Compounds for use in the treatment of phenylketonuria

Abstract

The present invention provides compounds of Formula (I), or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof, as well as methods for treating hyperphenylalaninemia (HPA), in particular phenylketonuria (PKU), using the compounds.

Description

[0001] Compounds for use in the treatment of phenylketonuria

[0002] Field of the invention

[0003] The present invention relates to compounds for use in the treatment of hyperphenylalaninemia (HPA), in particular phenylketonuria (PKU). These compounds act as pharmacological chaperones of the enzyme phenylalanine hydroxylase.

[0004] Background

[0005] Phenylalanine hydroxylase (PAH, EC 1.14.16.1) catalyzes the tetrahydrobiopterin (BH4)-dependent conversion of L-phenylalanine (L-Phe) to L-tyrosine (L-Tyr). This is the initial and the rate-limiting step in phenylalanine catabolism, consuming in humans about 75% of the phenylalanine input from the diet. PAH is primarily present in the liver, where removal of excess L-Phe occurs. Mutations in the human PAH gene lead to increased neurotoxic levels of L-Phe in the blood and to the appearance in urine of metabolites that arise from the transamination of L-Phe to phenylpyruvate. This is the hallmark of the HPAs, of which PKU (OMIM 261600) is the most severe. About 13,000 new patients are diagnosed with PKU each year (averaged figures worldwide 1:10,000).

[0006] PKU patients are normally classified in three (phenotypic) groups depending on their off-diet blood phenylalanine (Phe) levels; mild HPA (MHP; 120-600 pmol / l), mild PKU (600-1200 pmol / l) and classic PKU (> 1200 pmol / l). Most PAH variants are missense variants (58.5%), followed by deletions (15.9%), splice-site variants (13.7%), nonsense variants (6.0%) and insertions (3.1%). Missense mutations, as well as small deletions and insertions are mainly associated with PAH misfolding and instability. Wettstein, S et al. in Eur. J. Hum. Genet., 2015, 23(3): pp 302-9 describes the association of mutations with particular patient groups. The intracellular quality control mechanisms eliminate the defective mutant protein via degradation through polyubiquitin-dependent processes such as proteasome- mediated degradation and / or selective autophagy, resulting in a partial or complete lack of associated PAH function.

[0007] The accumulation of Phe in blood and the brain and the subsequent disturbance in brain neurotransmitters lead to neurological symptoms including mental retardation, purposeless movements and depression. The dietary intake of Phe must therefore be strictly controlled in PKU patients and the established treatment is a Phe- restricted diet and, recently, supplementation with preparations of the natural BH4 cofactor (i.e. the FDA-approved Kuvan®) also shows effectiveness for about 20- 30% patients, who can follow a less strict Phe-free diet. BH4 acts to increase PAH activity as it is one of the cosubstrates in the reaction (the “Michaelis-Menten” effect). In addition it has a PAH stabilizing, chaperone effect.

[0008] Pharmacological chaperones are the focus of increasing interest as an approach with therapeutic potential aiming to correct protein misfolding. The decrease in PAH protein stability is the main molecular pathogenic mechanism in PKU.

[0009] A new class of compounds acting as pharmacological chaperones for PAH has previously been discovered, as described in WO 2017 / 029202. However, it would be desirable to provide further and / or improved compounds which can act as pharmacological chaperones. In particular, it would be desirable to provide compounds which can stabilize PAH and many disease-associated mutations thereof, and which can increase PAH activity in cells expressing the enzyme.

[0010] Summary

[0011] The present invention provides a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof:

[0012] Formula (I) wherein

[0013] R1 and R2 are independently selected from the group consisting of H and C1-C3 alkyl;

[0014] Y is a 5- or 6-membered heteroaryl which may be substituted by one or more R3 groups; each R3 is independently selected from the group consisting of halogen, N(Ry)2, ORy, SRy, Ci-Ce alkyl, C2-C6 alkenyl, and a 5- or 6-membered carbocyclic or heterocyclic group, wherein each Ryis independently H or C1-6 alkyl and wherein each R3 group may be substituted by one or more R4 groups; or two R3 groups together form a 5- or 6-membered carbocyclic or heterocyclic group which may be substituted by one or more R4 groups; and

[0015] R4 is selected from the group consisting of halogen, C1-3 alkyl, =0, -OH and -NH2.

[0016] In embodiments, the compound is not

[0017] The present invention also provides a compound of the invention (e.g. a compound of Formula (I)) or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof, for use in the treatment of hyperphenylalaninemia (HPA), in particular phenylketonuria (PKU).

[0018] The present invention also provides a method of treating hyperphenylalaninemia (HPA), in particular phenylketonuria (PKU), comprising administering to a patient in need thereof an effective or therapeutically effective amount of a compound of the invention (e.g. a compound of Formula (I)) or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof.

[0019] The present invention also provides the use of a compound of the invention (e.g. a compound of Formula (I)) or a solvate, stereoisomer, tautomer, or salt thereof, in the manufacture of a medicament for use in the treatment of hyperphenylalaninemia (HPA), in particular phenylketonuria (PKU). The condition that is treated by the compounds described herein may be mild HPA, mild PKU or classic PKU, optionally in a subject exhibiting > 5% or > 10% of wildtype PAH activity.

[0020] Also provided herein is the use of a compound of the invention (e.g. a compound of Formula (I)) or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof, as a pharmacological chaperone. Also provided is a compound as defined herein (e.g. a compound of Formula (I)) or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof, for use as a pharmacological chaperone.

[0021] Also provided herein is the in vitro use of a compound of the invention (e.g. a compound of Formula (I)) or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof, to stabilise phenylalanine hydroxylase (PAH).

[0022] Figures

[0023] Figure 1 shows the effect of three compounds on stable PAH protein level in a HEK293 cell line with permanent expression of PAH-R261Q. A dose-dependent increase of PAH levels is shown. PAH levels have been detected and quantified by the In-cell Western method and normalized against cell number (DRAQ5). PAH levels have been normalized from 0 to 100%. Results are shown as mean + / -SD, n=3 (compound 53) or n=4 (PBAS499 and PBAS552).

[0024] Figures 2A-2I show the effect of a compound of the invention in a HEK293 cell line with transient expression of PAH mutations. Dose-dependent increase of PAH levels have been detected and quantified by the In-cell Western method and normalized against cell number (DRAQ5). PAH levels have been normalized from 0 to 100%. Results are shown as mean + / -SD, n=4.

[0025] Figure 3 shows the in vitro stabilization of PAH as measured by the melting temperature.

[0026] Figures 4A-4D show the effect of a compound of the invention in the Pah-R261Q mouse model. Figure 4A shows blood Phe levels after Phe-challenge in placebo (top) and PBAS499-treated (bottom) Pah-R261Q mice. Phe (200 pg / g body weight) was administered by i.p. at time 0, after the mice had been treated by either placebo (vehicle) or 20 mg / kg PBAS499 four times a day for 1.5 days (n=6 mice in each group). Figure 4B shows the area under curve (AUC) for the time dependence of Phe concentration between 0 and 180 minutes. Data has been normalized against AUC for placebo. Figure 4C shows the relative PAH specific activity in liver lysates from PBAS499-treated mice normalized to activity in liver lysates from placebo-treated mice (n=3 mice in each group). Figure 4D shows PAH protein levels in liver lysates normalized against GAPDH loading control. PAH levels in PBAS499-treated mice were then normalized to placebo-treated mice (set to 100%), (n=3 mice in each group). Data in each of Figures 4A-4D are presented as mean + / - SD, individual values are presented as circles.

