Therapeutic compound

Abstract

There are provided compounds, agents, compositions and preparations which may be used to treat conditions such as colorectal cancer. Activity may be achieved through agonism of Peroxisome Proliferator-Activated Receptors. An exemplary compound is Formula (I):

Description

[0001] P / 90574.W001

[0002] THERAPEUTIC COMPOUND

[0003] The present invention relates to a compound or agent and its use in therapy. The compound or agent may be used in a variety of therapeutic applications, such as in treatment of damaged or dysfunctional tissue. For example, the compound or agent may be useful in the treatment of cancer such as colorectal cancer or other conditions of the gastrointestinal tract. The present invention also relates to pharmaceutical compositions comprising the compound and agent, and methods of synthesising the compound or agent.

[0004] Colorectal cancer (CRC) is the second leading cause of cancer-related deaths globally. If detected and treated at an early stage, survival rates can exceed 90%. However, survival rates drop to 10% - 20% when cancer is diagnosed late.

[0005] CRC develops from polyps, which are overgrowths of intestinal mucosa that have undergone a series of gene mutations. Only 5% to 10% of adenomas progress to cancer, and this process typically takes 10 to 15 years. The progression depends on several factors, such as the size and type of precancerous polyp (adenoma), degree of dysplasia (abnormal cell growth) and contributing environmental factors such as diet and obesity.

[0006] National bowel screening programs serve a crucial role in reducing CRC incidence and mortality. A simple home test, typically starting at age 55, identifies 3% to 6% of the screened population who are referred for a colonoscopy. Colonoscopy procedures, which can range from $500 to $3,000, diagnose approximately 7% of these individuals with CRC, while 20% are identified as being at high risk for developing the disease. Despite regular colonoscopy screenings every 1 to 3 years for high-risk individuals, current standards of care still miss 10% to 15% of polyps.

[0007] While adenomatous polyps provide an important early biomarker for predicting CRC, their relatively low rate of progression, slow development into cancer, and the limitations of current surveillance methods underscore the need for a therapeutic approach to reduce both polyp formation and their progression to CRC. Developing an effective drug to target these mechanisms could significantly improve prevention and reduce the burden of CRC.

[0008] It is also desirable to develop active pharmaceutical ingredients which, in addition to treating polyps and CRC, may have properties that make them effective in treating other indications which may have biological pathways in common, or similar biological pathways. This may include other gastrointestinal conditions, as well as damaged or defective tissue in other areas of the body. According to the invention, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer (e.g. enantiomers or diastereomers) thereof:

[0009] (I)

[0010] The compound of the invention may also be referred to herein as “ULI262”.

[0011] The term “pharmaceutically acceptable salt” refers to a pharmaceutically acceptable organic or inorganic salt of the compound of the invention prepared from pharmaceutically acceptable non-toxic bases or acids, which include organic or inorganic bases and organic or inorganic acids. Examples of pharmaceutically acceptable salts may be found in “Handbook of Pharmaceutical Salts Properties, Selection, and use” (2011 ) by P. Heinrich Stahl and Camille G. Wermuth ISBN: 978-3-906-39051 -2.

[0012] The term “solvate” includes compounds formed by solvation, for example as a combination of solvent molecules with molecules or ions of a solute. Well known solvent molecules include water, alcohols and other polar organic solvents. Alcohols include methanol, ethanol, n- propanol, isopropanol, n-butanol, isobutanol, and t-butanol. Alcohols also include polymerized alcohols such as polyalkylene glycols (e.g., polyethylene glycol, polypropylene glycol). If the solvent is water, solvate compounds formed by solvation with water are termed hydrates.

[0013] The compound of the invention may be isolated or extracted. For example, it may be isolated or extracted from a natural source, such as a plant. A suitable method of isolation or extraction is provided, for instance, in Example 10. The plant from which the compound is isolated or extracted may be from the genus Lindera. For example, it may be isolated or extracted from Lindera aggregata.

[0014] According to the invention, there is provided a plant extract enriched with a compound of the invention. For example, the plant extract may have a higher concentration of the compound of the invention than is present in the natural source. The compound may be said to be purified. The plant extract may be from the genus Lindera. For example, it may be from Lindera aggregata. The compound of the invention may be synthetic. In other words, the compound may be chemically synthesised rather than being isolated from a natural source, such as a plant. The compound may thus be artificial, or artificially-produced. It may be preferable to artificially produce the compound rather than extract it from a natural source. Chemically synthesising the compound in vitro may, for example, make it more amenable for inclusion in a pharmaceutical composition. A suitable synthesis pathway for forming a compound of Formula (I) is provided, for instance, in Example 11 .

[0015] According to the invention, there is provided an isolated compound of Formula (I) or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof.

[0016] According to the invention, there is provided a synthetic compound of Formula (I) or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof.

[0017] The compound of the invention may be in the form of formula (la), or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer (e.g. enantiomer or diastereomer) thereof.

[0018] (la)

[0019] Compounds of the invention may bind to, and thus be a ligand for, a Peroxisome Proliferator- Activated Receptor (PPAR). PPARs are a family of nuclear receptor proteins that act as transcription factors regulating gene expression in areas such as cellular differentiation, development and metabolism. Compounds of the invention may modulate a PPAR. For example, compounds of the invention may activate or agonise a PPAR. Preferably, the PPAR is a human PPAR. Alternatively, the PPAR may be a non-human PPAR (such as an animal, or mammalian, PPAR). For example, the PPAR may be a pig PPAR. For example, the PPAR may be a mouse PPAR.

[0020] There are three main types, or isoforms, of PPAR: PPAR-a (also referred to as PPARA); PPAR- (3 / 6 (also referred to as PPARB or PPARD); and PPAR-y (also known as PPARG). A compound of the invention may modulate (e.g. activate or agonise) one of more of PPAR-a, PPAR-p / b and PPAR-y. For example, the compound may modulate PPAR-a, the compound may modulate PPAR-p / b, the compound may modulate PPAR-y, the compound may modulate PPAR-a and PPAR-p / b; the compound may modulate PPAR-a and PPAR-y, the compound may modulate PPAR-p / b and PPAR-y, or the compound may modulate PPAR-a, PPAR-p / b and PPAR-y. Preferably, the compound modulates PPAR-a, PPAR-p / 5 and PPAR-y and so may be termed a pan-PPAR modulator or pan-PPAR agonist.

[0021] A UniProt accession number for human PPAR-a is Q07869. Human PPAR-a may comprise the following amino acid sequence (SEQ ID NO: 1 ): MVDTESPLCPLSPLEAGDLESPLSEEFLQEMGNIQEISQSIGEDSSGSFGFTEYQYLGSCPGS DGSVITDTLSPASSPSSVTYPVVPGSVDESPSGALNIECRICGDKASGYHYGVHACEGCKGFF RRTIRLKLVYDKCDRSCKIQKKNRNKCQYCRFHKCLSVGMSHNAIRFGRMPRSEKAKLKAEILT CEHDIEDSETADLKSLAKRIYEAYLKNFNMNKVKARVILSGKASNNPPFVIHDMETLCMAEKTLV AKLVANGIQNKEAEVRIFHCCQCTSVETVTELTEFAKAIPGFANLDLNDQVTLLKYGVYEAIFAM LSSVMNKDGMLVAYGNGFITREFLKSLRKPFCDIMEPKFDFAMKFNALELDDSDISLFVAAIICC GDRPGLLNVGHIEKMQEGIVHVLRLHLQSNHPDDIFLFPKLLQKMADLRQLVTEHAQLVQIIKKT ESDAALHPLLQEIYRDMY. A compound or agent of the invention may bind to a polypeptide that has at least: 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; or 99% amino acid identity along its entire length with the sequence of SEQ ID NO: 1 .

[0022] A UniProt accession number for human PPAR-p / 5 is Q03181 . Human PPAR-p / 5 may comprise the following amino acid sequence (SEQ ID NO: 2): MEQPQEEAPEVREEEEKEEVAEAEGAPELNGGPQHALPSSSYTDLSRSSSPPSLLDQLQMGC DGASCGSLNMECRVCGDKASGFHYGVHACEGCKGFFRRTIRMKLEYEKCERSCKIQKKNRN KCQYCRFQKCLALGMSHNAIRFGRMPEAEKRKLVAGLTANEGSQYNPQVADLKAFSKHIYNA YLKNFNMTKKKARSILTGKASHTAPFVIHDIETLWQAEKGLVWKQLVNGLPPYKEISVHVFYRC QCTTVETVRELTEFAKSIPSFSSLFLNDQVTLLKYGVHEAIFAMLASIVNKDGLLVANGSGFVTR EFLRSLRKPFSDIIEPKFEFAVKFNALELDDSDLALFIAAIILCGDRPGLMNVPRVEAIQDTILRAL EFHLQANHPDAQYLFPKLLQKMADLRQLVTEHAQMMQRIKKTETETSLHPLLQEIYKDMY. A compound or agent of the invention may bind to a polypeptide that has at least: 90%; 91 %; 92%; 93%; 94%; 95%; 96%; 97%; 98%; or 99% amino acid identity along its entire length with the sequence of SEQ ID NO: 2.

[0023] A UniProt accession number for human PPAR-y is P37231 . Human PPAR-y may comprise the following amino acid sequence (SEQ ID NO: 3): MGETLGDSPIDPESDSFTDTLSANISQEMTMVDTEMPFWPTNFGISSVDLSVMEDHSHSFDIK PFTTVDFSSISTPHYEDIPFTRTDPVVADYKYDLKLQEYQSAIKVEPASPPYYSEKTQLYNKPHE EPSNSLMAIECRVCGDKASGFHYGVHACEGCKGFFRRTIRLKLIYDRCDLNCRIHKKSRNKCQ YCRFQKCLAVGMSHNAIRFGRMPQAEKEKLLAEISSDIDQLNPESADLRALAKHLYDSYIKSFP LTKAKARAILTGKTTDKSPFVIYDMNSLMMGEDKIKFKHITPLQEQSKEVAIRIFQGCQFRSVEA VQEITEYAKSIPGFVNLDLNDQVTLLKYGVHEIIYTMLASLMNKDGVLISEGQGFMTREFLKSLR KPFGDFMEPKFEFAVKFNALELDDSDLAIFIAVIILSGDRPGLLNVKPIEDIQDNLLQALELQLKLN HPESSQLFAKLLQKMTDLRQIVTEHVQLLQVIKKTETDMSLHPLLQEIYKDLY. A compound or agent of the invention may bind to a polypeptide that has at least: 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; or 99% amino acid identity along its entire length with the sequence of SEQ ID NO: 3.

[0024] A UniProt accession number for pig PPAR-a is Q9N135. Pig PPAR-a may comprise the following amino acid sequence (SEQ ID NO: 4) MVDTESPICPLSPLEADDLESPLSEEFLQEMGTIQEISQSIGEDSSGSFSFTDYQYLGSGPGSD GSVITDTLSPASSPSSVTYPVAPAGADESPSVALNIECRICGDKASGYHYGVHACEGCKGFFR RTIRLKLVYDKCDRSCKIQKKNRNKCQYCRFHKCLSAGMSHNAIRFGRMPRSEKAKLKAEILTC EHDLEDAETADLKSLAKRIYEAIKKNFNMNKVKARVILAGKASNNPAFCIRDMETLCMAEKTLVA KLVANGTQNKEAEVRIFHCCQCTSDVSVTELTEFAKSIXGFASLDLNDQVTLLKYGVYDAIFAM LSSVMNKDGMLVAYGNGFITREFLKSLRKPFCDIMEPKFDFAMKFNALELDDSDLSLFVAAIICC GDRPGLLNVGHIERMQEGIVHVLKLHLQTNHPDDVFLFPKLLQKLADLRQLVTEHAQLVQVIKK TEADAALHPLLQEIYRDMY. A compound or agent of the invention may bind to a polypeptide that has at least: 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; or 99% amino acid identity along its entire length with the sequence of SEQ ID NO: 4.

[0025] A UniProt accession number for pig PPAR-p / 5 is Q866Q2. Pig PPAR-p / 5 may comprise the following amino acid sequence (SEQ ID NO: 5): MEQPPEEAPEVREEEKKKEVAEAEGGPELNGGPEHSLPSSSCTDLSQSCSPPALLDQLQMG CDGASCGGLSMECRVCGDKASGFHYGVHACEGCKGFFRRTIRMKLEYEKCERICKIQKKNRN KCQYCRFQKCLALGMSHNAIRFGRMPEAEKRKLVAGLTANEGSQHNPQVADLKAFSKHLYSA YLKNFNMTKKKARAILTGKASHTAPFVIHDIETLWQAEKGLVWKQLVNGLPPYKEISVHVFYRC QCTTVETVRELTEFAKSIPSFDHFFLNDQVTLLKYGVHEAIFAMLASIVNKDGLLVANGTGFVTR EFLRSIRKPFSDIIEPKFEFAVKFNALELDDSDLALFIAAIILCGDRPGLMNVSQVEAIQDTILRALE FHLQANHPDAQYLFPKLLQKMADLRQLVTEHAQMMQRIKKTETETSLHPLLQEIYKDMY. A compound or agent of the invention may bind to a polypeptide that has at least: 90%; 91 %; 92%; 93%; 94%; 95%; 96%; 97%; 98%; or 99% amino acid identity along its entire length with the sequence of SEQ ID NO: 5.

[0026] A UniProt accession number for pig PPAR-y is 062807. Pig PPAR-y may comprise the following amino acid sequence (SEQ ID NO: 6):

[0027] MGETLGDSLIDPESDAFDTLSANISQEVTMVDTEMPFWPTNFGISSVDLSVMDDHSHSFDIKPF TTVDFSSISTPHYEDIPFPRADPMVADYKYDLKLQDYQSAIKVEPVSPPYYSEKTQLYNKPHEE PSNSLMAIECRVCGDKASGFHYGVHACEGCKGFFRRTIRLKLIYDRCDLNCRIHKKSRNKCQY CRFQKCLAVGMSHNAIRFGRMPQAEKEKLLAEISSDIDQLNPESADLRALAKHLYDSYIKSFPLT KAKARAILTGKTTDKSPFVIYDMNSLMMGEDKIKFKHITPLQEQSKEVAIRIFQGCQFRSVEAVQ EITEYAKNIPGFVNLDLNDQVTLLKYGVHEIIYTMLASLMNKDGVLISEGQGFMTREFLKSLRKP FGDFMEPKFEFAVKFNALELDDSDLAIFIAVIILSGDRPGLLNVKPIEDIQDNLLQALELQLKLNHP ESSQLFAKLLQKMTDLRQIVTEHVQLLQVIKKTETDMSLHPLLQEIYKDLY. A compound or agent of the invention may bind to a polypeptide that has at least: 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; or 99% amino acid identity along its entire length with the sequence of SEQ ID NO: 6.

[0028] A UniProt accession number for mouse PPAR-a is P23204. Mouse PPAR-a may comprise the following amino acid sequence (SEQ ID NO: 7) MVDTESPICPLSPLEADDLESPLSEEFLQEMGNIQEISQSIGEESSGSFGFADYQYLGSCPGSE GSVITDTLSPASSPSSVSCPVIPASTDESPGSALNIECRICGDKASGYHYGVHACEGCKGFFRR TIRLKLVYDKCDRSCKIQKKNRNKCQYCRFHKCLSVGMSHNAIRFGRMPRSEKAKLKAEILTCE HDLKDSETADLKSLGKRIHEAYLKNFNMNKVKARVILAGKTSNNPPFVIHDMETLCMAEKTLVA KMVANGVEDKEAEVRFFHCCQCMSVETVTELTEFAKAIPGFANLDLNDQVTLLKYGVYEAIFT MLSSLMNKDGMLIAYGNGFITREFLKNLRKPFCDIMEPKFDFAMKFNALELDDSDISLFVAAIICC GDRPGLLNIGYIEKLQEGIVHVLKLHLQSNHPDDTFLFPKLLQKMVDLRQLVTEHAQLVQVIKKT ESDAALHPLLQEIYRDMY. A compound or agent of the invention may bind to a polypeptide that has at least: 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; or 99% amino acid identity along its entire length with the sequence of SEQ ID NO: 7.

[0029] A UniProt accession number for mouse PPAR-p / 5 is P35396. Mouse PPAR-p / 5 may comprise the following amino acid sequence (SEQ ID NO: 8): MEQPQEETPEAREEEKEEVAMGDGAPELNGGPEHTLPSSSCADLSQNSSPSSLLDQLQMGC DGASGGSLNMECRVCGDKASGFHYGVHACEGCKGFFRRTIRMKLEYEKCDRICKIQKKNRNK CQYCRFQKCLALGMSHNAIRFGRMPEAEKRKLVAGLTASEGCQHNPQLADLKAFSKHIYNAYL KNFNMTKKKARSILTGKSSHNAPFVIHDIETLWQAEKGLVWKQLVNGLPPYNEISVHVFYRCQS TTVETVRELTEFAKNIPNFSSLFLNDQVTLLKYGVHEAIFAMLASIVNKDGLLVANGSGFVTHEF LRSLRKPFSDIIEPKFEFAVKFNALELDDSDLALFIAAIILCGDRPGLMNVPQVEAIQDTILRALEF HLQVNHPDSQYLFPKLLQKMADLRQLVTEHAQMMQWLKKTESETLLHPLLQEIYKDMY. A compound or agent of the invention may bind to a polypeptide that has at least: 90%; 91 %; 92%; 93%; 94%; 95%; 96%; 97%; 98%; or 99% amino acid identity along its entire length with the sequence of SEQ ID NO: 8.

