lipid, and chromatin pathways, indicating it can act by recalibrating maladaptive trained immunity and immunometabolism. These findings highlight compound 176’s therapeutic potential.

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 long COVID or a symptom thereof.

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):

9. The compound for use according to any one of claims 1-8, wherein the compound is represented by structure (Vila) or (VII b) :(Vila) (VII b).

10. The compound for use according to any one of claims 1 -9, wherein the compound is represented by structure (Vila):(Vila).11 . The compound for use according to any one of claims 1 -10, wherein the method is for treatment of a symptom of long COVID, wherein the symptom is post-exertional malaise, fatigue, shortness of breath, difficulty concentrating, cognitive impairment, dysautonomia, palpitations, tachycardia, postural orthostatic tachycardia syndrome, dizziness, nausea, loss of taste, loss of smell, distorted smell, sleep difficulties, brain fog, seizures, persistent cough, diarrhea, chest pain, joint pain, muscle pain, exercise-induced muscle damage, hair loss, increased risk of stroke, increased risk of pulmonary embolism, increased risk of myocardial infarction, increased risk of diabetes, persistent fever, sore throat, difficulty sleeping, disrupted fertility, disrupted menstrual cycle, perimenopausal symptoms, gonadal function disorders, or ovarian insufficiency.

12. The compound for use according to any one of claims 1 -11 , wherein the method is for treatment of post-exertional malaise.

13. The compound for use according to any one of claims 1 -12, wherein the treatment improves the outcome of the NeuroQoL short form Fatigue questionnaire, or improves exercise capacity, or reduces mental fatigue, or reduces physical fatigue, or heightens the post-exertional malaise threshold, or shortens post-exertional malaise duration, or reduces post-exertional malaise severity.

14. The compound for use according to any one of claims 1 -13, wherein the subject does not have a primary mitochondrial disease, preferably wherein the subject does not have an m.3243A>G mutation.

15. The compound for use according to any one of claims 1 -14, wherein the subject is 6 to 18 years of age.

16. The compound for use according to any one of claims 1 -15, wherein the treatment comprises reduction of immune cell infiltration in skeletal muscle of the subject.

17. The compound for use according to any one of claims 1 -16, wherein the compound is for reducing a hyperresponsive immune cell phenotype associated with long COVID.

18. The compound for use according to any one of claims 1 -17, wherein the compound is for treating post-viral chronic fatigue associated with long COVID.

19. A method for treating or preventing post-acute infection syndromes (PAIS) such as long COVID or a symptom thereof, the method comprising the step of administering a compound as defined in claim 1 to a subject.

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