[0027] Figures 5A-5D show the effect of a compound of the invention in combination with BH4 in the Pah-R261Q mouse model. Figure 5A shows blood Phe levels after Phe- challenge in placebo (top), BH4 (middle) or PBAS499 and BH4-treated (bottom) Pah-R261Q mice. Phe (200 pg / g body weight) was administered by i.p. at time 0, after the mice had been treated by either placebo (vehicle) or 20 mg / kg PBAS499 four times a day for 1.5 days. BH4 (20 mg / kg) was only given in the last dose before Phe injection (n=6 mice in each group) Figure 5B shows the area under curve (AUC) for the time dependence of Phe concentration between 0 and 180 minutes. Data has been normalized against AUC for placebo. Figure 5C shows the relative PAH specific activity in liver lysates from BH4 (middle) or PBAS499 and BH4-treated mice (right) normalized to activity in liver lysates from placebo-treated mice (left) (n=4 mice in each group). Figure 5D shows PAH protein levels in liver lysates normalized against GAPDH loading control. PAH levels in PBAS499- and BH4-treated mice were normalized to placebo-treated mice (set to 100%), (n=4 mice in each group). Data in each of Figures 5A-5D are presented as mean + / - SD, individual values are presented as circles.

[0028] Figures 6A-6F show the effect of a compound of the invention in a HEK293 cell line with transient expression of PAH mutations. PAH protein levels were normalized against GAPDH loading control. PAH levels in PBAS499-treated cells were then normalized to control / DMSO-treated cells (set to 100%), (n=3 parallels). Data are presented as mean + / - SD, individual values are presented as circles.

[0029] Detailed description According to one aspect, the present invention provides a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof:

[0030] Formula (I) wherein

[0031] Ri and R2 are independently selected from the group consisting of H and C1-C3 alkyl;

[0032] Y is a 5- or 6-membered heteroaryl which may be substituted by one or more R3 groups; each R3 is independently selected from the group consisting of halogen, N(Ry)2, ORy, SRy, Ci-Ce alkyl, C2-C6 alkenyl, and a 5- or 6-membered carbocyclic or heterocyclic group, wherein each Ryis independently H or C1-6 alkyl and wherein each R3 group may be substituted by one or more R4 groups; or two R3 groups together form a 5- or 6-membered carbocyclic or heterocyclic group which may be substituted by one or more R4 groups; and

[0033] R4 is selected from the group consisting of halogen, C1-3 alkyl, =0, -OH and

[0034] -NH2.

[0035] In embodiments, the compound of compound of Formula (I), or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof is not

[0036] Definitions

[0037] As used herein, the term "alkyl" refers to straight and branched saturated aliphatic hydrocarbon chains. Example alkyl groups include, but are not limited to, methyl (Me), ethyl (Et) and propyl (e.g. n-propyl or isopropyl).

[0038] As used herein, the term "alkenyl" refers to straight and branched hydrocarbon chains having one or more, preferably one or two, carbon-carbon double bonds. Examples of alkenyl groups include, but are not limited to, ethenyl, 1-propenyl,

[0039] 2-propenyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl,

[0040] 3-hexenyl, 4-hexenyl, 5-hexenyl, 2-methyl-2-propenyl, and 4-methyl-3-pentenyl.

[0041] As used herein, the term "heteroaryl" refers to aromatic hydrocarbons that include at least one heteroatom ring member such as sulfur, oxygen, or nitrogen. Examples of 5-membered heteroaryl groups include, but are not limited to, furan, thiophene, imidazole, thiazole, pyrrole, oxazole, isoxazole, pyrazole, triazole, tetrazole, 1,2,4- thiadiazole and isothiazole. Examples of 6-membered heteroaryl groups include, but are not limited to, pyridine, pyrimidine, pyrazine, pyridazine and triazine. Preferred heteroaryl groups which may be used in the compounds of the invention include furan, thiophene, pyrrole and pyridine.

[0042] As used herein, the term “carbocyclic” refers to a cyclic group formed of carbon atoms, such as a cycloalkyl, cycloalkenyl, or aryl group (i.e. cyclic aromatic hydrocarbons). Examples of 5- or 6-membered carbocyclic groups include, but are not limited to, cyclopentyl, cyclohexyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl or phenyl.

[0043] As used herein, the term “heterocyclic” refers to a cyclic group that include at least one heteroatom ring member such as sulfur, oxygen, or nitrogen, such as a heteroaryl group. Examples of 5- or 6-membered heterocyclic groups include, but are not limited to, tetrahydrofuran, pyrrolidine, tetrahydrothiophene, piperidine, oxane, thiane, diazinane, morpholine, thiomorpholine, oxathiane, dioxane, dithiane, triazine, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, dioxolane, dithiolane, and the 5- or 6-membered heteroaryl groups listed above.

[0044] As used herein, the term “salts” means pharmaceutically acceptable salts. Suitable salts include organic or inorganic salts. Preferred salts include citrates, fumarates, tartarates, malates, formates, acetates, maleates, chlorides, bromides, sulphites, and sulfates.

[0045] As used herein, the term “stereoisomer” means a diastereomer or an enantiomer. More preferably, the stereoisomer is an enantiomer. The present invention generally encompasses stereoisomers of the compounds of the invention, with the exception of where specific stereochemistry is defined.

[0046] Ri and R2

[0047] R1 and R2 are independently selected from the group consisting of H and C1-C3 alkyl.

[0048] Preferably, R1 and R2 are each independently selected from the group consisting of H and CH3.

[0049] More preferably R1 and R2 are both H.

[0050] Y group

[0051] Y is a 5- or 6-membered heteroaryl which may be substituted by one or more R3 groups. Preferably, Y may be substituted by one or two R3 groups. More preferably,

[0052] Y is substituted by one or two R3 groups, and most preferably by one R3 group.

[0053] Y may be any 5- or 6-membered heteroaryl, such as those listed above. In one embodiment, Y is selected from the group consisting of pyrrole, furan, thiophene, pyridine, pyrimidine, pyridazine and pyrazine. In one embodiment, Y is selected from the group consisting of pyrrole, furan, thiophene, pyridine, pyridazine and pyrazine. In one embodiment Y contains 1 heteroatom, which may be N, O or S, preferably N or S.

[0054] Preferably, Y is selected from the group consisting of furan, thiophene, pyrrole and pyridine. More preferably, Y is selected from the group consisting of furan, thiophene, and pyridine. Even more preferably, Y is thiophene or pyridine.

[0055] In one embodiment, Y is thiophene.

[0056] In some embodiments, Y is not unsubsidised imidazole when Ri and R2 are both H. In some embodiments, Y is not unsubsidised imidazole.

[0057] In some embodiments, Y is not imidazole when R1 and R2 are both H. In some embodiments, Y is not imidazole.