[0030] A UniProt accession number for mouse PPAR-y is P37238. Mouse PPAR-y may comprise the following amino acid sequence (SEQ ID NO: 9):

[0031] MGETLGDSPVDPEHGAFADALPMSTSQEITMVDTEMPFWPTNFGISSVDLSVMEDHSHSFDIK PFTTVDFSSISAPHYEDIPFTRADPMVADYKYDLKLQEYQSAIKVEPASPPYYSEKTQLYNRPH EEPSNSLMAIECRVCGDKASGFHYGVHACEGCKGFFRRTIRLKLIYDRCDLNCRIHKKSRNKC QYCRFQKCLAVGMSHNAIRFGRMPQAEKEKLLAEISSDIDQLNPESADLRALAKHLYDSYIKSF PLTKAKARAILTGKTTDKSPFVIYDMNSLMMGEDKIKFKHITPLQEQSKEVAIRIFQGCQFRSVE AVQEITEYAKNIPGFINLDLNDQVTLLKYGVHEIIYTMLASLMNKDGVLISEGQGFMTREFLKNLR KPFGDFMEPKFEFAVKFNALELDDSDLAIFIAVIILSGDRPGLLNVKPIEDIQDNLLQALELQLKLN HPESSQLFAKVLQKMTDLRQIVTEHVQLLHVIKKTETDMSLHPLLQEIYKDLY. A compound or agent of the invention may bind to a polypeptide that has at least: 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; or 99% amino acid identity along its entire length with the sequence of SEQ ID NO: 9).

[0032] Sequence identity is frequently measured in terms of percentage identity; the higher the percentage, the more similar the two sequences are. Homologs or variants of a given polypeptide or protein will possess a relatively high degree of sequence identity when aligned using standard methods. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. AppL Math. 2:482, 1981 ; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237- 244, 1988; Higgins and Sharp, CABIOS 5:151 -153, 1989; Corpet etal., Nucleic Acids’ Research 16:10881 -10890, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988. Altschul etal., Nature Genet. 6:119-129, 1994. The NCBI Basic Local Alignment Search Tool (BLAST™) (Altschul et al., J. Mol. Biol. 215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs BLASTP, blastn, blastx, tblastn and tblastx.

[0033] Sequence identity between amino acid sequences, can be determined by comparing an alignment of the sequences. When an equivalent position in the compared sequences is occupied by the same amino acid, then the molecules are identical at that position. Scoring an alignment as a percentage of identity is a function of the number of identical nucleotides or amino acids at positions shared by the compared sequences. When comparing sequences, optimal alignments may require gaps to be introduced into one or more of the sequences to take into consideration possible insertions and deletions in the sequences. Sequence comparison methods may employ gap penalties so that, for the same number of identical molecules in sequences being compared, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. Calculation of maximum percent identity involves the production of an optimal alignment, taking into consideration gap penalties.

[0034] Suitable computer programs for carrying out sequence comparisons are widely available in the commercial and public sector. Examples include MatGat (Campanella et aL, 2003, BMC Bioinformatics 4: 29; program available from http: / / bitincka.com / ledion / matgat), Gap (Needleman & Wunsch, 1970, J. Mol. Biol. 48: 443-453), FASTA (Altschul et aL, 1990, J. Mol. Biol. 215: 403-410; program available from http: / / www.ebi.ac.uk / fasta), Clustal W 2.0 and X 2.0 (Larkin et aL, 2007, Bioinformatics 23: 2947-2948; program available from http: / / www.ebi.ac.uk / tools / clustalw2) and EMBOSS Pairwise Alignment Algorithms (Needleman & Wunsch, 1970, supra; Kruskal, 1983, In: Time warps, string edits and macromolecules: the theory and practice of sequence comparison, Sankoff & Kruskal (eds), pp 1 -44, Addison Wesley; programs available from http: / / www.ebi.ac.uk / tools / emboss / align). All programs may be run using default parameters.

[0035] For example, sequence comparisons may be undertaken using the “needle” method of the EMBOSS Pairwise Alignment Algorithms, which determines an optimum alignment (including gaps) of two sequences when considered over their entire length and provides a percentage identity score. Default parameters for amino acid sequence comparisons (“Protein Molecule” option) may be Gap Extend penalty: 0.5, Gap Open penalty: 10.0, Matrix: Blosum 62.

[0036] Percent identity between a query amino acid sequence and a subject amino acid sequence may be the "Identities" value, expressed as a percentage, calculated by the BLASTP algorithm when a subject amino acid sequence has 100% query coverage with a query amino acid sequence after a pair-wise BLASTP alignment is performed. Such pair-wise BLASTP alignments between a query amino acid sequence and a subject amino acid sequence are performed by using the default settings of the BLASTP algorithm available on the National Center for Biotechnology Institute's website with the filter for low complexity regions turned off. A query amino acid sequence may be described by an amino acid sequence identified herein.

[0037] The sequence comparison may be performed over the full (or entire) length of the reference sequence.

[0038] Surprisingly, it has been found that a pan-PPAR agonist may be particularly beneficial for use as a medicament, and particularly in the uses and applications described herein. For example, other PPAR agonists which modulate PPAR-a and / or PPAR-y may not be as effective in polyp reduction in comparison to a compound or agent of the invention (See e.g. Fig. 24).

[0039] Pan-PPAR activity may restore or promote tissue homeostasis. Pan-PPAR modulation may promote the establishment and differentiation of gastrointestinal crypt organoids in a dosedependent, biphasic manner, supporting epithelial regeneration at low concentrations while limiting overgrowth at higher concentrations. Agents of the invention may provide a dual mode of action, simultaneously stimulating epithelial cell production and inducing antioxidant and antiinflammatory pathways, thereby reducing inflammatory mediators such as TNF-a.

[0040] According to the invention, there is provided an agent which is a PPAR agonist, preferably a human PPAR agonist. The agent may be an agonist of PPAR-a. The agent may be an agonist of PPAR-y. The agent may be an agonist of PPAR-a and PPAR-p / b. The agent may be an agonist of PPAR-a and PPAR-y. The agent may be an agonist of PPAR-p / b and PPAR-y. The agent may be an agonist of PPAR-a, PPAR-p / b and PPAR-y. Preferably, the agent is an agonist of at least PPAR-p / b, most preferably an agonist of PPAR-a, PPAR-p / b and PPAR-y.

[0041] Although the agent may be a compound of Formula (I) or (la), or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof, it could be any other agent which acts as a PPAR agonist. PPAR agonists may be identified, or confirmed, using techniques known in the art. For example, this may include cell-based reporter gene assays that measure transcriptional activation via luciferase or other reporter proteins under the control of a PPAR response element (PPRE). An example of an assay includes Human PPAR Receptor Assays Panel; PPARa, PPAR 5, PPAR y (Indigo Biosciences (PA 16801 , USA); product description #IB00131 - 32P) which is carried out in accordance with the protocol described in Indigo Biosciences Technical Manual (version 7.2M; incorporated herein by reference). Such an assay may allow determination of EC50 for an agent of the invention.

[0042] An agent of the invention may have an EC50 of less than or equal to 10-7M, less than or equal to 10-8M or less than or equal to 10-9M or less than or equal to 10-10M. For example, in respect of a PPAR-a assay (such as the Indigo assay #IB00131 -32P), an agent of the invention may have an ECso of less than or equal to 10-7M, less than or equal to 10-8M, less than or equal to 10-9M or less than or equal to 10-10M. In respect of a PPAR-p / b assay (such as the Indigo assay #l BO0131 -32P), an agent of the invention may have an EC50 of less than or equal to 10-7M, less than or equal to 10-8M, less than or equal to 10-9M or less than or equal to 10-10M. In respect of a PPAR-y assay (such as the Indigo assay #IB00131 -32P), an agent of the invention may have an ECso of less than or equal to 10-7M, less than or equal to 10-8M, less than or equal to 10-9M or less than or equal to 10-1°M.

[0043] An agent of the invention may demonstrate potency at low concentrations. For example, it has been shown that a compound of the invention can enhance pig polyp-derived organoids at concentrations of 1 nM (see Example 12, Fig. 43c).

[0044] Agonist activity may also be assessed using direct ligand binding assays, such as scintillation proximity assays or time-resolved fluorescence resonance energy transfer (TR-FRET), to directly assess binding of a to PPAR receptors.

[0045] For example, the agent may be a peptide or a protein, such as an antigen binding protein. An antigen binding protein may include an immunoglobulin, such as an antibody or functional fragment thereof. Antibodies or functional fragments thereof may include, but are not limited to, Fab, Fab’, and F(ab’)2 fragments, or Fvs, domain antibodies and scFvs.

[0046] The agent may specifically bind a PPAR, for example it may specifically bind PPAR-a, PPAR- (3 / 6 and PPAR-y. An agent may specifically bind a PPAR when it exhibits essentially background binding to non-PPAR molecules. A PPAR agonist that specifically binds PPAR may, however, cross-react with PPARs from different species, or with different PPAR isotypes.

[0047] An agent of the invention may bind PPAR-a with a dissociation constant (Kd) of less than or equal to 10-7M as measured by a surface plasmon resonance technique (e.g., BIACore, GE- Healthcare Uppsala, Sweden) or Kinetic Exclusion Assay (KinExA, Sapidyne, Boise, Idaho). An agent of the invention may bind PPAR-p / b with a Kd of less than or equal to 10-7M. An agent of the invention may bind PPAR-y with a Kd of less than or equal to 10-7M. Preferably, an agent of the invention binds PPAR-a, PPAR-p / b and PPAR-y with a Kd of less than or equal to 10-7M.

[0048] A compound or agent of the invention may activate or upregulate Retinoid X receptor alpha (RXRA or RXR-a). A PPAR may form a heterodimer with RXRA to form a PPAR-RXR transcriptional complex, which may modulate gene expression through peroxisome proliferator response elements (PPREs).

[0049] The compound or agent of the invention may be in a composition, such as a pharmaceutical composition. The pharmaceutical composition may comprise a pharmaceutically acceptable excipient.

[0050] According to the invention, there is provided a composition comprising an agent which is a PPAR agonist.

[0051] According to the invention, there is provided a composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof.

[0052] According to the invention, there is provided a pharmaceutical composition, or a medicament, comprising an agent which is a PPAR agonist, and a pharmaceutically acceptable excipient.

[0053] According to the invention, there is provided a pharmaceutical composition, or a medicament, comprising a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof, and a pharmaceutically acceptable excipient.

[0054] According to the invention, there is provided a composition that modulates (e.g. agonises) PPAR-a, PPAR-p / b and PPAR-y. Such a composition may comprise an agent which is a pan- PPAR agonist. In other words, there may be a single agent which can agonise PPAR-a, PPAR- (3 / 6 and PPAR-y (such as a compound of Formula I or Formula la). Alternatively, the composition may comprise a plurality of (at least two, possibly three) distinct PPAR agonists which, in combination, agonise PPAR-a, PPAR-p / b and PPAR-y. Such a composition may be a fixed dose combination. Each agent may therefore agonise up to two PPAR isotypes (possibly only one PPAR isotype), but not all three PPAR isotypes. For example, the composition may comprise a first agent which agonises PPAR-a, a second agent which agonises PPAR-p / b and a third agent which agonises PPAR-y. In another example, the composition may comprise a first agent which agonises PPAR-a and PPAR-p / b, and a second agent which agonises PPAR- y. In another example, the composition may comprise a first agent which agonises PPAR-p / b and PPAR-y, and a second agent which agonises PPAR- a. In another example, the composition may comprise a first agent which agonises PPAR- a and PPAR-y, and a second agent which agonises PPAR-p / b. According to the invention, there is provided a composition that modulates (e.g. agonises) PPAR-a, PPAR-p / 5 and PPAR-y, and a pharmaceutically acceptable excipient.

[0055] If there are a plurality of distinct agents which, in combination, agonise PPAR-a, PPAR-p / b and PPAR-y, at least one of the agents may be in a composition, such as a unit dose form, which is packaged separately. For example, at least one agent may be in a separate composition, which may be sourced from different suppliers, and then used in the methods or uses of the invention, as described herein. For example, there may be a first composition which agonises PPAR-a (e.g. comprises a first agent which is a PPAR-a agonist), a second composition which agonises PPAR-p / b (e.g. comprises a second agent which is a PPAR-p / b agonist) and a third composition which agonises PPAR-y (e.g. comprises a third agent which is a PPAR-p / b agonist). In another example, there may be a first composition which agonises PPAR-a and PPAR-p / b (e.g. comprises a first agent which is a PPAR-a agonist and a PPAR-p / b agonist), and a second composition which agonises PPAR-y (e.g. comprises a second agent which is a PPAR-y agonist). In another example, there may be a first composition which agonises PPAR- (3 / 6 and PPAR-y (e.g. comprises a first agent which is a PPAR-p / b agonist and a PPAR-y agonist), and a second composition which agonises PPAR- a (e.g. comprises a second agent which is a PPAR- a agonist). In another example, there may be a first composition which agonises PPAR-a and PPAR-y (e.g. comprises a first agent which is a PPAR-a agonist and a PPAR-y agonist), and a second composition which agonises PPAR-p / b (e.g. comprises a second agent which is a PPAR-p / b agonist). Each agent may therefore agonise up to two PPAR isotypes (possibly only one PPAR isotype), but not all three PPAR isotypes. Each composition preferably includes a pharmaceutically acceptable excipient.

[0056] If there are a plurality of agents which, in combination, agonise PPAR-a, PPAR-p / b and PPAR- y, they may be in a combined preparation in which separate compositions (e.g. unit dosage forms) are packaged together, such as in a kit of parts. At least two distinct PPAR agonists may be in separate compositions. For example, three distinct PPAR agonists may be in separate compositions. For instance, there may be a first composition which agonises PPAR-a (e.g. comprises a first agent which is a PPAR-a agonist), a second composition which agonises PPAR-p / b (e.g. comprises a second agent which is a PPAR-p / b agonist) and a third composition which agonises PPAR-y (e.g. comprises a third agent which is a PPAR-p / b agonist). In another example, there may be a first composition which agonises PPAR-a and PPAR-p / b (e.g. comprises a first agent which is a PPAR-a agonist and a PPAR-p / b agonist), and a second composition which agonises PPAR-y (e.g. comprises a second agent which is a PPAR-y agonist). In another example, there may be a first composition which agonises PPAR- (3 / 6 and PPAR-y (e.g. comprises a first agent which is a PPAR-p / b agonist and a PPAR-y agonist), and a second composition which agonises PPAR- a (e.g. comprises a second agent which is a PPAR- a agonist). In another example, there may be a first composition which agonises PPAR-a and PPAR-y (e.g. comprises a first agent which is a PPAR-a agonist and a PPAR-y agonist), and a second composition which agonises PPAR- / 5 (e.g. comprises a second agent which is a PPAR- / 5 agonist). Each agent may therefore agonise up to two PPAR isotypes (possibly only one PPAR isotype), but not all three PPAR isotypes. Each composition preferably includes a pharmaceutically acceptable excipient. References herein to methods and uses of compositions of the invention also apply to methods and uses of such combined preparations.

[0057] The term “pharmaceutically acceptable excipient” includes a solid or liquid filler, diluent, lubricant, binder, glidant, carrier, solvent or encapsulating substance which does not interfere with the effectiveness or the biological activity of the compound and which is not toxic to the subject (human or animal), to which it is administered. Depending upon the particular route of administration, a variety of pharmaceutically acceptable excipients, such as those well known in the art, may be used.

[0058] The excipient may include a filler or diluent. Suitable fillers or diluents may include mannitol, microcrystalline cellulose, lactose, starch, dibasic calcium phosphate anhydrous, tribasic calcium phosphate, kaolin, sucrose, calcium carbonate, sorbitol, maltodextrin, powdered cellulose or micro crystalline cellulose.

[0059] The excipient may include a lubricant. Suitable lubricants may include stearic acid, sodium stearyl fumarate, polyethylene glycol, magnesium stearate, calcium stearate, talc, zinc stearate, hydrogenated castor oil, silica, colloidal silica, cornstarch, calcium silicate, magnesium silicate or silicon hydrogel.

[0060] The excipient may include a binder. Suitable binders may include polyvinylpyrrolidone, hydroxypropyl methylcellulose, acacia, alginic acid, hydroxy propyl cellulose, carboxymethylcellulose sodium, compressible sugar, ethylcellulose, gelatin, liquid glucose, methylcellulose or pregelatinized starch.

[0061] The excipient may include a glidant. Suitable glidants may include colloidal silicon dioxide, colloidal silica, cornstarch, talc, calcium silicate, magnesium silicate, colloidal silicon or silicon hydrogel.

[0062] The excipient may include a solvent. A suitable solvent may be an non-aqueous solvent, such as methanol, acetone, methylene chloride. A suitable solvent may be an aqueous solvent, such as water. Suitable aqueous vehicles may include Ringer's solution or saline (such as isotonic sodium chloride). The pharmaceutical composition may comprise water, such as purified, sterile, aseptic or pyrogen-free water. The pharmaceutical composition may comprise a buffered medium, such as a buffered solution (such as a phosphate-buffered solution), or saline. The excipient may be an excipient other than water.

[0063] The excipient may include a polymer. The polymer may include a natural polymer derived from a natural source, such as a plant, animal, or microorganism. Examples of natural polymers include cellulose, starch, alginate, and collagen. The polymer may include a synthetic polymer. Examples of synthetic polymers include poly (ethylene glycol), poly (lactic acid), poly (lactic acid), poly (glycolic acid), poly(caprolactone) and poly (methyl methacrylate). Synthetic polymers may include polymers produced by chemically modifying natural polymers, such as cellulose acetate and hydroxypropyl methylcellulose. The polymer may be a biodegradable polymer which can be broken down by enzymes or other biological processes in the body. Examples of biodegradable polymers include poly(lactic acid), poly (glycolic acid), and poly(caprolactone).

[0064] The polymer may include a film-forming polymer. Examples of film-forming polymers include ethylcellulose, hydroxypropyl methylcellulose, hydroxypropylcellulose, polyvinyl alcohol, polyvinyl acetate methylcellulose, carboxymethyl cellulose, hydroxymethylcellulose, hydroxyethylcellulose, cellulose acetate, hydroxypropyl methylcellulose phthalate, cellulose acetate phthalate, cellulose acetate trimellitate; waxes; methacrylic acid polymers. Polymers may be used as an encapsulating substance.

[0065] The excipient may include a carbohydrate. Suitable carbohydrates may include a sugar, starch, cellulose or a cellulose derivatives such as hydroxymethyl cellulose, or hydroxyethyl cellulose.

[0066] Other examples of a suitable excipient include a salt (such as sodium chloride or calcium sulfate), vegetable oil, synthetic oil, polyol, alginic acid, or emulsifier.

[0067] The pharmaceutical composition may be sterile, such that it is substantially free of viable microorganisms. Sterility may be assessed according to US Pharmacopeia Chapter <71 > (USP <71 >) as of September 2024.