[0058] In some embodiments, Y is not pyrimidine substituted by -OH, -NH2 and -NH2 when R1 and R2 are both H. In some embodiments, Y is not pyrimidine substituted by -OH, -NH2and -NH2.

[0059] In some embodiments, Y is not pyrimidine substituted by three R3 groups when R1 and R2 are both H. In some embodiments, Y is not pyrimidine substituted by three R3 groups.

[0060] In some embodiments, Y is not substituted pyrimidine when R1 and R2 are both H. In some embodiments, Y is not substituted pyrimidine substituted by three R3 groups.

[0061] In some embodiments, Y is not pyrimidine when R1 and R2 are both H. In some embodiments, Y is not pyrimidine.

[0062] R3

[0063] Each R3 is independently selected from the group consisting of halogen, N(Ry)2, ORy, SRy, C1-C6 alkyl, C2-C6 alkenyl, and a 5- or 6-membered carbocyclic or heterocyclic group, wherein each Ryis independently H or C1-6 alkyl and wherein each R3 group may be substituted by one or more R4 groups. Alternatively, two R3 groups together form a 5- or 6-membered carbocyclic or heterocyclic group which may be substituted by one or more R4 groups.

[0064] When two R3 groups together form a 5- or 6-membered carbocyclic or heterocyclic group, the Y and R3 groups may together form, for example, an 11- or 12- membered heteroaryl such as, but not limited to, 7-azaindole, 4-azaindole, 5- azaindole, 6-azaindole, 7-azaindazole, purine, and indolizine, preferably 7- azaindole.

[0065] Preferably, each R3 may be substituted by one or two R4 groups, or two R3 groups together form a 5- or 6-membered carbocyclic or heterocyclic group which may be substituted by one or two R4 groups.

[0066] In one embodiment, each R3 is a 5- or 6-membered carbocyclic or heterocyclic group which may be substituted by one or two R4 groups; or two R3 groups together form a 5- or 6-membered carbocyclic or heterocyclic group which may be substituted by one or two R4 groups.

[0067] Preferably, each R3 is a 5-membered heterocyclic group which may be substituted by one or two R4 groups; or two R3 groups together form a 5-membered heterocyclic group which may be substituted by one or two R4 groups.

[0068] More preferably, each R3 is a 5-membered heteroaryl (e.g. furan, thiophene, pyrrole, pyrazole, imidazole or triazole) which may be substituted by one or two R4 groups; or two R3 groups together form a 5-membered heteroaryl which may be substituted by one or two R4 groups.

[0069] In one embodiment, R3 is selected from the group consisting of furan, thiophene, pyrrole, pyrazole, imidazole and triazole, preferably pyrazole, each of which may be substituted by one or two R4 groups.

[0070] In one embodiment, R3 is unsubstituted pyrazole.

[0071] R4 R4 is selected from the group consisting of halogen, C1-3 alkyl, =0, -OH and -NH2.

[0072] Preferably, each R4 is independently selected from the group consisting of halogen, C1-3 alkyl, -OH and -NH2.

[0073] More preferably, each R4 is independently selected from the group consisting of Cl, Br and C1-3 alkyl.

[0074] More preferably, each R4 is independently selected from the group consisting of Cl and C1-3 alkyl.

[0075] More preferably, each R4 is independently selected from the group consisting of Cl and CH3.

[0076] When two R4 groups are present in the same compound, one may be a halogen (preferably Cl), and the other may be C1-3 alkyl (preferably CH3).

[0077] Preferred compounds

[0078] In one embodiment, the compound of Formula (I) is a compound of Formula (Ila): wherein X is NH, O or S, preferably O or S and more preferably S, and R1, R2and R3 are as defined above. For clarity, the 5-membered ring shown in Formula (Ila) may be substituted by 0, 1 or 2 R3 groups, in any appropriate and chemically viable position.

[0079] In one embodiment, the compound of Formula (I) is a compound of Formula (lib):

[0080] Formula (lib) wherein X is NH, O or S, preferably O or S and more preferably S, and R1, R2 and R3 are as defined above.

[0081] In one embodiment, the compound of Formula (I) is a compound of Formula (He):

[0082] Formula (He) wherein X is NH, O or S, preferably O or S and more preferably S, and Ri, R2 and R3 are as defined above. Preferably, R3 is selected from the group consisting of furan, thiophene, pyrrole, pyrazole, imidazole and triazole, more preferably pyrazole, each of which may be substituted by one or two R4 groups, but is preferably unsubstituted.

[0083] In one embodiment, the compound of Formula (I) is a compound of Formula (lid):

[0084] Formula (lid) In another embodiment, the compound of Formula (I) is a compound of Formula (Illa): wherein each Y is independently C or N, preferably C, and R1, R2 and R3 are as defined above. Preferably, at most two Y groups are N. More preferably at most one Y group is N. Most preferably, all Y groups are C.

[0085] For clarity, the right hand ring shown in Formula (Illa) may be substituted by 0, 1 or 2 R3 groups, in any appropriate and chemically viable position. For the avoidance of doubt, any Y group which is C and which is not substituted by an R3 group will be substituted instead by H, as would be immediately apparent to the skilled person.

[0086] In the compounds of Formula (Illa), preferably two R3 groups are present and together form a 5- or 6-membered carbocyclic or heterocyclic group (preferably a 5- membered heteroaryl) which may be substituted by one or more R4 groups.

[0087] In another embodiment, the compound of Formula (I) is a compound of Formula (lllb):

[0088] Formula (lllb) wherein Rs is H or R4, preferably R4, and wherein R1, R2 and R4 are as defined above. In another embodiment, the compound of Formula (I) is a compound of Formula (lllc):

[0089] Formula (lllc) wherein R4 is as defined above.

[0090] In another embodiment, the compound of Formula (I) is a compound of Formula

[0091] (Hid):

[0092] Formula (Hid)

[0093] PKU treatment

[0094] The compounds described herein are particularly of use in the treatment of MHP and mild PKU but also patients with classic PKU, optionally who retain > 10% of wild-type PAH activity. However, interestingly, the compounds described herein can unexpectedly be used in the treatment of patients with classic PKU even where the patients retain < 10% of wild-type PAH activity, for example such as with the PAH variants R408W, R243Q and P281 L.

[0095] The compounds of the invention are not useful in the treatment of so-called “atypical PKU”. In atypical PKU, the accumulation of phenylalanine (L-Phe) is not associated with a deficiency of the PAH enzyme. Instead, atypical PKU is the consequence of a deficiency of tetrahydrobiopterin (BH4), which is the cofactor of PAH hydroxylation. The type of the disease depends on the metabolic defect of synthesis or regeneration of the cofactor. Atypical PKU is generally caused by mutations in enzymes involved in the recycling of the cofactor of PAH tetrahydrobiopterin (BH4), most probably dihydropteridine reductase (DHPR). Thus, references herein to the treated condition, such as HPA or PKU, do not include atypical PKU. That is, the treated condition as described herein (for example in relation to uses of the compounds or methods of treatment) is not atypical PKU.