[0068] Various pharmaceutical dosage forms, or unit dosage forms, are contemplated, according to the invention. This may include, for example, a solution, suspension, powder, tablet, capsule, lozenge, caplet suppository, buccal product, cream, ointment, gel, film (such as a transmucosal film), or patch.

[0069] The composition may be formulated in discrete dosage units. The dosage units may be for oral administration, such as capsules, tablets, lozenges and caplets.

[0070] Capsules for oral administration include hard and soft gelatin capsules. To prepare hard gelatin capsules, compounds of the invention may be mixed with a solid, semi-solid, or liquid diluent. Soft gelatin capsules may be prepared by mixing the compound of the invention with water, an oil (such as peanut oil or olive oil), liquid paraffin, a mixture of mono and di-glycerides of short chain fatty acids, polyethylene glycol, and / or propylene glycol.

[0071] Liquids for oral administration may be in the form of suspensions, solutions, emulsions or syrups, or may be lyophilized or presented as a dry product (such as a powder) for reconstitution with water or other suitable vehicle before use. Such liquid compositions may optionally contain: pharmaceutically-acceptable excipients such as suspending agents (for example, sorbitol, methyl cellulose, sodium alginate, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminium stearate gel and the like); non-aqueous vehicles, e.g., oil (for example, almond oil or fractionated coconut oil), propylene glycol, ethyl alcohol, or water; preservatives (for example, methyl or propyl p-hydroxybenzoate or sorbic acid); wetting agents, such as lecithin; or flavouring or colouring agents. Liquids for oral administration may be in discrete dosage units, such as in discrete vials or containers.

[0072] The composition may be in the form of an aqueous solution or suspension. The solution or suspension may include a buffer.

[0073] The composition may be in the form of an non-aqueous solution or suspension.

[0074] The composition may contain pharmaceutical grade, or pharmacopeia grade ingredients. Consequently, one or more, or all of the components, may be approved by the Food and Drug Administration (FDA) for use in humans or animals or for which a chemical purity standard has been established by the United States Pharmacopeia-National Formulary (USP-NF), or British Pharmacopeia (BP). For instance, purity may be assessed as described in US Pharmacopeia Chapter <891 > as of September 2024.

[0075] The purity of the compound or agent, used in a composition of the invention may be (by weight) at least 90%, at least 95%, at least 97.5%, at least 98%, or at least 99%. So, methods of the invention may comprise adding the compound in such high purity, with a pharmaceutically acceptable excipient. For instance, the compound or agent, with a purity of at least 90%, at least 95%, at least 97.5%, at least 98%, or at least 99%, may be added to, or mixed with a pharmaceutically acceptable excipient, to form a composition of the invention.

[0076] Although the compound or agent of the invention could potentially be derived or extracted from a plant, it is preferred that compositions of the invention do not comprise, or are substantially free of, other plant-derived components or components that are found in plants. Plant-derived components, or components found in plants, include lactones, flavonoids, alkaloids, essential oils, phenolics, and tannins.

[0077] Consequently, compositions of the invention are preferably free of lactones, or sesquiterpene lactones, other than the compound of the invention. Compositions of the invention may be substantially free of flavonoids.

[0078] Compositions of the invention may be substantially free of alkaloids.

[0079] Compositions of the invention may be substantially free of essential oils.

[0080] Compositions of the invention may be substantially free of phenolics.

[0081] Compositions of the invention may be substantially free of tannins.

[0082] According to the invention, there is provided a composition comprising a compound or agent of the invention, wherein the compound or agent is present in an amount (by weight) of at least 0.5%, at least 1%, at least 2% at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% of the composition.

[0083] The compound or agent, or the composition of the invention, may be for use as a medicament, or for use in therapy.

[0084] Various modes of administration are contemplated, according to the invention.

[0085] For example, administration may be enteral or parenteral. Enteral administration may be preferred for treating diseases or disorders of the gut.

[0086] Administration may be oral, rectal, subcutaneous, intradermal, intravenous, intra-arterial, intramuscular, intrathecal, epidural, intracistemal, intraperitoneal, transdermal, topical, transmucosal, buccal, sublingual, transmucosal, inhalation, intranasal, intra-atricular, intranasal, rectal or ocular routes. The composition may, in particular, be administered orally.

[0087] Administration may be by injection or infusion.

[0088] Administration may be systemic. Alternatively, administration may be direct, or local, to the tissue or organ being treated.

[0089] Administration may be intravenous or intra-arterial. For example, the composition may be administered by intravenous infusion or injection. The compositions may be administered through an intravenous catheter or intra-arterial catheter.

[0090] The composition of the invention may be present in, or administered using, a syringe (or other injection device) or an intravenous drip bag. Suitable unit-dose forms may include an ampule or an injection device (such as a disposable injection device), or in a solid form or pre-concentrate that can be used to prepare an injectable formulation.

[0091] References herein to “treat”, “treatment”, “treating” or “therapy” may, depending on the condition, refer to prevention, prophylaxis or reducing the risk of, a disease, disorder, or medical condition. It may include treatment of an established disease, disorder, or medical condition.

[0092] Treatment of an established disease, disorder, or medical condition may include amelioration of that disease, disorder, or medical condition. Treatment of an established disease, disorder, or medical condition may include reduction or elimination of one or more symptoms of the disease, disorder, or medical condition.

[0093] The term “effective amount” refers to that amount of the compound or composition that is being administered or that is to be administered, which is sufficient to treat the disease, disorder, or medical condition. Effective amounts or doses of the compounds of the present invention may be ascertained by routine methods such as modelling, dose escalation studies or clinical trials, and by taking into consideration routine factors, such as the mode or route of administration, the pharmacokinetics of the compound, the severity and course of the disease, disorder, or condition, the subject's previous or ongoing therapy, the subject's health status and response to drugs, and the judgment of the treating physician.

[0094] A suitable dose of a compound or agent of the invention may be between 0.01 mg to 100 mg per day, such as 0.1 mg and 10 mg per day.

[0095] According to the invention, the treatment may be for a human or animal subject. Preferably, the treatment is for a human subject.

[0096] For example, the compound, agent or composition of the invention may be for use in a method of treating cancer.

[0097] The invention may thus provide a method of treatment of cancer in a subject in need thereof, the method comprising administering an effective amount of the compound, agent or composition of the invention to the subject, thereby to treat the cancer.

[0098] The invention may thus provide use of the compound, agent or composition of the invention in the manufacture of a medicament for treatment of cancer.

[0099] The cancer may be a cancer of the gastrointestinal tract, such as colorectal cancer (CRC). Treatment of other cancers are envisaged, particularly as PPARs have wide tissue distributions throughout the body. It is therefore envisaged that compounds, agents and compositions of the invention could be used to treat cancers in endodermal, ectodermal and mesodermal tissues. Examples of such tissues are described in more detail below. The progression of, or the precursor to, the cancer may be characterised by one or more mutations in a tumour suppressor gene, such as the APC (adenomatous polyposis coli) gene. Examples of mutations in the APC gene which may lead to cancer are provided, for example, in Zhang, L. and Shay, J.W. J Natl Cancer Inst. 2017 Aug; 109(8): djw332. APC mutations may occur in intestinal crypt cells, such as intestinal crypt stem cells.

[0100] The compound, agent or composition of the invention may treat precancerous cells. For example, the compound, agent or composition of the invention may reduce or eliminate precancerous cells. Precancerous cells may have mutations in at last one tumour suppressor gene and / or at least one oncogene (or proto-oncogene). For example, there may be a mutation in one or more of APC, BRAF, KIT, SRC, MET, JAK2 / 3, GNAS, PDGFRA, HNF1A, FLT3, KRAS, TP53, NOTCH1 , FGFR2, PDGFRA, RET, CTNNB1 , PTPN11 , and ABL1 .

[0101] The compound, agent or composition of the invention may redirect precancerous tissues or organs to healthy, branched tissues or organs.

[0102] The compound, agent or composition of the invention may be for use in a method of treating Familial adenomatous polyposis (FAP).

[0103] The invention may thus provide a method of treatment of FAP in a subject in need thereof, the method comprising administering an effective amount of the compound, agent or composition of the invention to the subject, thereby to treat the FAP.

[0104] The invention may thus provide use of the compound, agent or composition of the invention in the manufacture of a medicament for treatment of FAP.

[0105] FAP may be characterised by growths, polyps or adenomas in the gastrointestinal tract, such as in the large intestine and possibly also in the small intestine. If they are not treated, they may develop into cancer. FAP may also affect other areas of the body. For example, it may lead to CHRPE (congenital hypertrophic retinal pigment epithelium), bony osteomas, sebaceous cysts, or desmoid tumours, In some circumstances, it may lead to lead to cancers in other areas of the body besides the gastrointestinal tract.

[0106] The compound, agent or composition of the invention may be for use as an anti-inflammatory. For example, the compound or composition of the invention may be for use in a method of treating inflammation or in conditions caused by inflammation.

[0107] The compound, agent or composition of the invention may modulate levels of the proinflammatory cytokine TNF-a, such as levels of this cytokine in the plasma. For example, the compound, agent or composition of the invention may reduce levels of TNF-a, or lessen an increase in TNF-a. TNF-a may be associated with polyp progression to cancer. The compound, agent or composition of the invention may reduce pro-inflammatory signals through pathways involving NF-KB and / or IKK. The compound, agent or composition of the invention may reduce inflammation markers CXCL2 and / or TRAF5.

[0108] Conditions caused by inflammation may include conditions of the gastrointestinal tract, such as chronic gut inflammation or inflammatory bowel disease (including Crohn’s disease or colitis, such as ulcerative colitis). Inflammation may also leads to cancer. For example, chronic gut inflammation may be associated with driving the development and progression of polyps to colorectal cancer.

[0109] The invention may thus provide a method of treatment of inflammation, or a condition caused by inflammation, in a subject in need thereof, the method comprising administering an effective amount of the compound, agent or composition of the invention to the subject, thereby to treat the inflammation, or conditions caused by inflammation.

[0110] The invention may thus provide use of the compound, agent or composition of the invention in the manufacture of a medicament for treatment of inflammation, or a condition caused by inflammation.

[0111] The compound, agent or composition of the invention may have antioxidative properties, and so may be for use as an antioxidant, in treating oxidative stress, or in a condition caused by oxidative stress.

[0112] For example, the compound, agent or composition of the invention may activate an oxidative stress response pathway. The compound or composition of the invention may upregulate one or more of the genes HMOX1 , KEAP1 , NQO1 , AKR1 C2, AKR1 C1 , AKR1 C3, ABHD3 and OAS1 . The compound or composition of the invention may downregulate one or more of the genes STMN1 , COLGALT2, and LARGE2.

[0113] The compound, agent or composition of the invention may enhance the anti-oxidant response via KEAP1 and / or NRF2 activation, or increased expression of KEAP1 and / or NRF2. The compound, agent or composition of the invention may downregulate the expression of genes involved in inflammation and / or may induce differentiation genes in the NF-kB / IKK pathway. The response may be as illustrated in Fig. 32.

[0114] Reactive oxygen species may induce pro-inflammatory signals through pathways involving NF- KB and IKK, so the compound, agent or composition of the invention may counteract reactive oxygen species-induced inflammation.

[0115] The invention may thus provide a method of treatment of oxidative stress, or a condition caused by oxidative stress, in a subject in need thereof, the method comprising administering an effective amount of the compound, agent or composition of the invention to the subject, thereby to treat the oxidative stress, or the condition caused by oxidative stress.

[0116] The invention may thus provide use of the compound, agent or composition of the invention in the manufacture of a medicament for treatment of oxidative stress, or conditions caused by oxidative stress.

[0117] The compound, agent or composition of the invention may be for use in treating damaged, defective or dysfunctional tissue. This may include tissue that has become damaged, defective or dysfunctional by disease (including genetic disease), infection, medication, or physical trauma. The compound, agent or composition of the invention may thus be used for tissue regeneration. For example, it may be used for epithelial regeneration.

[0118] The invention may thus provide a method of treatment of damaged, defective or dysfunctional tissue, in a subject in need thereof, the method comprising administering an effective amount of the compound, agent or composition of the invention to the subject, thereby to treat the damaged, defective or dysfunctional tissue.

[0119] The invention may thus provide use of the compound, agent or composition of the invention in the manufacture of a medicament for treatment of damaged, defective or dysfunctional tissue.

[0120] Treatment of damaged, defective or dysfunctional tissue may be achieved, for example, by promoting differentiation of stem cells and / or progenitor cells into healthy cells. The damaged, defective or dysfunctional tissue could be, for example, gastrointestinal tissue. If the tissue is gastrointestinal tissue, the compound, agent or composition of the invention may be used to treat, for example, chronic gut inflammation, inflammatory bowel disease or colorectal cancer. Inflammatory bowel disease may include ulcerative colitis or Crohn’s disease. Ulcerative colitis may only affect the colon (large intestine), whereas Crohn's disease may affect any part of the digestive system, from the mouth to the anus.

[0121] The damaged, defective or dysfunctional tissue may be gastrointestinal mucosa. For example, the tissue may include intestinal crypts. The tissue may comprise defective crypt stem cells.

[0122] The compound, agent or composition of the invention may treat dysplasia. The damaged, defective or dysfunctional tissue may comprise abnormal cell growth such as a polyp, tumour or cyst. The polyps may be present in the gastrointestinal tract, such as the mouth, oesophagus, stomach, small intestine, duodenum, jejunum, ileum, large intestine, rectum, or anal canal or may be present elsewhere in the body.

[0123] Compounds, agents or compositions of the invention may thus provide multiple therapeutic actions. For example, they may promote the removal of damaged or defective tissue (for example by necrosis), such as promoting necrosis of precancerous polyps, whilst healing and restoring normal tissue function, such as restoring gut mucosal function.

[0124] In the context of promoting restoration of gut function, compounds, agents or compositions of the invention may promote production of Paneth cells, Tuft cells and / or enterocytes. Compounds, agents or compositions of the invention may downregulate gene expression of the essential stem cell self-renewal and maintenance genes, for example in crypt base columnar (CBC) stem cells.

[0125] Compounds, agents or compositions of the invention may assist in healing the mucosal layer in the gut. The compounds or compositions may thus treat a hyperpermeable (or “leaky”) gut. Compounds, agents or compositions of the invention may thus treat diseases or disorders associated with a hyperpermeable gut, or diseases or disorder where a hyperpermeable gut may lead to disease initiation and progression. Such diseases may include FAP, IBD, irritable bowel syndrome (IBS), metabolic diseases (such as Type 2 diabetes, obesity, non-alcoholic fatty liver disease), Parkinson’s disease, Neurological and psychiatric disorders (such as autism, depression, Alzheimer’s disease), Cardiovascular diseases (such as Atherosclerosis, hypertension), skin disorders (such as psoriasis, acne), chronic fatigue syndrome, fibromyalgia and bacterial infections.

[0126] Compounds, agents or compositions of the invention may treat damaged, defective or dysfunctional tissue throughout the body. This may include treatment of damaged, defective or dysfunctional endodermal, mesodermal or ectodermal tissue. For example, compounds, agents or compositions of the invention may promote growth and differentiation of such tissue. This may be achieved, for example, by promoting differentiation of stem cells, such as pluripotent stem cells, multipotent stem cells and / or progenitor cells.

[0127] WO2024 / 170911 A1 , WO2024 / 170912A1 and WO2024 / 170913A1 disclose stem cells in the anorectal transition zone (ATZ) which are able to produce mature cells in endodermal, mesodermal and ectodermal developmental lineages. Surprisingly, it has been found that compounds, agents or compositions of the invention can promote or enhance growth and differentiation of stem cells, such as ATZ stem cells, into various tissue cells.

[0128] Endodermal tissue includes intestine, pancreas, liver and lung, pharynx, tonsils, thyroid, parathyroid, thymus, larynx, trachea, gastrointestinal tract, urinary bladder, vagina and urethra.

[0129] For example, compounds, agents or compositions of the invention may treat lung diseases. Lung diseases include asthma, chronic obstructive pulmonary disease (COPD), infections (e.g., influenza, pneumonia, and tuberculosis), occupational lung diseases, lung cancer, Idiopathic Pulmonary Fibrosis (IPF), Acute Respiratory Distress Syndrome (ARDS), Cystic Fibrosis (CF), Pulmonary Hypertension, Bronchopulmonary Dysplasia (BPD), Emphysema, Interstitial Lung Diseases (ILDs), Lung Transplantation or Acute Lung Injury (ALI). For example, compounds, agents or compositions of the invention may be used to treat liver diseases. Liver diseases may include chronic liver disease. Chronic liver disease includes alcoholic liver disease, non-alcoholic steatohepatitis (NASH), or hepatitis C infection. These diseases may be associated with cirrhosis of the liver, a form of irreversible fibrotic scarring.

[0130] Examples of mesodermal tissue include kidney, heart, muscle, cartilage, connective tissue, bone, blood, bone marrow, lymphoid tissue, epithelia of blood vessels, lymphatic vessels, body cavities, kidneys, ureters, gonads, genital ducts, and adrenal cortex.

[0131] Compounds, agents or compositions of the invention may be used to treat acute cardiac disease, chronic cardiac disease, chronic muscle diseases, chondrocyte diseases or chronic kidney disease.

[0132] Acute cardiac disease includes myocardial infarctions and acute heart failure. Chronic cardiac disease includes coronary artery disease and congestive heart failure. Chronic muscle diseases includes several skeletal muscle diseases including muscular dystrophy, myasthenia gravis, polymyositis. Chondrocyte diseases includes osteoarthritis, rheumatoid arthritis, and chondrosarcoma. Chronic kidney disease includes diabetic nephropathy, polycystic kidney disease, and Alport syndrome.

[0133] Examples of ectodermal tissue include nervous tissue (central and peripheral), skin, lens or corneal epithelium of the eye, or the epithelia of: nasal cavity; sinuses; mouth (including tooth enamel); and the anal canal.

[0134] Compounds, agents or compositions of the invention may be used to treat traumatic brain injury (TBI), stroke, seizures, Alzheimer’s disease, multiple sclerosis, Parkinson’s disease, spinal cord injuries or skin wounds.