[0096] Different misfolding mutations can result in PAH having more or less residual enzymatic activity. The Examples herein describe a suitable assay of PAH activity.

[0097] In certain preferred embodiments the compounds of the invention do not have a strong inhibitory effect on dihydrofolate reductase (DHFR) e.g. they inhibit (human) DHFR less than trimethoprim (TMP), preferably exhibiting at least 20% or 50% or 70% or 90% less inhibition. Preferably, the compounds of the invention have no, or essentially no, inhibitory effect on DHFR. Essentially no inhibitory effect implies not measurable and / or of no physiological significance.

[0098] Preferably the uses and methods of the present invention do not employ TMP.

[0099] HPA and PKU treatable according to the present invention are caused by reduced PAH activity and the compounds of the invention act as pharmacological chaperones to mutant forms of PAH, for example but not limited to those forms with > 10% residual activity, increasing their stability and therefore their catalytic activity, i.e. ability to convert L-Phe to L-Tyr. Different mutant forms of PAH are known in the art, resulting in PAH protein variants which may have their stability improved according to the present invention, including the following: R408W, R261Q, R243Q, P281 L, R158Q, Y414C, L48S, I65T, A300S, V388M, E280K, E390G, L348V and R68S, and in particular R261Q, Y414C, L48S, A300S, V388M, E390G, L348V, and R68S (PAH mutation defined by native amino acid using the single letter code, followed by location and then by the substituting amino acid).

[0100] In embodiments, the compounds of the invention may be used in the treatment of PKU in a subject having a missense mutant form of PAH. In embodiments, the compounds of the invention may be used in the treatment of PKU in a subject having a R408W, R261Q, R243Q, P281 L, R158Q, Y414C, L48S, I65T, A300S, V388M, E280K, E390G, L348V or R68S mutant form of PAH.

[0101] In embodiments, the compounds of the invention may be used in the treatment of PKU in a subject having a R261Q, Y414C, L48S, A300S, V388M, E390G, L348V or R68S mutant form of PAH.

[0102] In embodiments, the compounds of the invention may be used in the treatment of PKU in a subject having a R408W, R243Q, P281 L, R158Q, I65T or E280K mutant form of PAH.

[0103] In embodiments, the compounds of the invention may be used in the treatment of PKU in a subject having a R408W, R261Q or R243Q mutant form of PAH.

[0104] In embodiments, the compounds of the invention may be used in the treatment of PKU in a subject having a R408W mutant form of PAH.

[0105] In embodiments, the compounds of the invention may be used in the treatment of mild HPA, mild PKU or classic PKU, optionally in a subject exhibiting > 10% of wildtype PAH activity. In embodiments, the compounds of the invention may be used in the treatment of classic PKU in a subject exhibiting less than 10% or less than 7% wild-type PAH activity, such as 0-5% or 0-1%.

[0106] The Examples provide suitable in vitro methods using wild-type or mutant forms of PAH which may be used to confirm efficacy, estimate dosage etc. of the claimed compounds.

[0107] The inventors have unexpectedly found that the presently claimed compounds have a greater effect as pharmacological chaperones to PAH than known compounds, such as those disclosed in WO 2017 / 029202. For example, it has been found that the presently claimed compounds unexpectedly have a significantly increased ability (as measured by EC50 values) to stabilize PAH.

[0108] Treatment includes prophylaxis in that the patient may not have detectable symptoms of HPA or PKU; nevertheless, most patients treated in accordance with the present invention will have been diagnosed with HPA or PKU or suspected of having HPA or PKU. Methods of diagnosis of these conditions are known in the art. Treatment and prophylaxis may not be absolute but will result in a measurable improvement in one or more parameters (e.g. symptom or biochemical marker) associated with HPA or PKU, or in the case of prophylaxis, prevention or reduction of the otherwise expected development of traits of HPA / PKU.

[0109] In one aspect the present invention provides a compound of the invention as defined herein, or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof, for use in the treatment of hyperphenylalaninemia (HPA), in particular phenylketonuria (PKU). As discussed above, the treated condition is not atypical PKU.

[0110] In one aspect the present invention provides a method of treating a subject having or suspected of having HPA, in particular PKU, comprising administering to the subject an effective or therapeutically effective amount of a compound of the invention as defined herein or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof.

[0111] A therapeutically effective amount will be determined based on the clinical assessment and can be readily monitored.

[0112] Likewise, the present invention provides a method of treating HPA, in particular PKU, in a subject by administering to the subject in need thereof a compound of the invention as defined herein or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof.

[0113] Alternatively viewed the present invention provides a method of treating HPA, in particular PKU, in a subject by administering thereto an effective amount of a compound of the invention as defined herein or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof.

[0114] In a further aspect, the present invention provides a compound of the invention as defined herein, or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof, for use in increasing the stability of PAH. Also provided is the use, which may be in vitro or in vivo, of a compound of the invention as defined herein, or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof, to increase the stability of PAH.

[0115] Alternatively viewed, the present invention provides a method of increasing the stability of PAH, comprising contacting with or administering a compound of the invention as defined herein or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof. An increase in PAH stability typically leads to an increase in cellular PAH levels and / or in PAH activity.

[0116] The present invention further provides use of a compound of the invention as defined herein, or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof, in the manufacture of a medicament for the treatment of HPA, in particular PKU.

[0117] In each of the uses and methods described herein, the compound of the invention can be used or administered alone, or can be used or administered as part of a composition, such as a composition comprising a compound of the invention or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof and a pharmaceutically acceptable carrier, diluent or excipient.

[0118] The compound is administered according to a therapeutically effective dosage regimen which may require multiple administrations, e.g. 2 to 100, typically at least 10 or 20 doses, where such doses may conveniently be administered daily. The compound may also be administered less frequently, for example using 1 to 10, 1 to 5, or 1 or 2 doses per day. The compound may also be administered less than once per day.

[0119] Typical daily doses may be between about 0.1 and about 200 mg / kg / day, such as from about 1 to about 60 mg / kg / day, from about 5 to about 50 mg / kg / day, from about 10 to about 40 mg / kg / day, or from about 15 to about 25 mg / kg / day. A therapeutically effective dosage regimen will be one to effect treatment or prophylaxis, as defined above.

[0120] Effective amounts and effective dosage regimens are able to cause measurable and physiologically relevant increases in PAH activity in the subject, e.g. an observed improvement in Phe metabolism. The compounds of the invention may be formulated for administration in any convenient way, typically in a composition with a physiologically or pharmaceutically acceptable carrier, diluent or excipient.

[0121] The compounds and compositions may be administered to the subject in any convenient form or by any convenient means, e.g. by topical, oral, parenteral, enteral, parenteral routes or by inhalation. Oral or parenteral routes are preferred.

[0122] The skilled person will be able to formulate the compounds of the invention into pharmaceutical compositions that are adapted for these routes of administration according to any of the conventional methods known in the art and widely described in the literature.

[0123] The active ingredient may be incorporated, optionally together with other active agents, with one or more conventional carriers, diluents and / or excipients, to produce conventional galenic preparations such as tablets, pills, powders (e.g. inhalable powders), lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, soft and hard gelatine capsules, suppositories, sterile injectable solutions, sterile packaged powders, and the like.