[0135] Traumatic brain injury (TBI) refers to an injury to the brain caused by external trauma to the head (e.g., physical impact, whiplash) or an internal trauma (e.g., stroke, hematoma, aneurysm, oxygen deprivation).

[0136] Stroke refers to a medical emergency that occurs when blood flow to the brain is disrupted, causing brain damage. Damage caused by a stroke can lead to the brain becoming impaired.

[0137] Seizures refers to a medical emergency sudden and abnormal electrical activity in the brain.

[0138] Alzheimer's disease refers to a progressive brain disorder that affects memory, thinking, and behaviour.

[0139] Multiple sclerosis refers to an autoimmune disease that affects the nervous system and causes damage to the protective covering of nerve fibres. Parkinson's disease refers to a neurodegenerative disorder that primarily affects movement, characterized by symptoms such as tremors, stiffness, and difficulty with balance and coordination.

[0140] Spinal cord injuries refers to often permanent changes in strength, sensation, and other body functions below the site of injury.

[0141] Skin wounds includes acute and chronic wounds caused by physical damage and diseases such as diabetes.

[0142] A compound agent, or composition of the invention may provide a comprehensive antiinflammatory effect, and a pan-PPAR modulatory effect may suppress inflammation more than agents which modulate only one or two PPAR isotypes.

[0143] A compound, agent or composition of the invention may have anti-fibrotic action, which reduces fibrosis by modulating multiple signalling pathways.

[0144] A compound, agent or composition if the invention may normalise metabolic dysfunctions linked to polyp and cancer risk.

[0145] A compound, agent or composition of the invention may decrease the number and size of polyps, helping prevent cancer in various tissues.

[0146] A compound, agent or composition of the invention may provide balanced efficacy with potentially fewer side effects than agonists which modulate only one or two PPAR isotypes.

[0147] A compound, agent or composition of the invention may redirect cell production from precancerous cells, such as cystic cells, to healthy cells. This may occur in the gastrointestinal tract.

[0148] A compound, agent or composition of the invention may promote cell cycle arrest or blockade, and may reduce stem cell proliferation. For example, it may reduce proliferation of CBC cells and increase differentiation of enterocytes.

[0149] PPARs are expressed in a wide range of tissues, and so compounds or agents of the invention may be expect to act in a similarly wide range of tissues, as indicated herein.

[0150] Downstream activation of PPAR pathways by compounds, agents or compositions of the invention may includes upregulation of lipid metabolic and membrane-stabilising genes (e.g., FABP1 , FABP2, SCP2), mitochondrial p-oxidation genes (e.g., CPT1A, ACAA1 , ACOX1 ) that sustain energy balance, and antioxidant and stress-response genes (e.g., ANGPTL4, HMGCS2). Collectively, these PPAR-regulated mechanisms may account for the observed protective effect by maintaining membrane integrity, reducing oxidative stress, and preventing anoikis in the post-thaw recovery phase.

[0151] Compounds, agents or compositions of the invention may be co-administered with cells, such as stem cells (e.g. isolated stem cells), to treat damaged, defective or dysfunctional tissue. Coadministration may be concurrent or sequential. For example, stem cells may be administered to the damaged, defective or dysfunctional tissue first, followed by administration of the compound or composition of the invention, or vice versa.

[0152] According to the invention, there is provided a composition (e.g. a cellular composition) or combined preparation including a compound or agent of the invention, and cells. The cells may include ATZ cells (e.g. isolated ATZ cells). The cells may include stem cells such as pluripotent stem cells and / or or multipotent stem cells, and / or progenitor cells.

[0153] As used herein, the term "cellular composition" refers to a preparation of cells, which preparation may include, in addition to the cells, non-cellular components such as cell culture media, e.g., proteins, amino acids, nucleic acids, nucleotides, co-enzyme, antioxidants, metals and the like. Furthermore, the cellular composition can have components which do not affect the growth or viability of the cellular component, but which are used to provide the cells in a particular format, e.g., as polymeric matrix for encapsulation or a pharmaceutical preparation. Cellular compositions may also include buffers, salts (such as calcium chloride, potassium chloride, sodium chloride, sodium lactate), polymers and preservatives. Cellular compositions may include albumin, such as human serum albumin.

[0154] Cells may be administered with an exogenous biocompatible scaffold. As used herein, the term “exogenous biocompatible scaffolds” refers to three-dimensional porous, fibrous, or permeable volume-retaining biomaterials intended to provide physical or mechanical support while permitting diffusion or transport of body liquids and gases, allowing for cellular interactions. Preferably, such scaffolds cause limited or minimal inflammation and toxicity. Optionally, such scaffolds are biodegradable. Examples of scaffolds can include a biological scaffold (e.g., a laminin or collagen-based scaffold) and synthetic scaffolds (e.g., non-biological, or synthetic, polymers such as PGA, PLA, PLGA). Scaffolds may be in the physical form of a thread, sheet, paste, powder, or liquid. Scaffolds can be used in vitro and in vivo.

[0155] As used herein, the term “anorectal transition zone" or “ATZ” refers to the intestinal epithelium interposed between the uninterrupted squamous epithelium of the anoderm and dentate (or pectinate) line below and the uninterrupted rectal columnar epithelium above. The dentate line is the junction between the superior and inferior anal canal. There are many differences between these 2 regions, including their embryological origins, innervation, venous and arterial supply, and lymphatic supply. Above the dentate line, the epithelium of the anal canal has an endodermal origin and is lined by simple columnar epithelia. Below the dentate line, the epithelium of the anal canal has an ectodermal origin and is predominantly lined by stratified squamous epithelium. The epithelium of the ATZ is typically 1 -4 mm wide and can easily be identified and biopsied by those skilled in the art.

[0156] As used herein, the term “anorectal transition zone cells" or “ATZ cells” refers to a mixed population of cells derived from anorectal transition zone epithelial tissue comprising crypts, submucosal glands, and other epithelial cells.

[0157] As used herein, the term “isolated ATZ cells” refers to a mixed population of cells physically isolated (e.g., by biopsy sample) from the ATZ epithelium. Optionally, the ATZ tissue can be physically minced or enzymatically digested to isolate ATZ cells.

[0158] The cells may include crypt cells. As used herein, the term “crypt cells” refers to the cells of the crypts of Lieberkuhn, structures below the surface of the intestinal mucosal lining, and comprising stem cells that are responsible for continuously regenerating intestinal mucosa throughout life.

[0159] The cells may include ATZ crypt cells. “ATZ crypt cells” refers to the crypt cells of the ATZ, which include multipotent stem cells (i.e. , cells that have the capacity to self-renew by dividing and to develop into multiple specialised cell types present in a specific tissue or organ), progenitor cells, and mature crypt cells. Multipotent ATZ cells are stem cells that have the capacity to differentiate into multiple somatic cell lineages including endodermal cells (e.g., intestinal mucosa), mesodermal cells (e.g., blood vessels), and / or ectodermal cells (e.g., skin).

[0160] As used herein, the term “isolated ATZ crypt cells” refers to a mixed population of cells (e.g., multipotent stem cells, progenitor cells, fully differentiated or mature cells) forming the crypts that have been physically isolated (e.g., by dissection) from the ATZ epithelium. Alternatively, or in addition, the ATZ crypts can be physically, chemically, or enzymatically dissociated to further isolate the ATZ crypt cells.

[0161] As used herein, the term “stem cells” refers to undifferentiated or partially differentiated cells that can differentiate into various types of cells and proliferate indefinitely to produce more of the same stem cell (i.e., self-renewing). As used herein, “self-renewal” is the process by which stem cells divide to make more stem cells, perpetuating the stem cell pool throughout life. Selfrenewal is division with maintenance of the undifferentiated state. This requires cell cycle control and often maintenance of multipotency or pluripotency, depending on the stem cell.

[0162] As used herein, the term “progenitor cells” refers to stem cells with the potential to differentiate into a single cell type or lineage, and the term “progenitor ATZ cells” means progenitor cells derived from the ATZ crypt cells. As used herein, the term “multipotent cells” refers to stem cells with the potential to differentiate into at least 2 cell types or lineages, and the term “multipotent ATZ cells” means multipotent cells derived from the ATZ crypt cells.

[0163] As used herein, the term “pluripotent cells” refers to stem cells with the potential to differentiate into each of the three primary groups of cells, i.e. , ectoderm, mesoderm and endoderm, and the term “pluripotent ATZ cells” means pluripotent cells derived from the ATZ crypt cells.

[0164] As used herein, the terms "proliferation" and “proliferating” refer to an increase in cell number by mitosis.

[0165] The stem cells (e.g. ATZ stem cells) in compositions of the invention may be allogenic or autologous to the subject.

[0166] The stem cells (e.g. ATZ stem cells) in compositions of the invention may not express significant levels of CD45.

[0167] The stem cells (e.g. ATZ stem cells) in compositions of the invention may express markers CD34, CD117 and CD184.

[0168] The stem cells (e.g. ATZ stem cells) in compositions of the invention may express markers NANOG and OCT4A.

[0169] As used herein, the term "differentiation" refers to the formation of cells expressing markers known to be associated with cells that are more specialised and closer to becoming terminally differentiated cells incapable of further division or differentiation. For example, in a haematological context, differentiation can be seen in the production of functional cells of multiple cellular lineages (e.g., red blood cells, platelets, granulocytes, macrophages). The terms "further" or "greater" differentiation refers to cells that are more specialised and closer to becoming terminally differentiated cells incapable of further division or differentiation than the cells from which they were cultured.

[0170] The stem cells according to the invention may be expanded to create an expanded population of stem cells that can be generated in suspension or adherent 2D cultures (e.g., individual cells) (e.g., clusters) or 3D cultures consisting of complex structures (e.g., organoids). The expanded population of cells may retain the ability to differentiate into one or more cell types.

[0171] Stem cells may be expanded to produce expanded stem cells. If ATZ stem cells are expanded, that may be referred to as “eATZ stem cells”. The stem cells (e.g. eATZ stem cells) may retain at least one marker of stem cells selected from the group consisting of CD34, CD117 (KIT), CD184 (CXCR4), OCT4, NANOG, SOX17, Brachyury (TBXT), PAX6 and NESTIN. Compounds, agents or compositions of the invention may be used to promote growth, differentiation and / or development of cells in vitro or ex vivo. For example, compounds, agents or compositions of the invention may be used to promote growth, differentiation and / or development of an organoid.

[0172] Compounds, agents or compositions of the invention may assist in the rescue of cryopreserved cells and organoids, enabling survival under conditions where cryodamage may typically induce anoikis and apoptosis. These effects may provide protection against the consequences of cryoinjury, including e.g. membrane and organelle disruption from ice crystal formation, osmotic shock, and ROS, thereby supporting viability during recovery after cryopreservation.

[0173] Compounds, agents or compositions of the invention may prevent or reduce anoikis or apoptosis in cells in vitro or ex vivo.

[0174] According to the invention, there is provided a cell or tissue culture medium comprising a compound or agent of the invention. The cell or tissue culture medium may be sterile. The cell or tissue culture medium may contain one or other factors, such as a growth factor or an inhibitor. For instance, the culture medium may contain a ROCK inhibitor, such as Y27 (Y- 27632). The culture medium may contain amino acids, vitamins, inorganic salts, and / or a carbon source, such as glucose

[0175] As used herein, the term “organoid” refers to a multiplicity of cells grown in culture which selforganize to form a three-dimensional structure. Organoid structures may be a simple solid mass of cells, a hollow mass (e.g., a cystic organoid), a tube, or a more complicated structure (e.g., a crypt-like or follicle-like structure). Organoids may then be transplanted into a subject, for example to treat damaged, defective or dysfunctional tissue. Organoids may include, for example, intestinal organoids, pancreatic organoids, hepatic organoids or lung organoids.

[0176] Stem cells (e.g. ATZ stem cells) may be differentiated in a lineage-specific medium (LSM), a cell culture medium which provides the nutrients and growth factors necessary for stem cells (e.g. pluripotent stem cells and / or multipotent stem cells) to undergo differentiation toward specific-lineage cells, and to result in a decreased absolute number of pluripotent and / or multipotent stem cells. The lineage-specific differentiation may be partial (e.g., toward endoderm, mesoderm or ectoderm progenitors) or terminal (e.g., toward goblet cells, cardiac muscle, neurons). The differentiation in the LSM may generate a variety of organoids, depending on the nature of the LSM.

[0177] An endodermal lineage-specific medium (LSM) can be purchased from commercial vendors and can include: intestinal (e.g., IntestiCultTM, Human Intestinal Protocols, Bio-Techne); pancreas (e.g. PancreaCultTM); liver (HepatiCultTM); lung (e.g., STEMdiff™ Branching Lung Organoid Kit, or PneumaCult™ Airway Organoid Kit, or PneumaCult™ Apical-Out Airway Organoid Medium, or PneumaCult™ Alveolar Organoid Medium, all from STEMCELL Technologies, Inc; Human Lung Organoid Culture Protocols, Bio-Techne; 3dGRO™ Lung Organoid Branching Medium3dGRO™and Lung Organoid Maturation Medium, Merck).

[0178] A mesodermal lineage-specific medium (LSM) can be purchased from commercial vendors (e.g., StemDiff KidneyTM Organoid Kit, STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit, MyoCultTM, DiffMesenCult™-ACF Chondrogenic Differentiation Kit, Vancouver, CA).

[0179] An ectodermal lineage- specific medium (LSM) can be purchased from commercial vendors (e.g., STEMdiff™ Cerebral Organoid Kit, STEMCELL Technologies, Vancouver, CA; NeurobasalTM-A Medium and Gibco™Keratinocyte-SFM Medium, both from Thermo Fisher Scientific, Waltham, Massachusetts; DermaCultTM Keratinocyte Expansion Medium, STEMCELL Technologies).

[0180] According to the invention, there is provided a method of producing an organoid comprising contacting stem cells (e.g. ATZ stem cells) with a growth medium, the growth medium comprising a compound or agent of the invention. For example, the growth medium may be a LSM comprising a compound of the invention. The method may be carried out in vitro or ex vivo. The stem cells may be isolated.

[0181] According to the invention, there is provided a composition comprising one or more growth factors and a compound or agent of the invention. The composition may be a LSM comprising a compound of the invention. The composition may also include one or more stem cells, such that the composition may be a cell culture.

[0182] According to the invention, there is provided a method of treating damaged, defective or dysfunctional tissue in a subject, comprising: a) obtaining a sample of stem cells (e.g. ATZ stem cells) that are autologous or allogeneic to the subject; b) expanding the stem cells, including contacting the stem cells with a compound, agent or composition of the invention; and c) introducing a therapeutically effective amount of the stem cells into the damaged, defective or dysfunctional tissue in the subject.

[0183] According to the invention, there is provided a method of treating damaged, defective or dysfunctional tissue in a subject, comprising: a) obtaining a sample of stem cells (e.g. ATZ stem cells) that are autologous or allogeneic to the subject; b) expanding the stem cells, including contacting the stem cells with a compound, agent or composition of the invention, to promote at least partial differentiation of the stem cells to produce an expanded population of ectodermal, endodermal or mesodermal progenitor cells; and c) introducing a therapeutically effective amount of the stem cells and ectodermal, endodermal or mesodermal progenitor cells into the damaged, defective or dysfunctional tissue in the subject.

[0184] According to the invention, there is provided a method of synthesising a compound of the invention. The method may comprise the following synthesis step, which may be termed ‘step 1 ’, to form a compound of formula int-1 :

[0185] This step may thus comprise a 1 ,4 conjugate addition. According to the invention, there is provided a composition comprising the compound of formula int-1 . The composition may be used in a method of synthesising a compound of Formula (I).

[0186] The method may comprise the following synthesis step, which may be termed ‘step 2’, to form a compound of formula int-2: This step may thus comprise ketal formation.

[0187] According to the invention, there is provided a composition comprising the compound of formula int-2. The composition may be used in a method of synthesising a compound of Formula (I).

[0188] The method may comprise the following synthesis step, which may be termed ‘step 3’, to form a compound of formula int-3:

[0189] This step may thus comprise amide formation, such as Weinreb amide formation.

[0190] According to the invention, there is provided a composition comprising the compound of formula int-3. The composition may be used in a method of synthesising a compound of Formula (I). The method may comprise the following synthesis step, which may be termed ‘step 4’, to form a compound of formula int-4: int-3 int-4

[0191] This step may thus comprise allylation.

[0192] According to the invention, there is provided a composition comprising the compound of formula int-4. The composition may be used in a method of synthesising a compound of Formula (I).

[0193] The method may comprise the following synthesis step, which may be termed ‘step 5’, to form a compound of formula int-5: int-4 int-5

[0194] This step may thus comprise a reduction, such as a Luche reduction.

[0195] According to the invention, there is provided a composition comprising the compound of formula int-5. The composition may be used in a method of synthesising a compound of Formula (I).

[0196] The method may comprise the following synthesis step, which may be termed ‘step 6’, to form a compound of formula int-6: int-5 int-6

[0197] This step may thus comprise a ketal deprotection. According to the invention, there is provided a composition comprising the compound of formula int-6. The composition may be used in a method of synthesising a compound of Formula (I).

[0198] The method may comprise the following synthesis step, which may be termed ‘step 7’, to form a compound of formula int-7:

[0199] This step may thus comprise a lactone annulation.

[0200] According to the invention, there is provided a composition comprising the compound of formula int-7. The composition may be used in a method of synthesising a compound of Formula (I).

[0201] The method may comprise the following synthesis step, which may be termed ‘step 8’, to form a compound of formula int-8: int-7 int-8

[0202] This step may thus comprise a ring-closing metathesis.

[0203] According to the invention, there is provided a composition comprising the compound of formula int-8. The composition may be used in a method of synthesising a compound of Formula (I). The method may comprise the following synthesis step, which may be termed ‘step 9’, to form a compound of formula int-9: int-9

[0204] This step may thus comprise an oxidation, such as an alcohol oxidation.

[0205] According to the invention, there is provided a composition comprising the compound of formula int-9. The composition may be used in a method of synthesising a compound of Formula (I). The method may comprise the following synthesis step, which may be termed ‘step 10’, to form a compound of formula int-10: int-9 int-10

[0206] This step may thus comprise an epoxydation.

[0207] According to the invention, there is provided a composition comprising the compound of formula int-10. The composition may be used in a method of synthesising a compound of Formula (I).

[0208] The method may comprise the following synthesis step, which may be termed ‘step 11 ’, to form a compound of formula int-11 : int-11 (Linderolide H)

[0209] This step may thus comprise an epoxide elimination. According to the invention, there is provided a composition comprising the compound of formula int-11 , or Linderolide H. The composition may be used in a method of synthesising a compound of Formula (I). The method may comprise the following synthesis step, which may be termed ‘step 12’, to form a compound of formula int-12: int-11 (Linderolide H)

[0210] This step may thus comprise an alcohol protection..

[0211] According to the invention, there is provided a composition comprising the compound of formula int-12. The composition may be used in a method of synthesising a compound of Formula (I).

[0212] The method may comprise the following synthesis step, which may be termed ‘step 13’, to form a compound of formula int-13: int-12 int-13

[0213] This step may thus comprise olefination, such as a Wittig olefination.

[0214] According to the invention, there is provided a composition comprising the compound of formula int-13. The composition may be used in a method of synthesising a compound of Formula (I).

[0215] The method may comprise the following synthesis step, which may be termed ‘step 14’, to form a compound of formula int-14:

[0216] This step may thus comprise hydroxylation. According to the invention, there is provided a composition comprising the compound of formula int-13. The composition may be used in a method of synthesising a compound of Formula (I).

[0217] The method may comprise the following synthesis step, which may be termed ‘step 15’, to form a compound of the invention. int-14

[0218] This step may thus comprise alcohol deprotection.

[0219] Methods for synthesising compounds of the invention may include one or more of steps 1 to 15, depending on the starting compound.

[0220] Examples of the present invention may be provided in the following numbered clauses. 1 . A compound of Formula (I) or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof:

[0221] 2. The compound of clause 1 , wherein Formula (I) is in the form of Formula (la):

[0222] (la)

[0223] 3. An isolated compound of formula (la):

[0224] (la)

[0225] 4. An agent which is an agonist of PPAR-a, PPAR-p / 5 and PPAR-y, optionally wherein the agent is a compound as defined in any of clauses 1 to 3.

[0226] 5. A pharmaceutical composition comprising the compound or agent of any preceding clause, and a pharmaceutically acceptable excipient.

[0227] 6. A composition comprising the compound or agent of any of clauses 1 to 4, in an amount of at least 0.5% by weight of the composition. 7. The composition according to clause 6, wherein the compound or agent is present in the composition in an amount of at least 1%, at least 2% at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, or at least 50%, by weight.

[0228] 8. A composition that agonises PPAR-a, PPAR-p / 5 and PPAR-y, optionally wherein the composition comprises a plurality of distinct PPAR agonists which, in combination, agonise PPAR-a, PPAR-p / 5 and PPAR-y.9. The composition according to any of clauses 6 to 8, comprising a pharmaceutically acceptable excipient.

[0229] 10. The composition according to any of clauses 5 to 9, which is sterile.

[0230] 11 . The composition according to any of clauses 5 to 10, in the form of a solution, suspension, powder, tablet, capsule, lozenge, suppository, buccal product, cream, ointment, gel, film or patch. 12. The composition according to any of clauses 5 to 11 , which is substantially free of flavonoids, alkaloids, essential oils, phenolics and / or tannins.

[0231] 13. The composition according to any of clauses 5 to 12, which, aside from the compound Formula (I) or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof, comprises substantially no other lactone or comprises substantially no other sesquiterpene lactone.

[0232] 14. The composition according to any of clauses 5 to 13, comprising cell growth factors.

[0233] 15. The composition according to clause 14, comprising a lineage-specific medium (LSM).

[0234] 16. The composition according to any of clauses 5 to 15, comprising isolated cells.

[0235] 17. The composition according to clause 16, wherein the cells comprise stem cells.

[0236] 18. The composition according to clause 16 or clause 17, comprising pluripotent stem cells, multipotent stem cells and / or progenitor cells.

[0237] 19. The composition according to any of clauses 16 to 18, wherein the cells are anorectal transition zone (ATZ) cells.

[0238] 20. The composition according to any of clauses 16 to 19, comprising an exogenous biocompatible scaffold.

[0239] 21 . A combined preparation that agonises PPAR-a, PPAR-p / 5 and PPAR-y, comprising at least a first PPAR agonist which is in separate composition to a second, distinct PPAR agonist.

[0240] 22. The compound, agent, composition or preparation of any preceding clause, for use as a medicament.

[0241] 23. The compound, agent, composition or preparation for the use of clause 22, for oral, subcutaneous, intradermal, intravenous, intra-arterial, intramuscular, intrathecal, epidural, intracistemal, intraperitoneal, transdermal, topical, transmucosal, buccal, sublingual, transmucosal, inhalation, intranasal, intra-atricular, intranasal, rectal or ocular administration.

[0242] 24. The compound, agent, composition or preparation for the use of clause 22 or clause 23, for use in treating cancer.

[0243] 25. The compound, agent, composition or preparation for the use of clause 24, wherein the cancer is colorectal cancer. 26. The compound, agent, composition or preparation for the use of clause 24 or clause 25, wherein the cancer is characterised by at least one mutation in the adenomatous polyposis coli (APC) gene.

[0244] 27. The compound, agent, composition or preparation for the use according to clause 22 or clause 23, for use in treating Familial adenomatous polyposis (FAP).

[0245] 28. The compound, agent, composition for the use according to clause 22 or clause 23, for use in treating inflammation.

[0246] 29. The compound, agent, composition or preparation for the use according to clause 22 or clause 23, for use in treating oxidative stress.

[0247] 30. The compound, agent, composition or preparation for the use according to clause 22 or clause 23, for use in treating damaged, defective or dysfunctional tissue.

[0248] 31 . The compound, agent, composition or preparation for the use according to clause 30, wherein the damaged, defective or dysfunctional tissue is endodermal tissue.

[0249] 32. The compound, agent, composition or preparation for the use according to clause 30, wherein the damaged, defective or dysfunctional tissue is mesodermal tissue.

[0250] 33. The compound, agent, composition or preparation for the use according to clause 30, wherein the damaged, defective or dysfunctional tissue is ectodermal tissue.

[0251] 34. The compound, agent, composition or preparation for the use according to clause 22 or clause 23, for use in treating a disease or disorder of the gastrointestinal tract.

[0252] 35. The compound, agent, composition or preparation for the use of clause 34, wherein the disease or disorder is cancer, Crohn’s disease, chronic gut inflammation or inflammatory bowel disease.

[0253] 36. The compound, agent, composition or preparation for the use of any of clauses 22 to 35, for use in treating human subjects.

[0254] 37. The compound, agent or composition for the use of any of clauses 22 to 35, for use in treating non-human subjects, such as animal or mammalian subjects.

[0255] 38. The compound, agent, composition or preparation of any of clauses 1 to 21 , for use in a method of treating damaged, defective or dysfunctional tissue in a subject, comprising: a) obtaining a sample of stem cells that are autologous or allogeneic to the subject; b) expanding the stem cells, including contacting the stem cells with the compound, agent, composition or preaparation; and c) introducing a therapeutically effective amount of the stem cells into the damaged, defective or dysfunctional tissue in the subject.

[0256] 39. The compound, agent, composition or preparation of any of clauses 1 to 21 , for use in a method of treating damaged, defective or dysfunctional tissue in a subject, comprising: a) obtaining a sample of stem cells that are autologous or allogeneic to the subject; b) expanding the stem cells, including contacting the stem cells with the compound, agent, composition or preparation to promote at least partial differentiation of the stem cells to produce an expanded population of ectodermal, endodermal or mesodermal progenitor cells; and c) introducing a therapeutically effective amount of the stem cells and ectodermal, endodermal or mesodermal progenitor cells into the damaged, defective or dysfunctional tissue in the subject.

[0257] 40. The compound for the use according to clause 38 or clause 39, wherein the stem cells are ATZ stem cells.

[0258] 41 . A method comprising contacting a cell with a compound, agent, composition or preparation as defined in any of clauses 1 -21 , in vitro or ex vivo.

[0259] 42. The method of clause 41 in which the cell is a stem cell, optionally an ATZ stem cell.

[0260] 43. A method for producing an organoid, comprising contacting isolated stem cells with a growth medium, the growth medium comprising the compound, agent, composition or preparation of any of clauses 1 to 21 .

[0261] 44. A method of synthesising a compound according to any of clauses 1 to 3.

[0262] 45. The method of clause 44, comprising the following step:

[0263] 46. The method of clause 44 or clause 45, comprising the following step: 47. The method of any of clauses 44 to 46, comprising the following step:

[0264] 48. The method of any of clauses 44 to 47, comprising the following step: 49. The method of any of clauses 44 to 48, comprising the following step:

[0265] 50. The method of any of clauses 44 to 49, comprising the following step:

[0266] 51 . The method of any of clauses 44 to 50, comprising the following step: int-6 int-7 The method of any of clauses 44 to 51 , comprising the following step: int-7 int-8 The method of any of clauses 44 to 52, comprising the following step: int-9 The method of any of clauses 44 to 53, comprising the following step: int-9 int-10 The method of any of clauses 44 to 54, comprising the following step: int-11 (Linderolide H) 6. The method of any of clauses 44 to 55, comprising the following step: int-11 (Linderolide H) 7. The method of any of clauses 44 to 56, comprising the following step: int-12 int-13 8. The method of any of clauses 44 to 57, comprising the following step: int-13 int-14 9. The method of any of clauses 44 to 58, comprising the following step: SPECIFIC EXAMPLES

[0267] Embodiments of the invention are now described, by way of example only, with reference to the accompanying drawings.

[0268] FIG. 1 shows crypts and crypt-derived organoids: (a) illustrates the anatomical structure of the intestinal crypt, and (b) shows a graphic and example of a crypt-derived organoid, highlighting in the insert the replication of key structural and functional aspects of actual organ tissues. FIG. 1 (c) shows bright field images of the isolation of healthy porcine crypts throughout the gastrointestinal tract (Gl) from the oesophagus to the rectum in the upper panel and the establishment of crypt organoids in the lower panel.

[0269] FIG. 2 shows brightfield images of 4 types of morphologies of FAP patient crypt-derived organoids: (a) ring structures for developing organoids (Morphology I) ; (b) normal organoids with branched structures (Morphology II); (c) cystic organoid structures (Morphology III); and (d) poorly differentiated branched organoids (Morphology IV). FIG. 2(e) shows a graphic summary comparing compositions of different organoid morphologies among FAP patients and a non- FAP healthy individual.

[0270] FIG. 3 shows protein expression by immunocytochemistry on FAP patient (FAP115) duodenal cryp organoids: (a) lysozyme, (b) mucin, (c) villin (magnification, 40X). Arrows identify protein expression.

[0271] FIG. 4 shows the distribution of pathogenic mutations in individual crypt-derived organoids (n=43) established from 3 FAP patients (FAP115, FAP134, and FAP125) and 1 non-FAP healthy individual (HP115). The top bar plot indicates the tumour mutational burden (TMB) for each sample. The grayscale squares within the oncoplot represent various mutation types: splice site mutations (light grey); missense mutations (medium grey); nonsense mutations (dark grey); in frame insertions (very light grey); frameshift deletions and frameshift insertions (dark grey); multi-hit mutations (black). The percentages next to each gene indicate the mutation frequency across the samples.

[0272] FIG. 5 illustrates the initiation and progression of ORC development and the therapeutic potential of ULI262 to intervene early to intervene in disease progression: (a) shows the transition from a healthy state to cancer with mutations in APC, KRAS, and SMAD2 / 4 genes, and loss of p53, progressing through stages of mutation, polyp formation, and cancer; (b) illustrates ULI262 redirecting cell production in vitro of precancerous cystic APC+ / - crypt organoids to healthy, branched healthy crypt organoids; (c) illustrates the production of healthy intestinal mucosa after ULI262 treatment. FIG. 6 shows an example of bright field images of: (a) cystic, precancerous FAP patient crypt organoids; and (b) healthy branched crypt organoids after treatment with ULI262-A.

[0273] FIG. 7 illustrates a graphic summary of ULI262-A induced differentiation of FAP cystic, precancerous crypt organoids to healthy, branched crypt organoids in all 12 patients tested, regardless of the position of the mutation in the APC gene.

[0274] FIG. 8 shows bright field images of ULI262-A in vitro activity inducing precancerous, cystic crypt to healthy branched crypt organoids throughout the Gl tracts of (a) FAP patients and (b) ApcMin / + mice.

[0275] FIG. 9 shows a graphic summary of ULI262-A experiments on crypt organoids throughout the Gl tract of FAP patients and ApcMin / + mice in preparation to in vivo translation to mice.

[0276] FIG. 10 is an anatomical map illustrating the locations of adenomas within the gastrointestinal tract of the ApcMin / + mouse, primarily in the small intestine and more rare in the stomach, cecum, colon, and anus.

[0277] FIG. 11 shows body weight changes, as expressed as percentages of initial weights, in ApcMin / + mice during Study 1 while treated by oral gavage from weeks 9 to 16 for male mice with (a) vehicle control or (b) ULI262-A or female mice with (c) vehicle control or (d) ULI262-A.

[0278] FIG. 12 shows the total number of polyps in each ApcMin / + mouse in Study 1 when treated with vehicle control or ULI262-A for (a) males or (b) females.

[0279] FIG. 13 shows body weight changes, as expressed as percentages of initial weights, in ApcMin / + mice during Study 2: treatment by oral gavage from weeks 6 to 15 with ppULI262-B of (a) males or (b) females; treatment with ULI262-A of (c) males or (d) females; or treatment with vehicle control of (e) males or (f) females.

[0280] FIG. 14 shows the total number of polyps in each ApcMin / + mouse in Study 2 with no treatment, vehicle control, ULI262-A or ppULI262-B for (a) males and (b) females.

[0281] FIG. 15 shows body weight changes, as expressed as percentages of initial weights, in ApcMin / + mice during Study 3 while treated by oral gavage from weeks 9 to 16 for male mice: with (a) ppULI262-B; (c) ULI262-A; or (e) control, or female mice with: (b) ppULI262-B (d) ULI262-A; or (f) control.

[0282] FIG. 16 shows (a) small and (b) large polyps at the termination of Study 3: photographic examples of methylene blue stained polyps (upper panel) and graphs of the total number of polyps per mouse in the vehicle, ULI262-A and ppULI262-B treatment groups (lower panel). FIG. 17 illustrates the distribution and the macroscopic properties of polyps in untreated FAP pigs: (a) an anatomical map identifying the locations of adenomas (dark grey) and hyperplastic polyps (light grey) throughout the gastrointestinal tract of an FAP pig; (b) photographs of polyps in situ (left panel) and biopsied (right panel) for progressive polyps (solid lined square) and regressive polyps (dashed line square); (c) PAS-AB and Ki76 stained immunohistochemistry sections of normal pig intestinal mucosa, progressive polyps and regressive polyps.

[0283] FIG. 18 shows (a) bright field images of aberrant cystic crypt organoid cultures of crypts derived from rectal polyps of the 4 FAP pigs in the study before treatment (1 Ox magnification). Washout debris from the rectum in preparation for the endoscopy after 1 month shows (b) bloody stools identified by black arrows in the untreated male FAP (2714) and (c) sheets of dead tissue identified by black arrows in the ULI262-A treated male FAP pig (2705). FIG. 18(d) shows photographs of the biopsied rectal progressive polyp and an example of a regressive polyps taken at 1 month.

[0284] FIG. 19 shows a graphic summary of the proportions of cystic and branched crypt organoids derived from untreated (a) male and (c) female FAP pigs and after 1 month of daily ULI262-A treatment of (b) male and (d) female FAP pigs.

[0285] FIG. 20 shows washout debris from the rectum in preparation for the endoscopy after 3.5 months of the study: (a) bloody stools identified by a black arrow in the untreated male FAP (2714) and (b) large sheets of dead tissue identified by the black arrow in the ULI262-A treated male FAP pig (2705). FIG. 20(c) shows a graphical representation of polyps distributed in the colorectal region of the male FAP pig (2705) after 3.5 months of daily oral ULI262-A treatment. FIG. 20(d) shows examples of biopsied polyps: the left panel shows necrotic polyps characterised by a disorganised, and loose structure; the few regressing polyps exhibit a small, whitish appearance. In contrast, the single biopsied progressive polyp in the right panel exhibits a distinctive broccoli-like structure. FIG. 20(e-f) shows H&E staining of histological sections prepared from necrotic and progressive polyps from the male FAP pig (2705) treated daily with ULI262-A; a healthy control polyp shown in FIG. 20(g).

[0286] FIG. 21 shows a graphic summary of the proportions of cystic, necrotic and branched crypt organoids derived from untreated the (a) untreated and (b) ULI262-A male FAP pigs and 3.5 months of the study.

[0287] FIG. 22(a) shows a graphical representation of polyps distributed in the colorectal region of the female FAP pig (2721 ) after 6 months of daily oral ULI262-A treatment. FIG. 22(b) shows a photographic example of regressed / healed intestinal mucosa (left panel) and the progressive polyp before biopsy. FIG. 23 shows serum TNF-a levels in (a) FAP patients, with and without an ileal pouch, and (b) male FAP pigs during the ULI262-A study.

[0288] FIG. 24 shows a graphical summary of the percentage polyp reduction over time for clinical trials. The therapeutic candidates included chemotherapeutics, natural products and NSAIDs / generics. Near complete polyp reduction is shown for ULI262-A for FAP pigs in 6 months.

[0289] FIG. 25 shows FAP patient crypt organoids from which mRNA was harvested for RNA sequencing: (a) pre-treatment, (b) PBS treatment for 48 hr, and (c) ULI262-A treatment for 48 hr.

[0290] FIG. 26 shows the workflow for the preparing mRNA for sequencing.

[0291] FIG. 27 shows a KEGG graph of cell cycle with gene changes identified by treatment of FAP crypt organoids by ULI262-A for 48 hours. Dark shaded boxes indicate upregulation of gene expression; light shaded boxes indicate downregulation of gene expression.