[0124] Examples of suitable carriers, excipients, and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, inert alginates, tragacanth, gelatine, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, water, water / ethanol, water / glycol, water / polyethylene, hypertonic salt water, glycol, propylene glycol, methyl cellulose, methylhydroxybenzoates, propyl hydroxybenzoates, talc, magnesium stearate, mineral oil or fatty substances such as hard fat or suitable mixtures thereof.

[0125] The compositions may additionally include lubricating agents, wetting agents, emulsifying agents, suspending agents, preserving agents, sweetening agents, flavouring agents, and the like.

[0126] Parenterally administrable forms, e.g. intravenous solutions, should be sterile and free from physiologically unacceptable agents, and should have low osmolarity to minimize irritation or other adverse effects upon administration and thus solutions should preferably be isotonic or slightly hypertonic, e.g. hypertonic salt water (saline). Suitable vehicles include aqueous vehicles customarily used for administering parenteral solutions such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection and other solutions such as are described in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co. The solutions can contain preservatives, antimicrobial agents, buffers and antioxidants conventionally used for parenteral solutions, excipients and other additives which are compatible with the active compounds and which will not interfere with the manufacture, storage or use of products.

[0127] Individual doses may contain 0.01 to 40 mg / kg, e.g. 0.02 to 2 mg / kg.

[0128] The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art, adjustments for systemic versus localized delivery, age body weight, general health, sex, diet, time of administration drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.

[0129] Subjects or patients treated in accordance with the present invention will preferably be humans but veterinary treatments are also contemplated.

[0130] The uses, treatments and compositions defined herein may also include the use of a second active agent, e.g. a second active agent for treating HPA or PKU.

[0131] The skilled person would be well-aware of active agents suitable for treating HPA or PKU from their common general knowledge. For example, nicotinamide adenine dinucleotide (NADH) is a known active agent which may be suitable for treating HPA or PKU, as disclosed in WO 2016 / 074641. Other active agents suitable for treating HPA or PKU include pegvaliase (sold under the brand name Palynziq®) and cofactors of PAH such as tetrahydrobiopterin.

[0132] Preferred combination treatments include co-administration of a compound of the invention with a compound which acts as a cofactor of PAH, e.g. tetrahydrobiopterin (BH4) or analogues or precursors thereof. One example of such a cofactor is the commercially available product Kuvan®, which may preferably be administered with ascorbic acid.

[0133] The skilled person would be aware of suitable BH4 analogues and precursors. For example, sepiapterin and BH2 are known examples of precursors of BH4, which can be metabolised into BH4 via a salvage pathway (e.g. see Sawabe et al. J Pharmacol Sci. , 2004 Oct;96(2): 124-33, Sawabe et al., Chemistry and Biology of Pteridines and Folates, 2002, p199-204 and Crabtree et al. J Biol Chem., 2009, Oct 9;284(41):28128-36).

[0134] The present invention also provides a composition comprising a compound of the invention as defined herein or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof and a further agent effective in the treatment of HPA, in particular PKU.

[0135] The present invention also provides a pharmaceutical pack comprising, not in admixture but for simultaneous or sequential administration, a compound of the invention as defined herein, or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof, and a further agent effective in the treatment of HPA, in particular PKU. As used herein, the term “sequential” does not imply any particular order or proximity to the administration (temporally or spatially), simply that the two agents are part of a combination therapy where they are not simultaneously administered. Said packs or compositions preferably comprise BH4 or an analogue or precursor thereof, e.g. Kuvan®. Nevertheless, treatments according to the invention do not require a second active and in particular do not require BH4 or an analogue or precursor thereof.

[0136] Examples

[0137] Materials

[0138] Compounds

[0139] Compounds were synthesized by Charnwood Molecular Ltd (>95% purity). Stock solutions were prepared at 12 mM in 100% DMSO.

[0140] Enzymes Tetrameric full length wild-type (WT) human PAH (hPAH) was recombinantly expressed in E. coli fused to maltose-binding protein and purified by amylose- affinity chromatography and further cleaved and isolated to homogeneity essentially as described [Martinez, A., et al., Biochem. J., 1995. 306: p. 589-597], Protein concentration was measured in a NanoDrop spectrophotometer (Thermo Scientific) using the absorbance at 280 nm and the theoretical molar extinction coefficient of 49780 M'1cm-1for WT-hPAH.

[0141] Cell lines

[0142] HEK293 EBNA (Epstein-Barr virus) cells, with low endogenous PAH expression, were used for transfection and transient expression of PAH mutants. HEK293 EBNA cells were grown in DMEM, high glucose medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 50 ll / rnl penicillin, 50 pg / ml streptomycin (all from Merck) and 0.25 mg / ml geneticin (Fisher).

[0143] HEK293 cells with permanent expression of the PAH-R261Q mutant (HEK293 / PAH-R261Q, GenScript) were used to evaluate the effect on this mutant. HEK293 / PAH-R261Q cells were grown in DMEM, high glucose medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 50 ll / rnl penicillin, 50 pg / ml streptomycin (all from Merck) and 1 pg / ml puromycin (Fisher).

[0144] Mouse model

[0145] A constitutive knock-in mouse model containing the Pah-R261Q mutation was generated by Taconic Biosciences GmbH (Kbln, Germany) via CRISPR / Cas9- mediated gene editing as described in Aubi et al. 2021. The mouse is BH4 responsive, and its PAH protein level and activity level is 10-20% compared to WT mice, which corresponds to observations in humans bearing this mutation. Mice have a much higher level of PAH compared to humans, and they are therefore healthy and well-functioning even with a much lower percentage of active PAH. After a Phe challenge (Phe injection) the mice show a rapid increase in Phe levels corresponding to the Phe level in untreated humans with this mutation (600-1200 pM). Thus, the Pah-R261Q mouse model has been proven to be clinically relevant concerning both blood Phe levels and PAH activity and protein levels (Aubi et al. 2021).

[0146] Methods

[0147] Evaluating PAH stability (chaperone effect) in cell culture HEK293 / PAH R261Q: Cells were seeded in black well / clear bottom poly-D-lysine coated 96-well plates (0.16 x 105cells / well) (Corning, 354640) and grown in complete medium for 24h. Cells were washed with phosphate buffered saline (PBS) before addition of compounds diluted in DMEM high glucose supplemented with 2 mM L-glutamine (no FBS or antibiotics) and subsequent incubation for 24 h. DMSO (vehicle) concentration was adjusted to same level in all samples within experiments.

[0148] HEK293 EBNA: Cells were seeded as described above, or seeded in 6 well plates (5 x 105cells / well) and grown in complete medium for 24 h before transient transfection with PAH mutants (R408W, R261Q, R243Q, P281 L, R158Q, Y414C, L48S, I65T, A300S, V388M, E280K, E390G, L348V, R68S in pcDNA3 vector) with Lipofectamine LTX with PLUS reagent (Fisher) according to the manufacturers protocol. Cells were incubated with transfection mix for 5h before cells were washed with PBS and then incubated with compounds as described above. The cells were incubated with compounds for 19h to not exceed 24h incubation in medium without FBS.

[0149] Following compound incubation, the cells were fixed and subjected to In-Cell Western analysis, or harvested for WB analysis.

[0150] In-Cell Western analyses

[0151] Cells were fixed for 5 min with ice cold methanol (100 pl / well), followed by 3 rinses with PBS. The cells were subsequently incubated with blocking buffer (1% bovine serum albumin, 10% normal goat serum, 0.3M glycine, 0.1% Tween in PBS) for 1h at RT, and then primary antibodies against PAH (rabbit monoclonal anti-PAH, EPR12380, Abeam) diluted 1:100 in blocking buffer ON at 4 °C. The following day cells were incubated with secondary antibodies (goat anti-rabbit DyLight800 4X PEG conjugate, Cell Signaling Technology) diluted 1 :500 in blocking buffer for 90 min in RT and subsequently DRAQ5 (Fisher) diluted 1 :1000 in PBS for 30 min in RT.

[0152] Cells were washed with PBS for 4 x 5 min after each antibody incubation, and all incubations and washing steps after fixation were performed with gentle agitation. Fluorescence signal was detected using a Typhoon biomolecular imager (Amersham) and fluorescence intensities from the obtained images were quantified using Imaged software (Java). PAH signal from each well was normalized against the DRAQ5 (DNA binding dye) signal from the same well to correct for any differences in cell numbers between wells. PAH increase was then normalized from 0 to 100 using Graph Pad Prim’s Normalize analysis with 0 being defined as the smallest and 100 as the largest mean within each dataset. EC50s were estimated by nonlinear regression using a four parameter dose-response fit in Graph Pad Prism.

[0153] Cell lysates

[0154] Cells were incubated in lysis buffer (20 mM Hepes, pH 7.5, 125 mM NaCI, 1x protease inhibitor cocktail with EDTA) for 20 min at 4 °C before centrifugation at 16000 x g for 20 min at 4 °C. Supernatant was transferred to new tubes and incubated with 1% Triton X-100 at 4 °C for 1h with rotation. Immunoblotting was performed as described below for ex vivo PAH detection.

[0155] Differential scanning fluorimetry (DSF)

[0156] DSF was used to monitor the thermal denaturation of recombinant WT-hPAH protein in the presence of the fluorescent dye SYPRO Orange (Sigma-Aldrich). The experiments were carried out in a LightCycler 480 Real-Time PCR (RT-PCR) instrument (Roche Applied Science, Indianapolis, IN). Each well contained 18 pl of a hPAH protein solution containing 0.1 mg / ml (1.93 pM subunit) in 20 mM NaHepes pH 7.0, 200 mM NaCI and 5x SYPRO Orange, with final concentrations of 1 % DMSO and 10 pM compound. The thermal denaturation was monitored by following the expected increase in fluorescence intensity of the extrinsic probe SYPRO Orange (instrument filter settings: Aexc=465 nm; Aem=610nm) as a consequence of the unfolding / denaturation of the protein. Melting curves were registered from 20 to 95°C at a scan rate of 2.4 °C / min and the experimental data obtained allowed the extraction of values of Tm (midpoint melting temperature) by fitting, smoothing, normalization and analysis of the aforementioned unfolding curves using the software HTSDSF Explorer (Martin-Malpartida, et al. (2024) J. Mol. Biol. 434, 167372). Tmrepresents the temperature at which the fraction of unfolded (or folded) protein is 50% and it is calculated as the intersection between denaturation curve and fraction of unfolding xU =0.5. Control experiments with 1 % DMSO were performed in the same way. The Tm-values for hPAH in the presence of each compound was compared to the value for the control without compound but with 1% DMSO, and the shifts in Tm(ATm) were calculated. ATffl(= TmCompound - TmDMSO control).

[0157] Evaluating effect on blood Phe in vivo Mice were divided into groups (n=6, age between 3 and 6 months) with age and sex distributed evenly between the groups. They were all switched to Phe free food (Research Diets) 1 day before treatment started. The groups were administered 6 doses by oral gavage (20 mg / kg / dose PBAS 499 or a placebo solution of the compound vehicle) with 6 hours between each dose. 6 hours after the final dose was administered the mice were subjected to a Phe challenge (i.p injection of 200 ug / g body weight) followed by whole blood sampling from the saphenous vein at time points: 1 day before treatment (baseline level before treatment), 1 h before Phe challenge (baseline level after treatment), and 30, 90 and 180 minutes after injection. When BH4 was included in the treatment, it was administered orally at the same time as the sixth dose of PBAS499 (6 hours before Phe challenge).

[0158] Extraction and quantification of L-Phe from dried blood samples was performed by a protocol adapted from Lee et al. (Biotechniques, 2019. 67(5): p. 219-228) and Zoppa et al. (J Chromatogr B Analyt Technol Biomed Life Sci, 2006. 831 (1-2): p. 267-73). Mitra microsamplers (Neoteryx) with 10 pL dried blood was soaked in 50 pL ddhLO for 30 min with vigorous shaking. 200 pL methanol was then added and the tubes were shaken for 30 more min. For the L-Phe standards, the added methanol was spiked with known concentrations of L-Phe. After extraction, the samples were centrifuged at 16000 x g for 10 min at 4 °C and 150 pL of the supernatant was transferred to new tubes. The supernatant was frozen with liquid nitrogen and dried by vacuum evaporation overnight in an Alpha 1-4 LOC-1 freeze- drying system (Martin Christ). The dried samples were reconstituted in 50 pL 50 % ethanol, 1 % acetic acid and vortexed for 10 seconds before placed in a -20 °C freezer for at least 30 minutes to precipitate proteins. Samples were centrifuged at 16000 x g for 10 min at 4 °C and the supernatant was transferred to SureSTART™ 0.3 mL HPLC vials (Thermo Scientific). Analysis was performed using a 1200 series high performance liquid chromatography (HPLC) system (Agilent Technologies). 10 pL of sample was injected and chromatographic separation was achieved on an Agilent Zorbax 300-SCX column (4.6 mm ID x 150 mm) with an Agilent Zorbax 300- SCX guard column (4.6 mm ID x 12.5 mm). The flow rate was 1 mL / min and the mobile phase was 20 mM acetic acid and 1 % 1-propanol, pH 3.5. Fluorometric detection of L-Phe was done with Aex=265 nm and Aem=290 nm. Amount of L-Phe in each sample was calculated using a standard curve.

[0159] Ex vivo PAH protein and activity analysis Preparation of liver tissue: Mice were sacrificed in a carbon dioxide euthanize chamber and their liver was within minutes surgically excised and snap-frozen in liquid nitrogen. The liver tissue was then ground into a fine powder and stored in aliquots (-200 mg powder) at -80°C. Liver homogenates from the ground aliquots (-200 mg powder) were prepared by adding 800 pL of a lysis buffer solution containing 1* PBS and protease inhibitor cocktail (Roche), a 5mm diameter stainless steel bead (Qiagen), and a mechanical disruption step in a Tissue Lyser II (Qiagen) instrument (2 min 30 s, 20 Hz). Cellular debris was then removed through centrifugation (20 000 ref, 20 min) to obtain a clear supernatant. The total protein concentration of the liver lysates was determined in a Direct Detect infrared spectrometer (Millipore).