[0292] FIG. 28 shows expression of CBC stem cell genes in FAP patient crypt organoids treated with PBS vehicle control or ULI262-A at 6 hr (FIG. 28(a, c, e)) and 48 hr (FIG. 28(b, d, f)).

[0293] Normalised gene expression is shown for SOX9 (a, b), ASCL2 (c, d), and LGR5 (e, f).

[0294] FIG. 29 shows a box plot summarising Log2 FC in gene expression for ULI262-A treated versus untreated FAP crypt organoids after 48 hr of culture across various cell types: CBC stem cells, Paneth cells, transit amplifying cells, tuft cells, and enterocytes. Sample sizes are indicated for each cell type, revealing the variation in expression levels across these populations.

[0295] FIG. 30 shows a KEGG graph of reactive oxygen species with genes enriched by treatment of human crypt organoids by ULI262 for 48 hours. Dark shaded boxes indicate upregulation of gene expression; light shaded boxes indicate downregulation of gene expression.

[0296] FIG. 31 shows antioxidative gene expression significantly upregulated in FAP patient crypt organoids from the PBS vehicle control to ULI262-A as early as early as 6 hr (a, c, e) and after 48 hr (b, d, f). Normalised gene expression is shown for HMOX1 (a, b), KEAP1 (c, d), and NQO1 (e, f).

[0297] FIG. 32(a) illustrates 2 pathways, NF-kB / IKK and KEAP1 / NRF2, that respond to oxidative damage. Chronic NF-kB / IKK gene activation may result in sustained inflammation and oxidative stress, leading to increased polyp growth and progression to cancer. FIG. 32(b) illustrates that treatment of FAP patient cystic crypt organoids with ULI262-A downregulated the expression of genes involved in inflammation and induced differentiation genes in the NF-kB / IKK pathway and powerfully increased the expression of genes to respond to oxidative stress in the KEAP1 / NRF2 pathway.

[0298] FIG. 33 shows and example of aberrant morphology of crypt organoids derived from Crohn’s patients’ (a) apparent healthy ATZ tissue or (b) inflamed ATZ tissue.

[0299] FIG. 34 shows an example of (a) poor crypt ATZ crypt-derived organoid growth of a Crohn’s patient that was improved by the addition of a (b) neutralising antibody cocktail against proinflammatory cytokines (anti-TNF-a, anti-IFN-y, anti-IL6) or (c) ULI262-A.

[0300] FIG. 35 shows an example of (a) poor crypt ATZ crypt-derived organoid growth of a Crohn’s patient even in the presence of the neutralising antibody cocktail against proinflammatory cytokines. Addition of ULI262-A to cultures containing the antibody cocktail activated the growth of a large number of healthy crypt-derived patient organoids (FIG. 35(b)).

[0301] FIG. 36(a) shows human ATZ crypt-derived cultured in HepatiCultTM generated expected ring structures. Addition of synthesised ULI262 to cultures generated of organ tissue-like structures (b-d), morphologies not observed in HepatiCultTM or with other specialised medium alone.

[0302] FIG. 37 shows the drug activity assay used to isolate and characterise ULI262-B: (a) dose response curve and (b) consistency of the assay over time.

[0303] FIG. 38 shows a schematic diagram of the workflow for the isolation of the native ULI262-B small molecule from a plant extract.

[0304] FIG. 39 shows the structural characterisation of the isolated native ULI262 by (a) high resolution UHPLC-MS and (b) analysing1H NMR shifts.

[0305] FIG. 40 shows a summary of the synthesis of ULI262.

[0306] FIG. 41 shows GCMS fragment patterns of (a) synthetic ULI262 and (b) native ULI262-B. FIG. 41 (c) shows assignments of the ULI262 structure based on GCMS and native ULI262-B and synthetic ULI2621H NMR shifts summarised in TABLE 6.

[0307] FIG. 42 shows (a) human ATZ crypt organoid growth at day 5. An increased number of organoids was observed with the addition of (b) native ULI262-B and (c) synthetic ULI262. Figure 42(d) shows organoid generation for synthetic ULI262 at various concentrations.

[0308] FIG. 43. Establishment and propagation of FAP pig polyp-derived organoids with Y27 and ULI262. (a) Both Y27 and ULI262 were required to initiate organoid formation, with only a single organoid surviving in ULI262. (b) Passage of this organoid without additions generated small ring-shaped structures, while continued culture with 5 pM synthesised ULI262 produced differentiated organoids with normal morphology, (c) Culture of FAP pig polyp-derived organoids with 1 nM synthesised ULI262 enhanced organoid establishment, consistent with the biphasic response described in EXAMPLE 12.1 .

[0309] FIG. 44(a) shows principal component analysis (PCA) clustering of ileal samples from ApcMin / + mice treated with ULI262, demonstrating separation of early (days 2+3) and late (days 4+5) treatment groups. FIG. 44(b) shows increased expression of canonical PPAR target genes (Apoa4, Fabp2, 1118, 3Reg3) in the ileum of ULI262-treated ApcMin / + mice in the late treatment group (days 4+5).

[0310] 45(a) shows pull-down binding of biotinylated ULI262 to recombinant PPARa, PPARy, and PPAR5 proteins. Bound proteins were retained on ULI262-coated beads and released only under high-salt conditions (350-500 mM NaCI).

[0311] FIG. 46 shows RNA-Seq of FAP crypt-derived organoids treated with ULI262-A for 48 h. (a) Upregulation of PPARA, PPARD, PPARG, and RXRA. (b) Induction of downstream targets for metabolic support, antioxidant defence, and barrier conditioning.

[0312] EXAMPLE 1. Intestinal crypt organoid models for drug discovery.

[0313] Introduction

[0314] Intestinal crypts and in vitro organoid model development. Intestinal stem cell behaviour in mice was extensively characterised over three decades (1960s -1990s) by Professor Christopher Potten and colleagues (Patterson Institute, Christie Hospital, Manchester, UK). They described the stem cell niche located below the gut surface, called the crypt. Cell production works in an escalator-like fashion to continuously regenerate the single cell layer of the mucosa every 3-5 days in mice (FIG. 1(a)).

[0315] In vitro culture and characterisation of the intestinal cell crypt only became possible decades later. Professor Hans Clevers and his colleagues (University of Utrecht, Netherlands) reported the development of mouse and human crypt-derived organoids in 2009 and 2011 , respectively (Sato, T. et al (2009) “Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche,” Nature, 459: 262-265; Sato, T. et al (2011 ) “Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium,” Gastroenterology, 141 :1762-1772) (FIG. 1(b)).

[0316] The 3D “mini-gut” crypt organoid models have significantly facilitated drug discovery and development by providing more accurate, human-relevant systems to study disease mechanisms, screen drugs, and predict responses. Individual crypt stem cells recapitulate their healthy and cancerous in vivo counterparts in animal models and humans (de Wetering et al (2015), “Prospective derivation of a Living Organoid Biobank of colorectal cancer patients,” Cell 161 : 933-945 ).

[0317] Crypt organoid models used for ULI262 discovery and characterisation

[0318] Healthy porcine crypt organoids. Due to ethical limitations on access to freshly isolated human intestinal mucosa, healthy porcine crypt organoid models were developed throughout the gastrointestinal (Gl) tract. (FIG. 1(c)). Porcine crypt organoids were established and maintained by embedding intestinal crypts in Corning® Matrigel® Matrix (Corning, Inc, New York, US) with human organoid growth medium (IntestiCult™ Organoid Growth Medium, STEMCELL Technologies, Vancouver, CA).

[0319] Familial adenomatous polyposis (FAP) patients. Ethical approval was obtained from the UK Health Research Authority (HRA) to obtain biopsies taken from apparent healthy gut tissue, from the duodenum to the rectum of FAP patients. In a survey of crypt organoids derived from FAP patients and non-FAP healthy individuals organoid phenotypes were categorised in 4 distinct morphologies shown in the brightfield images in FIG. 2: (a) ring structures of developing organoids; (b) normal branched structures; (c) cystic organoid structures; and (d) poorly differentiated branched organoids.

[0320] FIG. 2(e) shows the summary of characterising the morphology of 4,119 crypt organoids established from 23 FAP patients and 145 crypt organoids from 3 non-FAP healthy individuals. The frequency of ring structures was lower in FAP patients but not statistically significant. The frequency of normal branched organoid structures was significantly higher in healthy individuals than in FAP patients (p=0.0062), and the frequency of cystic and poorly branched organoid structures was significantly higher in FAP patients (p=0.0054, 0.0125, respectively).

[0321] Expression of protein markers by immunocytochemistry for mature cell types in crypt organoids derived from the duodenum of FAP115 patient was confirmed for Paneth cells (lysozyme) (FIG. 3(a)), mucin-producing cells (mucin-2) (FIG. 3(b)), and microvilli (villin) (FIG. 3(c)).

[0322] FIG. 4 shows the distribution of pathogenic mutations in individual crypt organoids (n=43) derived from three FAP patients (FAP115, FAP125, FAP134) and one healthy individual (HP115). Heterozygous APC mutations (APC+ / -) were present in the FAP patients but absent in the healthy control. In FAP115 crypt organoids, a combination of mutations was observed, including RET, CTNNB1 , PTPN11 , and ABL1 (n=1 ). Organoids from FAP125 crypts exclusively exhibited deleterious splice site mutations in the APC gene (n=6). For FAP134, 5 crypt organoids displayed mutations in APC, NOTCH1 , and FGFR2 genes, indicating a clonal population. Additionally, other from FAP134 crypts contained mutations in BRAF (n=1 ), PDGFRA, KIT, SRC (n=1 ), and a separate organoid with mutations in MET, JAK2 / 3, GNAS, PDGFRA, HNF1A, and FLT3 (n=1 ). EXAMPLE 2. Identification of a therapeutic candidate in a plant extract to intervene early in CRC development.

[0323] Introduction Sporadic CRC develops through a stepwise progression (FIG. 5(a)) It starts with a mutation in one allele of the APC tumour suppressor gene (APC+ / -). This mutation disrupts the Wnt signalling pathway in colonic stem cells, leading to abnormal cell regulation. Subsequent loss of the remaining APC allele (APC- / -) leads to uncontrolled cell proliferation, contributing to the formation of adenomatous polyps. As the disease progresses, additional mutations in key oncogenes and tumour suppressor genes, such as KRAS and TP53, accumulate to promote the transition from benign polyps to CRC. . The period between the initial APC mutations in crypt stem cells and the development of CRC presents a compelling opportunity for early therapeutic intervention. By redirecting abnormal, precancerous cell production to healthy cells (FIG. 5(b)), tissue homeostasis can be restored (FIG. 5(c)). This strategy could prevent further mutations from accumulating, stopping the progression towards CRC.

[0324] Identifying a plant extract containing the therapeutic candidate ULI262. Plant extract components were initially prepared by incubating pulverised plant material in phosphate buffered saline (PBS) or water at a 10 g / 100 ml ratio in roller bottles for 2-3 days in a cold room. After that, the slurry was centrifuged, filtered, aliquoted, and stored at -20° C.

[0325] A plant extract containing native ULI262 small molecule (termed ULI262-A) induced in vitro differentiation of precancerous, cystic FAP patient crypt organoids (FIG. 6(a)) to healthy branched crypt organoids ((FIG. 6(b)).

[0326] Characterising ULI262-A on FAP patients’ crypt organoids. ULI262-A induced differentiation of aberrant crypt organoids in all 12 FAP patients tested, regardless of the mutation position in the APC gene (FIG. 7). The patients’ mutations included the most common and lethal (pathogenic) mutation at position 1309, one of which FAP115 served as the source for the drug activity assay (EXAMPLE 10). Most of the mutations occurred between the mutational cluster region (MCR) and 5' end of the APC gene. These gene mutations are more likely to disrupt critical domains of APC protein involved in cellular regulation, leading to more severe disease outcomes.

[0327] ULI262-A induced in vitro differentiation of FAP cystic crypt organoids throughout the Gl tract (FIG. 8(a)). The ability of ULI262-A to induce differentiation in duodenal is especially noteworthy, as FAP patients have a 100 to 300-fold increased risk of developing duodenal cancer, even after prophylactic colectomy (Offerhaus, GJA et al, “The Risk of Upper Gastrointestinal Cancer in Familial Adenomatous Polyposis,” (1992) Gastroenterology, 102:1960-1961 ). Surgical resection of duodenal cancer often requires a pancreaticoduodenectomy (Whipple procedure), which is a complex surgery involving the duodenum, gallbladder, bile duct, and part of the stomach, with a <50% survival rate for 5 years (Meijer, LL et al (2018) “Outcomes and Treatment Options for Duodenal Adenocarcinoma: A Systematic Review and Meta-Analysis,” Ann Surg Oncol 25:2681-2692). Lifelong treatment with ULI262 could offer a preventive strategy against this high-risk malignancy.

[0328] Preparing for in vivo safety and efficacy studies in mice. Establishing strong correlations between relevant surrogate in vitro assays and in vivo outcomes is crucial for successful drug development. Before assessing the safety and efficacy of ULI262-A in the mouse model for FAP (ApcMin / +), ULI262-A was shown to induce differentiation of cystic crypt organoids throughout the mouse small intestine; crypt organoids derived from the colon of ApcMin / + mice were already generally non-cystic (FIG. 9). The in vitro pattern of cystic organoids mirrored the in vivo localisation of the disease in ApcMin / + mice (FIG. 10).

[0329] EXAMPLE 3. ULI262-A treatment reduced polyps in ApcMin / + mice

[0330] In vivo studies were conducted in ApcMin / + mice to determine if ULI262-A’s in vitro redirection of precancerous cystic cell production toward healthy branched crypt organoids, as described in EXAMPLE 2, would translate to reduce polyps. Specifically, the studies were designed to test if 1 ) ULI262 reduced polyp formation in young ApcMin / + mice and 2) ULI262-A reduced established polyps in older ApcMin / + mice.

[0331] Male and female ApcMin / + were included in the studies. Polyp growth rates and disease progression are slower in female ApcMin / + mice. Previous studies have reported that castration of male ApcMin / + mice led to reduced polyp formation, suggesting that androgens contribute to promoting tumour growth (White, JP et al, “Characterization of the male ApcMin / + mouse as a hypogonadism model related to cancer cachexia,” (2013) Biol Open 2:1346-1353).

[0332] Materials and methods

[0333] Ethics approval. Studies were conducted by 2 contract research organisations after they obtained project license approvals from the UK Home Office.

[0334] ULI262 reagents. ULI262-A or partially purified native ULI262 (termed ppULI262-B). The amount of the purified native ULI262-B in the plant extract (ULI262-A) was estimated at 400 ng / ml.

[0335] Dose determination. The in vivo dose of ULI262-A or the equivalent for ppULI262-B for treating ApcMin / + mice was based on a common calculation for traditional Chinese medicines (TCMs), taking into consideration the body mass and surface area of mice:

[0336] • TCM extract: 1 g plant material per 10 ml water

[0337] • Typical TCM extract dose: 10 ml plant extract per 10 kg of body weight (1 ml / kg)

[0338] • Mouse equivalent dose: 12 times more than humans (from the literature) • Volume delivered to mice via gavage: 10 ml / kg or an estimated dose of 4 ug / kg

[0339] Treatment groups. One animal facility sourced all ApcMin / + mice for studies. An equal number of male and female mice were used in the studies: 5 mice per group in Study 1 and 6 mice per group in Study 2.

[0340] Schedule. ApcMin / + mice were dosed at the same time in the mornings on Mondays, Wednesdays, and Fridays.

[0341] Monitoring adverse effects and humane endpoints. The life-ending toxicities in ApcMin / + mice required careful monitoring of adverse effects as they age. Animal technicians monitored the appearance of pale feet due to anaemia, weight loss, and behavioural changes. Mice were killed when weight loss reached 20% compared to age-matched controls or if they lost more than 15% of body weight in 3 days.

[0342] Tissue and blood collection. Blood and gut tissue, from the stomach to the anorectum, was collected at termination.

[0343] Polyp analysis. The number and location of polyps were visually determined when mice were at termination, either at a pre-determined time or when reaching a humane endpoint.

[0344] EXAMPLE 3.1. ULI262-A treatment reduced polyp formation in young ApcMin / + mice

[0345] In Study 1 , ApcMin / + mice started treatments with ULI262-A or vehicle control at 9 weeks of age, while in Study 2, ULI262-A or vehicle control treatments began after weaning at 6 weeks. Both studies continued treatment until week 15, at which point the number of polyps plateaued in untreated mice and before the development of any signs of anaemia (e.g., pale feet) or weight loss.

[0346] Results

[0347] No adverse behaviour and similar growth rates. Throughout the treatment period, animal technicians reported no adverse behaviours associated with ULI262-A or ppULI262-B treatments. Similar growth rates were observed in Study 1 between mice treated with ULI262-A and vehicle control (FIG. 11) or in Study 2 with ULI262-A, ppULI262-B, vehicle control, and untreated mice (FIG. 13). Growth of male ApcMin / + mice treated with ULI262-A or ppULI262-B plateaued at a modestly lower level than all other treatment groups in both studies; however, no differences were observed otherwise in this group.

[0348] ULI262-A and ppULI262-B reduced polyp development. Fewer polyps developed in young ApcMin / + mice treated with ULI262-A in Study 1 and with ULI262-A or ppULI262-B in Study 2 compared to the vehicle controls or untreated mice. The reduction in polyp numbers was more pronounced in males (FIG. 12(a) and FIG. 14(a)) than in females (FIG. 12(b) and FIG. 14(b)), as anticipated (see above). In Study 2, at least one mouse in each of 5 out of 8 groups, regardless of treatment or control, exhibited a significantly higher polyp burden, a phenomenon that remained unexplained (FIG. 14).

[0349] Summary. Despite the presence of high-burden polyp outliers, treatment with ULI262-A and ppULI262-B effectively reduced the median number of polyps formed in young ApcMin / + mice of both sexes in both studies. The variation in the extent and location of polyp reduction between the 2 studies is likely attributable to differences in the age at treatment initiation and the environmental conditions at different animal facilities.