[0160] PAH enzymatic activity assay. Liver lysates were first loaded into 0.5 mL Zeba- Spin desalting columns (7.000 Da cutoff; ThermoFisher Scientific), previously equilibrated with 20 mM HEPES, pH 7.0, 200 mM NaCI, and protease inhibitor cocktail solution, and centrifuged (1 700 ref) for 2 min. PAH activity in the homogenates was measured at 25 °C using 5-20 pg of total protein in each assay, with 1 mM L-Phe in 20 mM Na-Hepes, 0.2M NaCI, pH 7.0, containing catalase (0.04 mg / ml). After 4 min preincubation at 25 °C, ferrous ammonium sulfate (100 pM) was added, and the reaction triggered after 1 min by adding 200 pM BH4 and 5mM DTT (final concentrations in the assay). The reactions were allowed to run for 2 min and stopped with 2% acetic acid in ethanol. Under these conditions, PAH activity was linear to the amount of protein in the extracts. L-Tyr formed was quantified by HPLC with fluorimetric detection.

[0161] Immunoblotting. Protein immunodetection was performed by Western blot. Total protein (20 pg / well for liver lysates and 10 pg / well for cell lysates) was separated using 10% polyacrylamide gel and immunodetected by using primary antibodies, 1:5000 for primary antibody a-PAH (1:5000; Millipore-MAB5278), 1 :2000 for a- glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1000; Abcam-ab9485), and 1 :5000 for secondary antibodies goat anti-mouse (GAM) (Bio-Rad Laboratories) and goat anti-rabbit (GAR) (Bio-Rad Laboratories), conjugated to horseradish peroxidase. Quantification of non-ubiquitinated and monoubiquitinated PAH and GAPDH proteins was performed by gel band densitometry.

[0162] Example 1 - Effect on PAH stability in cell culture Three compounds were tested in several concentrations in the HEK293 cells with permanent expression of PAH-R261Q to establish dose-response relationship and estimate EC50 values. The compounds tested were as follows: Compound 53 (comparative)

[0163] PBAS499 (also referred to herein as Formula (lid))

[0164] PBAS552 (also referred to herein as Formula (Hid))

[0165] The results are shown in Figure 1. As shown in Figure 1, it was surprisingly found that the compounds PBAS499 and PBAS552 showed significantly increased potency (EC50 1.93 and 4.79 pM, respectively) compared to previously a known compound having a similar structure, namely compound 53 (EC50 55.6 pM).

[0166] Example 2 - Effect of PBAS499 on additional PAH mutations

[0167] To investigate the effect of the compound PBAS499 on additional PAH mutations, HEK293 cells with low endogenous PAH expression were transiently transfected with selected PAH mutants. Missense mutations with high worldwide allele frequency were selected. PAH protein increase with a high dose a PBAS499 (50 pM, typically showing Emax) were determined and normalized against PAH levels in vehicle-treated cells. Dose-dependent PAH increase was demonstrated and EC50 values estimated for selected mutations.

[0168] The results are shown in Figures 2A-2I and 6A-6F, and Table 1 below.

[0169] Table 1 : EC50s and % PAH increase in response to PBAS499 in a HEK293 cell line with transient expression of PAH mutations. aMean PAH protein increase after incubation with 50 pM PBAS499 in HEK293 cells, compared to untreated cells ( 100%)bBH4 responsiveness of tested homozygous individuals according to the BIOPKU database (biopku.org)

[0170] CAPV (allelic phenotypic values) calculated based on the frequencies of the metabolic phenotype (i.e., cPKU, mPKU or MH P) for genotypes presenting in a functionally hemizygous state (APV = (%cPKU x 0 + %mPKU x 5 + %MHP x 10) / 100) according to the BIOPKU database (biopku.org). 0-2.7 is classic PKU, 2.8- 6.6 is mild PKU and 6.7-10.0 is mild HPAdWorldwide allele frequency according to Hillert et al. (Am J Hum Genet, 2020, 107(29) 234-250).

[0171] Transient transfection with PAH-R261Q was included and EC50 determined and compared to that obtained in the cell line with permanent expression of the same mutant. The EC50s were nearly identical: 1.93 pM in cell line with permanent and 2.02 pM in cell line with transient R261Q expression. EC50s for the mutations R261Q, P281 L L48S, I65T, V388M, L348V and R68S were in the range ~2 pM or below.

[0172] The compounds of the invention are therefore effective against a range of PAH variants.

[0173] Interestingly, PBAS499 was shown to be effective in stabilizing classic PAH mutants associated with low PAH activity such as R408W, R243Q and P281 L, where reports on BH4 responsiveness have shown that these patients do not respond (BIOPKU database, biopku.org). The compounds of the invention may therefore be used in the treatment of classic PKU, even in a subject not exhibiting > 10% of wild-type PAH activity. Thus, the compounds of the invention may be used in the treatment of PKU in a subject exhibiting low PAH activity, i.e. in a subject exhibiting less than 10% or less than 7% wild-type PAH activity, such as 0-5% or 0- 1%. The compounds of the invention may also be used in the treatment of PKU in a subject exhibiting more than 3% wild-type PAH activity. PAH activity may be measured in vitro in a COS cell line, as reported in Himmelreich, N et al. (Mol Genet Metab, 2018. 125(1-2): p. 86-95). R408W has 2 ± 0 % residual in vitro activity and R243Q has 6 ± 1 % residual in vitro activity relative to WT PAH in a COS cell line reported in Himmelreich, N et al. (Mol Genet Metab, 2018. 125(1-2): p. 86-95). P281 L has 0-1% residual in vitro activity relative to WT PAH in a COS cell line (Himmelreich et al., Mol Genet Metab, 2018. 125(1-2): p. 86-95).

[0174] Example 3 - Target engagement by DSF

[0175] To demonstrate direct binding and stabilization of PAH, the effect of PBAS499 and PBAS552 on the melting curves of WT hPAH was analyzed by DSF. The regulatory and catalytic domains of hPAH unfolds separately and therefore give two transitions. The results are shown in Figure 3 and Table 2 below.

[0176] Table 2: Increase of wt PAH Tm-values with PBAS499 and PBAS552

[0177] As shown in Figure 3 and Table 2, both melting temperatures were increased in the presence of PBAS499 and PBAS552. Increases were larger for PBAS499 than for PBAS552, in accordance with the higher potency (lower EC50) of PBAS499 in cell culture (1.93 pM for PBAS499 and 4.79 pM for PBAS552).

[0178] Example 4 - Effect on blood Phe in a mouse model for PKU

[0179] The compound PBAS499 was then tested in the Pah-R261Q mouse model.

[0180] Two groups of mice (placebo group and 20 mg / kg PBAS499 group (n=6 in both)) received 6 doses with 6 hours between each dose. 6 h after the last dose, the mice were injected with Phe (200 pg / g body weight).

[0181] Blood Phe levels were analyzed pre-treatment (t=-36 h), right before Phe injection (t=0) and 30, 90 and 180 min after Phe challenge, with the results shown in Figure 4A. The area under curve (AUG) for the time dependence of Phe concentration between 0 and 180 min was then normalized against AUG for the placebo. These results are shown in Figure 4B.