[0350] EXAMPLE 3.2. ULI262-A and ppULI262-B treatment reduced polyps in older ApcMin / + mice

[0351] In Study 3, older male and female mice ApcMin / + mice with established polyps were treated with ULI262-A or ppULI262-B with vehicle control and untreated ApcMin / + mice as controls. Treatments were administered from weeks 10 to 15 unless the mice reached their humane endpoints before the scheduled end of the study. Throughout the treatment period, animal technicians reported no adverse behaviours associated with treatments.

[0352] ULI262-A and DDULI262-B extended lifespans of ApcMin / + mice. ULI262-A and ppULI262-B treatment of older ApcMin / + mice in Study 3 extended lifespans up to 3 weeks (FIG. 15(a-d)) compared to the vehicle controls (FIG. 15(e-f)). The longest surviving ApcMin / + mice in the vehicle control groups lived to week 16 (day 117). Notably, both ULI262-A and ppULI262-B treatments maintained healthy body weights until the study’s endpoint at week 19 (day 135) for 2 of 6 male and 3 of 6 female ApcMin / + mice. These results indicate that native ULI262 in a plant extract promoted healthy mucosal cell production, compensating for some of the deleterious global consequences of the Ape gene mutation.

[0353] ULI262 reduced established polyps in older male ApcMin / + mice. In Study 3, the scoring of male ApcMin / + polyps visualised by methylene blue staining was categorized based on size and visual characteristics. Small polyps (<2 mm) had an intact mucosal layer, indicating newly forming polyps. In contrast, large polyps (>2 mm) had breached the mucosal layer.

[0354] Summary. Treatment with ULI262-A and ppULI262-B resulted in a reduction in the median number of polyps in older ApcMin / + mice. This reduction was modest for small polyps (FIG. 16(a)) but more pronounced for large polyps, particularly with ULI262-A treatment (FIG. 16(b)). These findings indicate that native ULI262 treatments were more noticeable with the reduction of larger polyps. EXAMPLE 4. ULI262-A treatment reduced polyps in FAP pigs

[0355] Introduction. The FAP pig model was developed at the Technical University of Munich (TUM) to elucidate the fundamental biology of intestinal polyp progression and regression and for its use in developing therapeutics to prevent bowel cancer. FAP pigs were created by targeting the APC tumour suppressor gene (APC1311) that is orthologous to the most common mutation in FAP patients (APC1309) (Flisikowska, T et al (2012) “A porcine model of familial adenomatous polyposis,” Gastroenterology, 143:1173 -1175).

[0356] FAP pigs serve as the most relevant animal model available for human FAP disease. Hundreds of polyps form throughout the colon and rectum, while relatively few form in the small intestine (FIG. 17(a)). FAP pigs do not develop ORC; additional gene mutations are required (e.g., KRAS, P53).

[0357] The FAP pig model may more closely reflect human disease than the ApcMin / + mouse model, as described in EXAMPLE 3, where polyps predominantly develop in the small intestine (FIG. 10), and ApcMin / + mice die from anaemia and weight loss by 4 months (FIG. 15).

[0358] The good general health of FAP pigs allows for longitudinal studies and frequent endoscopies, either not possible in mice or impractical in human patients.

[0359] Materials and methods

[0360] Ethics approval. Studies were conducted under contract from TUM after they obtained project license approval.

[0361] ULI262-A preparation. Bottles containing 200 ml slurries of ULI262-A were prepared for study and stored at -20° C. The amount of the purified native ULI262-B in the plant extract (ULI262-A) was estimated at 800 ug / day.

[0362] Dose determination.

[0363] • Pig and human gut physiologies are similar

[0364] • TOM extract: 1 g plant material per 10 ml water

[0365] • Typical TOM extract dose: 10 ml plant extract per 10 kg of body weight (1 ml / kg)

[0366] • FAP pig weights ranged from approximately 10 kg to 100 kg during the study

[0367] • FAP pigs treated daily with 200 ml ULI262-A extract as a slurry of the plant material (20 g plant material 1200 ml)

[0368] ■ For 10 kg FAP pig: 200 ml per 10 kg = 20 ml / kg (20x typical TOM) or an estimated 800 ug / 1 Okg = 80 ug / kg dosage = 800 ug / day

[0369] ■ For 100 kg FAP pig: 200 ml per 100 kg = 2 ml / kg (2x typical TOM) or an estimated 800 ug / 100kg = 8 ug / kg dosage = 800 ug / day Treatment groups. 2 males and 2 females, ULI262-A treated and untreated. FAP pigs with a higher polyp burden were chosen for treatment with ULI262-A.

[0370] Schedule. A bottle consisting of a ULI262-A slurry was added to FAP pigs’ food every morning during the study.

[0371] Tissue and blood collection. Blood and gut tissue from the stomach to the anorectum was collected.

[0372] Monitoring adverse effects and humane endpoints. Animal technicians observed no adverse behavioural or clinical effects of ULI262-A treatment throughout the study.

[0373] Study observations. Polyps were evaluated in treated and untreated FAP pigs after 1 , 3.5, and 6 months of the study.

[0374] Polyp analysis

[0375] Morphology classifications. Examples of progressive and regressive FAP pig polyps are shown in FIG. 17(b), in situ (left panel) and biopsied (right panel), and summarised in TABLE 1. For the current study, a 3rdclassification of “necrotic” was added, of which an example is shown in FIG. 20(d).

[0376] TABLE 1

[0377] FAP polyp type In vivo properties (polyps) In vitro properties (organoids)

[0378] Progressive Large, "brocoli-like", firm and bloody Cystic, undifferentiated organoids

[0379] Regresse to healed Small, whitish, soft Healthy, branched organoids

[0380] Necrotic Mostly dead or dying cells Mostly dead or dying cells in organoids

[0381] Histology. PAS-ab and Ki67 histology staining of normal pig gut mucosa and FAP progressive and regressive polyps is shown in FIG. 17(c). Compared to normal mucosal tissue, progressive polyps were characterised by increased dysplasia and architectural distortion, whereas regressive polyps show signs of fibrosis, reduced dysplasia, and decreased proliferative activity, often reflecting a benign or healing process.

[0382] Pre-treatment assessment of FAP polyps’ cellular health

[0383] FIG. 18(a) shows aberrant cystic organoid cultures established from crypts derived from rectal polyps of the 4 study FAP pigs before treatment. EXAMPLE 4.1 FAP pig polyp assessment at 1 month

[0384] Washing discharge indicates activity. The academic principal investigator who developed the FAP pig model noted differences in the washing discharge when flushing the rectum in preparation for endoscopies after 1 month (TABLE 2). Bloody stool was flushed from the untreated male FAP pig (2174), identified by arrows in FIG. 18(b). Apoptotic or dead mucosal tissue was flushed from the ULI262-A treated male FAP pig (2705), identified by arrows in FIG. 18(c). The untreated FAP pig had bloody mucosa in the stool, blood was detected in the stool of the ULI262-A treated female FAP pig (2721 ) (not shown).

[0385] TABLE 2

[0386] Polyp biopsies. FIG. 18(d) shows examples of a progressive polyp (left panel) and regressive polyp (right panel) taken via endoscopic biopsy from the rectum of the male FAP pig (2705). After the first month of the study, the polyps were similar in size to the examples in FIG. 17(b); their small size made accurate counting impossible.

[0387] Assess the health of FAP polyps after 1 month of treatment. Crypts were isolated from progressive and regressive polyps biopsied from the four FAP pigs after 1 month. Crypt organoids derived from polyps of untreated and ULI262-A treated FAP pigs consisted of a mixture of cystic and branched structures, summarised in FIG. 19: (a) 38% cystic organoids and 62% healthy branched organoids in untreated in the male FAP pig (2714); FIG. 19(c) 15% cystic organoids and 85% healthy branched organoids in the untreated female FAP pig (2722).

[0388] In contrast, crypt organoids derived from polyps biopsied from ULI262-A treated pigs after 1 month showed a marked reduction in cystic structures, summarised in FIG. 19: (b) only 2% cystic organoids and 98% healthy branched organoids in the ULI262-A treated male FAP pig (2705); (d) <1% cystic organoids and 99% healthy branched organoids in the ULI262-A treated female FAP pig (2721 ).

[0389] Summary of ULI262-A activity after 1 month of treatment. The principal investigator concluded that the flushed dead tissue resulted from the shedding of mucosal polyp tissue undergoing a transformation toward regression and healing. This interpretation was further supported by the observations that crypt derived from ULI262-A-treated male and female pigs produced normal, healthy crypt organoids. Taken together, these observations indicate that ULI262-A was already redirecting precancerous cell production toward healthy mucosal tissue within 1 month. In addition, FAP polyp derived served as a relevant biomarker for the status of cellular production.

[0390] EXAMPLE 4.2. FAP pig polyp assessment at 3.5 months

[0391] Washing discharge and endoscopy. Bloody debris was flushed from the untreated male FAP pig (2714), as identified by the arrow in FIG. 20(a) whereas sheets of dead tissue in the discharge were observed when flushing the rectum at 3.5 months in the male FAP pig (2705), identified by the arrow in FIG. 20(b). The academic principal investigator noted mostly necrotic polyps in the treated male treated FAP pig (2705) during rectal endoscopy. For this reason, the study on the male FAP pigs was stopped to characterise necrotic polyps.

[0392] Termination. Blood was collected for serum analysis; intestines were resected, flayed open, and thoroughly washed. Polyps were enumerated, and biopsies were taken for histological analysis and the establishment of crypt organoids.

[0393] Polyp assessment. FIG. 20(c) shows a graphical representation of polyps distributed in the colorectal region of the male FAP pig (2705) after 3.5 months of daily oral ULI262-A treatment. Enumeration of polyps of the whole large intestine and rectum noted 256 mostly necrotic and regressive polyps and 1 progressive polyp at 58 cm from the terminus.

[0394] Polyp biopsies. FIG. 20(d) shows examples of biopsied polyps: the left panel shows necrotic polyps characterised by a disorganised, and loose structure; the few regressing polyps exhibit a small, whitish appearance. In contrast, the single biopsied progressive polyp in the right panel exhibits a distinctive broccoli-like structure.

[0395] Histology on polyps. FIG. 20(e-f) shows H&E stained histological sections prepared from necrotic and progressive polyps from the male FAP pig (2705) treated daily with ULI262-A. with a healthy control polyp shown in FIG. 20(g).

[0396] FIG. 20(e) shows a polyp undergoing reduced cellular proliferation, characteristic of a regressive or involuting adenoma. The region on the right shows previously aberrant crypts, which have lost their defined structure. A high density of immune cells is present, indicating active immune involvement during tissue regression.

[0397] The histological structure of regressing polyps includes three distinct regions: a basal area resembling normal mucosa, a middle adenomatous area similar to that seen in progressing polyps, and a marginal region characterized by regressive features, such as atypical crypts and expanding stroma. In the marginal region, the atypical crypts exhibit significant changes in gland architecture, including deformed crypt structures, distorted crypt walls, and irregular gland edges. Crypt abscesses are occasionally observed. The epithelial cells in these crypts appear "compressed" and are accompanied by marked immune cell infiltration. These epithelial cells also show enlarged nuclei, frequent nuclear stratification, and a loss of nuclear polarity.

[0398] FIG. 20(f) shows a histological section of a progressive polyp from an APC1311 / + pig model. Large regions of the polyp display focal areas of high-grade adenomatous crypts, characterized by dense cellular proliferation and abnormal crypt architecture. In progressing polyps, the body is dominated by a uniform adenomatous structure, with crypts exhibiting a branching and crowded pattern, along with dilated Lieberkuhn glands. These features are typically driven by the accumulation of enlarged and elongated mutant epithelial cells.

[0399] The epithelial cells within these crypts frequently show a dense, stratified, or multilayered distribution, with irregularly enlarged hyperchromatic nuclei (marked by intense staining). This results in a thickened crypt wall and asymmetrical crypt architecture. Goblet cells within the crypts appear abnormally shaped and are difficult to distinguish, while the number of mitotic cells is notably higher.

[0400] Additionally, in the basal region, a small hump-like structure composed mainly of normal mucosa is observed. In larger progressive polyps, a stalk composed of submucosal tissues surrounded by elongated crypts is also frequently present.

[0401] FIG. 20(g) shows a histological section that displays healthy mucosa, characterized by regular crypt structures and an intact basal membrane. The tissue shows well-organized crypt architecture without signs of dysplasia or abnormal cell proliferation, indicative of normal colonic function. This serves as a baseline for comparison against adenomatous or polyposis-affected tissue.

[0402] Necrotic polyp-derived organoids dominated the regressive polyp-derived organoids. Crypt organoid cultures were established from the progressive, necrotic, and regressive polyps taken from the male FAP pig (2705) to assess the health status of cell production after 3.5 months. The proportions of organoid phenotypes are summarised in FIG. 21 : (a) organoids derived from a progressive polyp consisted of 70% cystic, 20% healthy, and 10% necrotic debris phenotypes; (b) crypt derived from an intact regressive polyp consisted of 27% cystic, 3% healthy, and 70% necrotic debris phenotypes.

[0403] Summary: ULI262-A induced terminal differentiation in FAP polyps. Taken together, the extensive sheets of dead tissue in the water flush discharge, along with the near-complete regression of polyps and polyp-derived organoids into a predominantly necrotic state after 3.5 months of daily oral ULI262-A treatment, indicated that ULI262-A induced terminal differentiation in FAP polyps. In addition, FAP polyp derived-organoids served as a relevant biomarker of the status of cellular production. EXAMPLE 4.3 FAP pig polyp assessment at 6 months

[0404] Female FAP pigs were terminated at 6 months. No washing discharge was recorded. FIG. 22(a) shows a graphical representation of polyps distributed in the colorectal region of the female FAP pig (2721 ) after 6 months of daily oral treatment with ULI262-A. A necropsy examination intestinal tract by the academic principal investigator enumerated 571 polyps of regressed and mostly healed polyps that had fused with the gut mucosa. FIG. 22(b) shows photographs of regressive polyps (left panel) and the single progressive polyp (right panel) identified 114 cm from the terminus.

[0405] Overall, the daily oral treatment in FAP pigs was safe and led to a significant reduction in polyps while promoting gut mucosa healing, beginning as early as 1 month and achieving full effects by 6 months.

[0406] EXAMPLE 5: Lower serum TNF-a levels in FAP pigs treated with ULI262-A

[0407] Introduction. Chronic gut inflammation is associated with driving the development and progression of polyps to CRC, particularly in patients with inflammatory bowel disease (Ullman and Itzkowitz (2011 ), “Intestinal Inflammation and Cancer,” Gastroenterology 140: 1807-1816)). Specifically, the pro-inflammatory cytokine TNF-a acts as a central regulator in the initiation and progression of colitis-associated colon carcinogenesis in a mouse model (Popivanova et al. (2008). “Blocking TNF-alpha in mice reduces colorectal carcinogenesis associated with chronic colitis,” J Clin Invest 2008 118: 560-570).

[0408] Serum TNF-a levels elevated in FAP patients. FIG. 23(a) shows elevated serum TNF-a in 68% of FAP patients with no pouch (11 of 19) and 84% with an ileal pouch (10 of 19) (Curileum analysis on FAP patients at St Mark’s Hospital, Harrow, UK). The range of serum TNF-a levels in healthy individuals is 2 pg / ml - 20 pg / ml.

[0409] Serum TNF-a levels were reduced in FAP pigs treated with ULI262-A. FIG. 23(b) shows that serum TNF-a levels in the untreated male FAP pig (2714) increased 6-fold over the 3.5 month study (58 pg / ml to 355 pg / ml), whereas serum TNF-a levels remained near baseline levels throughout the study (<30 pg / ml to a maximum of 53 pg / ml the second month). The range of serum TNF-a levels in healthy pigs is 2 pg / ml - 20 pg / ml.

[0410] EXAMPLE 6. Superior properties of ULI262-A over current clinical strategies to prevent bowel cancer

[0411] Competitive landscape. Current clinical strategies to reduce precancerous polyps primarily focus on reducing inflammation since this is recognised to drive polyps formation and progression to cancer. FIG. 24 shows a graphical summary of the percentage polyp reduction over time in clinical trials. The therapeutic candidates included chemotherapeutics, natural products and NSAIDs / generics. With one exception, polyps were reduced by <50% over 3-5 years of treatment.

[0412] Follow-up 12 months after completion of a randomised, placebo-controlled with aspirin demonstrated an increased colorectal polyp risk during post-trial surveillance, concluding that the rebound elevated neoplastic risk after short-term aspirin use has important implications for aspirin cessation driven by age-related bleeding risk (Downing, A et al “Colorectal polyp outcomes after participation in the seAFOod polyp prevention trial: Evidence of rebound elevated colorectal polyp risk after short-term aspirin use”. (2023) Aliment Pharmacol Ther 58: 562 - 572).

[0413] Near complete reduction of polyps by daily ULI262-A treatment. When compared to therapeutic candidates in clinical trials, ULI262-A was superior in >99% reduction of polyps at 3.5 months and healing by 6 months (FIG. 24). ULI262-A stands out from the competition by its superior safety and effectiveness, uniquely combining its anti-inflammatory properties reducing TNF-a (FIG. 23(b)) with activation of healthy cell production. This dual action promoted necrosis of precancerous polyps while simultaneously healing and restoring mucosal function.

[0414] EXAMPLE 7: In vitro biological mechanism of action (MoA) studies with ULI262-A

[0415] Study objective. In vitro studies mechanism of action (MOA) were conducted to profile the of gene expression in crypt-derived organoids, thereby identifying key intracellular signalling pathways involved in the responses to ULI262-A treatment.

[0416] Materials and methods. Bulk mRNA sequencing on an Illumina NextSeq500 System was employed to characterise transcriptional changes in response to ULI262-A’s redirection of in vitro precancerous FAP patient cystic crypt organoids to healthy branched crypt organoids. FIG. 25 shows FAP134 crypt organoids from which mRNA was harvested for RNA sequencing: a) pre-treatment, (b) PBS treatment for 48 hr, and (c) ULI262-A treatment for 48 hr. The workflow for the preparation of mRNA for sequencing is shown in FIG. 26.

[0417] Differential gene expression (DGE) analyses were performed comparing ULI262-A and PBS treated samples at 6 hr and 48 hr (3 independent biological replicates). DGE analyses were expressed as Log2 fold changes (Log2 FC) with a p-value (p-value) representing the p-value adjusted for multiple test corrections using the Benjamini-Hochberg method.