[0182] As shown in Figures 4A and 4B, using the Pah-R261Q mouse model PBAS499 has been shown to significantly reduce blood Phe by 49%. After the last blood sample, mice were sacrificed, and livers harvested. Analysis of liver lysates showed a 218% increase in PAH activity (Figure 4C) and 229% increase in PAH protein level (Figure 4D), demonstrating that PBAS499 has its mechanism of action as a pharmacological chaperone for PAH.

[0183] Example 5 - Combination with BH4

[0184] A corresponding experiment to Example 4 was also conducted to evaluate the combination of PBAS499 and BH4. The results are shown in Figures 5A-5D.

[0185] Three groups of mice (placebo, BH4 and combination PBAS499+BH4, n=6 per group) were given either placebo (vehicle) or 20 mg / kg PBAS499 four times a day for 1.5 days. BH4 (20 mg / kg) was only given in the last dose before Phe injection.

[0186] The results (see Figures 5A and 5B) demonstrate larger blood Phe decrease in mice which received the combination of PBAS499 and BH4 (62% blood Phe reduction) than in mice receiving only BH4 (31% blood Phe reduction) or only PBAS499 (49% blood Phe decrease, Figure 4B).

[0187] Analysis of liver lysates show increased PAH activity in mice treated with BH4 (Figure 5C, middle result), in accordance with the co-factor role of BH4. However, there was no detectable or notable increase of PAH protein levels after BH4 treatment in the Pah-R261Q mouse (Figure 5D, middle result). In mice simultaneously treated with both BH4 and PBAS499 a large increase in both PAH activity and PAH protein levels was detected (see Figure 5C and 5D, right hand results).

Claims

Claims1. A compound of Formula (I), or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof:Formula (I) whereinRi and R2 are independently selected from the group consisting of H and C1-C3 alkyl;Y is a 5- or 6-membered heteroaryl which may be substituted by one or more R3 groups; each R3 is independently selected from the group consisting of halogen, N(Ry)2, ORy, SRy, Ci-Ce alkyl, C2-C6 alkenyl, and a 5- or 6-membered carbocyclic or heterocyclic group, wherein each Ryis independently H or C1-6 alkyl and wherein each R3 group may be substituted by one or more R4 groups; or two R3 groups together form a 5- or 6-membered carbocyclic or heterocyclic group which may be substituted by one or more R4 groups; andR4 is selected from the group consisting of halogen, C1-3 alkyl, =0, -OH and -NH2; optionally wherein the compound of compound of Formula (I) is not2. The compound of claim 1, wherein Ri and R2 are each independently selected from the group consisting of H and CH3, preferably wherein R1 and R2 are both H.

3. The compound of any preceding claim, wherein Y is selected from the group consisting of pyrrole, furan, thiophene, pyridine, pyrimidine, pyridazine and pyrazine.

4. The compound of any preceding claim, wherein Y is thiophene or pyridine.

5. The compound of any preceding claim, wherein Y is substituted by one or two R3 groups.

6. The compound of any preceding claim, wherein each R3 may be substituted by one or two R4 groups, or two R3 groups together form a 5- or 6-membered carbocyclic or heterocyclic group which may be substituted by one or two R4 groups.

7. The compound of any preceding claim, wherein each R3 is a 5- or 6- membered carbocyclic or heterocyclic group which may be substituted by one or two R4 groups; or two R3 groups together form a 5- or 6-membered carbocyclic or heterocyclic group which may be substituted by one or two R4 groups.

8. The compound of any preceding claim, wherein each R3 is a 5-membered heteroaryl which may be substituted by one or two R4 groups; or two R3 groups together form a 5-membered heteroaryl which may be substituted by one or two R4 groups.

9. The compound of any preceding claim, wherein R3 is selected from the group consisting of furan, thiophene, pyrrole, pyrazole, imidazole and triazole, each of which may be substituted by one or two R4 groups, preferably wherein R3 is unsubstituted pyrazole.

10. The compound of any preceding claim, wherein each R4 is independently selected from the group consisting of Cl, Br and C1-3 alkyl, preferably wherein each R4 is independently selected from the group consisting of Cl and CH3.11 . The compound of any of claims 1 , 2 or 6-10, wherein the compound of Formula (I) is a compound of Formula (Ila) or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof:wherein X is NH, O or S, preferably O or S; preferably wherein the compound of Formula (Ila) is a compound of Formula (lib) or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof:Formula (lib)12. The compound of claim 11 , wherein the compound of Formula (lib) is a compound of Formula (He) or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof:Formula (He)preferably wherein the compound of Formula (He) is a compound of Formula (lid) or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof:Formula (lid)13. The compound of any of claims 1 , 2 or 6-10, wherein the compound ofFormula (I) is a compound of Formula (Illa) or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof:wherein each Y is independently C or N, preferably C; preferably wherein the compound of Formula (Illa) is a compound of Formula (lllb) or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof:Formula (lllb) wherein Rs is H or R4, preferably R4.

14. The compound of claim 13, wherein the compound of Formula (111 b) is a compound of Formula (I I Ic) or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof:Formula (lllc) preferably wherein the compound of Formula (lllc) is a compound of Formula (Hid) or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof:

15. A composition comprising a compound of any preceding claim or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof and a pharmaceutically acceptable carrier, diluent or excipient.

16. The composition of claim 15, further comprising a further agent effective in the treatment of HPA, in particular PKU.

17. A pharmaceutical pack comprising a compound as defined in any of claims 1-14, and a further agent effective in the treatment of HPA, in particular PKU.

18. The composition of claim 16 or the pharmaceutical pack of claim 17, wherein the further agent effective in the treatment of HPA is pegvaliase, tetrahydrobiopterin or an analogue or precursor thereof.

19. The composition or pharmaceutical pack of claim 18, wherein the further agent effective in the treatment of HPA is tetrahydrobiopterin, sepiapterin or dihydrobiopterin.

20. A compound according to any of claims 1-14 or a composition according to any of claims 15, 16 and 18, for use in the treatment of hyperphenylalaninemia (HPA), in particular phenylketonuria (PKU), optionally wherein the compound has essentially no inhibitory effect on dihydrofolate reductase (DHFR).

21. The compound or composition for use according to claim 20, wherein the treated condition is mild HPA, mild PKU or classic PKU, optionally in a subject exhibiting > 10% of wild-type PAH activity.

22. A method of treating hyperphenylalaninemia (HPA), in particular phenylketonuria (PKU), comprising administering to a patient in need thereof a therapeutically effective amount of compound according to any of claims 1-14 or a composition according to any of claims 15, 16 and 18.

23. The use of a compound according to any of claims 1-14 or a composition according to any of claims 15, 16 and 18, in the manufacture of a medicament for the treatment of hyperphenylalaninemia (HPA), in particular phenylketonuria (PKU).

24. The use of a compound according to any of claims 1-14 or a composition according to any of claims 15, 16 and 18, as a pharmacological chaperone.

25. The in vitro use of a compound according to any of claims 1-14 or a composition according to any of claims 15, 16 and 18, to stabilise PAH.

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