[0418] When evaluating gene set enrichment analysis (GSEA) of gene sets containing more than 50 genes, their collective expression changes were examined at 6 hr and 48 hr using KEGG and Reactome databases. Normalized Enrichment Scores (NES) were assigned for a positive score, indicating an increase in expression across pathways, or a negative NES score, indicating a decrease in expression across pathways. Results. At 6 hr, the most significantly upregulated gene by ULI262-A treatment was HMOX1 (Log2 FC = 3.68, p-value = 2.96E-114), a gene that reduces oxidative stress, and the most downregulated gene was ADRA2A (Log2 fold change: -2.18, p-value: 3.81 E-99). At 48 hr, the most significantly upregulated genes by ULI262-A treatment were AKR1 C1 (Log2 FC = 7.66, p- value = 0) and AKR1 C2 (Log2 FC = 4.2, p-value = 3.52E-251 ), 2 genes involved in protecting against reactive oxygen species, while CDCA7 (Log2 FC = -5.55, p-value = 8.14E-199), which has a key role in cellular differentiation, was highly downregulated.

[0419] KEGG databases identified increased cell differentiation (FIG. 27) and reduced reactive oxidative stress (ROS) (FIG.30) as 2 of the highest enriched gene set pathways (TABLE 3) .

[0420] TABLE 3

[0421] Time ID Description setSize NES p value

[0422] 6 hr hsa04110 Celt cycle (differentiation) 155 -2.19 2.02E-08

[0423] 6 hr hsa 05208 Reactive oxygen species (ROS) 205 2.20 2.00E-08

[0424] 48 hr hsa04110 Cell cycle (differentiation) 155 -2.19 3.90E-17

[0425] 48 hr hsa05208 Reactive oxygen species (ROS) 205 2.20 1.11E-10

[0426] EXAMPLE 7.1 Molecular characterisation of ULI262-A inducing healthy cell production

[0427] Introduction. The single-cell layer of the intestinal mucosa is replenished weekly from a stem niche below the surface called crypts in humans and pigs. As described in EXAMPLE 2, cell production works in an escalator-like manner, starting at the base with crypt base columnar (CBC) stem cells, expanding the number of cells in the transit amplifying zone to produce mature cells (FIG. 1(a)).

[0428] Confirmation that ULI262-A induced in vitro production of mature cells. CBC stem cell genes were significantly downregulated in FAP patient crypt organoids treated with ULI262-A when compared to the PBS vehicle control as early at 6 hr (FIG. 28(a, c, e)) and at 48 hr (FIG. 28(b, d, f)).

[0429] FIG. 28 shows expression of CBC stem cell genes in FAP patient crypt organoids treated with PBS vehicle control or ULI262-A: (a, b) SOX9 is essential for maintaining the population of intestinal stem cells located at the base of the CBCs, particularly LGRr5-expressing stem cells. LGR5 protein helps sustain stem cell identity by promoting the expression of stem cell markers and repressing expression of differentiation genes; (c, d) ASCL2 is a transcription factor that is essential for the maintenance of the CBC stem cell population. It activates a set of genes that define the identity and function of intestinal stem cells, including LGR5; (e, f) LGR5 is considered a marker of actively cycling adult CBC stem cells. The interaction between LGR5 stem cells and their niche is vital for ensuring balanced renewal and differentiation of intestinal cells.

[0430] FIG. 29 shows a box plot summarising Log2 FC in gene expression for ULI262-A treated versus PBS vehicle control treated FAP crypt organoids after 48 hr of culture across various cell types: CBC stem cells, Paneth cells, transit amplifying cells, tuft cells, and enterocytes. Sample sizes are indicated for each cell type, revealing the variation in expression levels across these populations.

[0431] • CBC stem cells. In addition to the significant downregulation gene expression of the essential stem cell self-renewal and maintenance genes shown in FIG. 27, related CBC stem cell-regulated genes were down regulated.

[0432] • Paneth cells. Expression levels increased with ULI262-A treatment, which is consistent with the production of more Paneth cells during periods of elevated stem cell activity, such as during differentiation and crypt expansion.

[0433] • Tuft cells. Expression levels increased with ULI262-A treatment. These rare, specialized chemosensory cells in the intestinal epithelium help to maintain gut homeostasis.

[0434] • Enterocytes. Expression levels increased with ULI262-A treatment. These water absorbing cells in the colon demonstrate that ULI262-A induced the production of healthy, mature cells from cystic precancerous FAP patient crypt organoids.

[0435] EXAMPLE 7.2 Antioxidative properties of ULI262-A

[0436] Gene set enrichment analysis of bulk mRNA sequencing using KEGG databases identified significant increases in anti-oxidant response element genes related to the KEAP1 / NRF2 pathway when comparing FAP patient cystic crypt organoids treated with ULI262-A compared to the PBS vehicle control at 6 hr and 48 hr (FIG. 30). Dark shaded boxes indicate upregulation of gene expression; light shaded boxes indicate downregulation of gene expression.

[0437] Gene expression of key antioxidative genes at 6 hr and 48 hr. FIG. 31 shows antioxidative gene expression significantly upregulated in FAP patient crypt organoids from the PBS vehicle control to ULI262-A as early as early as 6 hr (a, c, e) and after 48 hr (b, d, f).

[0438] HMOX1 gene expression was highly upregulated at 6 hr (Log2 FC of 3.7, p-value = 2.25E-110) (FIG. 31(a)) and at 48 hr (Log2FC = 5.3, p-value = 6E-128) (FIG. 31(b)). The HMOX1 protein reduces oxidative stress in the gut by producing antioxidants (bilirubin), modulating inflammation (via CO), and preventing further ROS generation through iron regulation. This protective mechanism is especially important in the context of polyps, where oxidative stress is elevated, helping to limit the risk of malignant transformation. KEAP1 gene expression was upregulated at 6 hr (Log2FC = 0.6, p-value = 9.42E-06) (FIG. 31(c)) and at 48 hr (Log2FC = 0.9, p-value = 1 .29E-26) (FIG. 31(d)). The KEAP1 protein helps to reduce oxidative stress in the gut by regulating the NRF2 pathway, which is crucial in the presence of polyps, where oxidative damage is often high.

[0439] NQO1 gene expression was upregulated at 6 hr (Log2FC = 0.9, p-value = 9.81 E-20) (FIG. 31(e)) and at 48 hours (Log2 FC = 2.0, p-value = 2.61 E-104). (FIG. 31(f). The NQO1 protein is essential for reducing oxidative stress by detoxifying harmful quinones, limiting the production of ROS, supporting the antioxidant defence system, and maintaining the integrity of the gut lining. NRF2 regulates NQO1 gene expression.

[0440] ROS role in inflammation and polyps. FIG. 32(a) illustrates 2 pathways, NF-kB / IKK and KEAP1 / NRF2, that respond to oxidative damage. Chronic NF-kB / IKK gene activation may result in sustained inflammation and oxidative stress, leading to increased polyp growth and progression to cancer. Binding and inhibiting NF-kB / IKK activity reduces inflammation (CXCL2, TRAF5, TNFA) but also removes the suppression of antioxidant response element genes in the KEAP1 / NRF2 pathway, which responds with the increase of antioxidative gene expression (HMOX1 and NQO1 ).

[0441] ULI262-A down regulated pathways activated in response to ROS. FIG. 32(b) illustrates that treatment of FAP patient cystic crypt organoids with ULI262-A downregulated the expression of genes involved in inflammation and induced differentiation genes in the NF-kB / IKK pathway and powerfully increased the expression of genes to respond to oxidative stress in the KEAP1 / NRF2 pathway. These in vitro results were consistent with the in vivo reduction of serum TNF-a in the male FAP pig (2705) treated daily with ULI262-A (FIG. 23(b)).

[0442] Biological mode of actions (MOAs) of ULI262-A: 2 distinct but complementary cell regulatory pathways.

[0443] 1. Activated cell production. Bulk mRNA sequencing confirmed morphologic changes in ULI262-A’s redirecting in vitro precancerous FAP patient cystic crypt organoids to healthy branched crypt organoids, initially described in EXAMPLE 2, and confirmed at the mRNA level in EXAMPLE 7.1 .

[0444] 2. Reduced inflammation. Molecular characterisation studies showed ULI262-A directly reduces inflammation by activating genes that respond to oxidative stress in FAP patient cystic crypt organoids within 6 hr and continued at 48 hr, as described in EXAMPLE 7.2. EXAMPLE 8. Broad therapeutic applications for ULI262 to treat chronic gut inflammation diseases.

[0445] Introduction. Chronic gut inflammation weakens the gut’s protective barrier by loosening tight junctions between cells in the mucosal lining, leading to increased gut permeability. This allows bacteria, toxins, and other harmful substances to pass through the intestinal lining into the bloodstream, which can further activate the immune system and worsen the inflammatory response.

[0446] EXAMPLE 8.1. ULI262 initiation and growth of healthy Crohn’s disease crypt organoids.

[0447] Inflammatory bowel diseases (IBD) , including Crohn's disease and ulcerative colitis, are characterised by an abnormal immune response that leads to chronic inflammation in the gut.

[0448] Poor growth of Crohn’s crypt organoids overcome by ULI262-A. Crypt organoids derived from Crohn’s patients’ anorectum were difficult to establish and maintain in standard organoid culture conditions, regardless of whether biopsies were taken from apparent healthy ATZ tissue (FIG. 33(a)) or inflamed ATZ tissue (FIG. 33(b)). Intestinal crypts derived from actively inflamed Crohn’s disease tissue frequently exhibit poor yield and limited ability to form or sustain organoids, reflecting reduced stem cell viability and distorted crypt architecture (Howell KJ et aL, World J Gastroenterol 2019;25:4125-4147).

[0449] Poor crypt ATZ crypt-derived organoid growth of a Crohn’s patient (FIG. 34(a)) was improved by the addition of a neutralising antibody cocktail against proinflammatory cytokines (anti-TNF- a, anti-IFN-y, anti-IL6, all from Thermo Fisher, Waltham, US) (FIG. 34(b)). Addition of ULI262-A to a Crohn’s disease patient’s cultures similarly improved crypt organoid development, such as initiation and growth (FIG. 34(c)).

[0450] In another Crohn’s disease patient, poor crypt organoid growth was observed even in the presence of the neutralising antibody cocktail against proinflammatory cytokines (FIG. 35(a)). However, the addition of ULI262-A to cultures containing the antibody cocktail activated the growth of a large number of healthy crypt-derived patient organoids (FIG. 35(b)).

[0451] Superior properties of ULI262-A to address major unmet medical needs in IBD. Cu rrent inadequate treatments for IBD result from an incomplete understanding of disease mechanisms, limited efficacy, and side effects of long-term treatment such as infections and cancer.

[0452] ULI262-A's potent anti-inflammatory properties, coupled with its ability to stimulate cell production, present a novel solution to prevent the symptoms of painful, chronic gut inflammation flare-ups in IBD patients by reducing inflammation, healing the mucosal layer (“sealing the barrier”), restoring healthy gut function. ULI262-A uniquely enables the initiation and propagation of Crohn’s disease-derived crypt organoids that otherwise fail to establish. In parallel, its anti-inflammatory activity and stimulation of epithelial regeneration provide a novel therapeutic approach to suppress inflammation, restore mucosal barrier integrity, and re-establish normal gut function in IBD

[0453] EXAMPLE 9. Broad regenerative medicine applications of ULI262 to promote repair and heal damaged tissues and organs

[0454] ULI262-A’s demonstrated properties to promote repair and healing of the gut mucosa has broad applications in regenerative medicine, including but not limited to organs and tissues derived from: endoderm (intestine, pancreas, liver, lung), mesoderm (blood vessels, kidney, cardiomyocytes, striated muscle), and ectoderm (skin, brain).

[0455] EXAMPLE 9.1. ATZ stem cells as relevant in vitro model systems to characterise ULI262’s therapeutic potential

[0456] Introduction. Since most organ and tissue stem cells are dormant, a novel source of adult multipotent stem cells for regenerative medicine was used to demonstrate ULI262’s broad therapeutic potential in regenerative medicine.

[0457] • Endoderm: International Patent Application PCT / GB2024 / 050415, published as WO2024 / 170911 A1 on 22 August 2024

[0458] • Mesoderm: International Patent Application PCT / GB2024 / 050416, published as WO2024 / 170912 A1 on 22 August 2024

[0459] • Ectoderm: International Patent Application PCT / GB2024 / 050417, published as WO2024 / 170913 A1 on 22 August 2024

[0460] Adult human and porcine multipotent stem cells at the junction of the endoderm (rectum) and ectoderm (anal skin) - called the anorectal transition zone (ATZ) - expressed pluripotent (NANOG, OCT4) and multipotent (LGR5, KIT, CD34) stem cell markers.

[0461] Specialised culture medium (STEMCELL Technologies) directly differentiated ATZ crypt- derived stem cells into organoids that exhibited in vitro morphologies and markers of differentiated cells of all 3 developmental lineages.

[0462] The in vivo regenerative capacity of allogeneic ATZ stem cells was first demonstrated in the porcine perianal fistula model, sealing a mechanical fistula with highly vascularised fibrotic tissue after 1 -3 months (International Patent Application PCT / IB2022 / 000456, published as WO 2023 / 017316 on 16 February 2023). Allogeneic transplantation of male porcine ATZ stem cells implanted into a female porcine pancreas and liver migrated from the injection site, engrafted, differentiated and produced insulin or albumin, respectively, within 5 weeks with no evidence of an immune reaction.

[0463] Elevated levels of tissue remodelling were observed in the pancreas (stellate and endothelial cells) and liver (MMP9, MMP2, LOXL2, COL8A2, and ACTA2), suggesting that ATZ cells remodelled the healthy tissue microenvironment.

[0464] EXAMPLE 9.2. ULI262-A promoted the growth and differentiation of human ATZ-derived liver organoids.

[0465] Human ATZ crypts cultured in a specialised medium to promote liver organoids (HepatiCult™, STEMCELL Technologies) exhibited the properties of liver-derived organoids. FIG. 36A shows the expected ring structures. Adding the isolated native ULI262 small molecule (described in EXAMPLE 10) to cultures promoted the growth of organ tissue-like structures (FIG. 36(b-d)), morphologies not observed in HepatiCult™ or with other specialised medium alone.

[0466] EXAMPLE 9.3. ULI262 promotes growth and differentiation of pancreatic, lung, kidney, cardiac muscle, striated muscle and skin.

[0467] ULI262’s demonstrated ability to promote the growth and differentiation of intestinal and liver organoids from human ATZ-derived crypts provides a relevant platform showing that ULI262’s properties extend to the growth and differentiation of additional tissues, including but not limited to pancreatic, lung, kidney, cardiac muscle, striated muscle, brain, and skin.

[0468] EXAMPLE 10. Isolation and characterisation of the native ULI262 small molecule from a extract

[0469] Druo . A robust, reproducible phenotypic assay using FAP patient crypt organoids as a cell source was developed to isolate and characterise the purified, active native small molecule (ULI262-B) from a plant extract (Lindera aggregata). Assay results were reported by the percentage of branched healthy organoids calculated by % branched organoids I (% branched organoids + % cystic organoids).

[0470] Test agents were diluted 5-fold over a 625-fold range (1 :10 to 1 :6,250 final concentration) covering cystic to branched phenotypes (FIG. 37(a)). The reference standard FAP organoid cell source (FAP115) and the native ULI262 containing reference standard extract (ULI262-A, lot PP03-09) were used throughout the isolation and characterisation of purified native ULI262- B. The half-maximal dose PP03-09 with an EC50 at a dilution of circa 1 :200 remained consistent throughout the isolation and characterisation of ULI262-B (FIG. 37(b)).

[0471] Materials and methods FIG. 38 shows a schematic diagram of the workflow for the isolation of the native ULI262-B small molecule from a plant extract. For several reasons, such as miscibility in the aqueous media used to grow organoids, an extraction solvent with some polarity was desired. After optimising the efficacy and viability of the extraction solvent using GCMS (TABLE 4) and the drug activity assay, 1 kg of the plant source was ground to 10 mesh (2.5 mm) and extracted with 3 x 1 ,500 ml aliquots of ethyl acetate. The aliquots were combined and evaporated under vacuum at 40°C to obtain 40 g of viscous oil.

[0472] TABLE 4

[0473] A second optimisation was conducted to determine the best HEMWat solvent system for a high- performance countercurrent chromatography (HPCCC) separation (Sutherland, IA and IJ Garrard, “Dynamic Extraction: A High-Speed, High-Capacity Purification Process That is Rapidly Scalable,” (2008) LCGC North America, 26: 424-438). Fractionation of the oil extract was done in multiple batches using a Dynamic Extraction HPCCC Midi Centrifuge (35° C, 1 ,400 rpm, 230 nm) with a mobile phase composed of 12.7% ethyl acetate, 28.1 % methanol, and 59.2% water, and a stationary phase composed of 37.4% hexane, 58.8% ethyl acetate, 3.3% methanol, and 0.5% water. A 2 g load of the oil extract in 50 ml of mobile phase was injected with a flow rate of 50 ml / min for each batch of sample after an 8 min equilibration. After 34 min, extrusion was performed at 200 ml / min. Fractions from each batch eluting between 10 and 11 min (termed FR10) of ppULI262-B were collected and evaporated to yield a 267 mg of powder.

[0474] A portion of the FR10 powder was first dissolved in 100% DMSO and subsequently diluted with water to achieve a final DMSO concentration of 10% for the drug activity assay. The remainder was dissolved in various solvents and analysed by various instrumental methods to isolate and characterise the physiochemical properties of the active molecule. Quantification of the active components (7%) in FR10 was accomplished using GCMS (Agilent 7890A GC and 5970 MS with a 30 m x 250 / zm x 0.25 / zm HP-5MS column using a temperature program 50° C to 300° C at 10° C / min) and LCMS (Waters ACQUITY UPLC-PDA-MS, 40oC BEH C18 column, 130A, 1 .7 pm, 2.1 mm X 50 mm, using a 12 min solvent program, 0.4 ml / min, 10% to 90% acetonitrile in water with 0.1 % formic acid). Most of the UPLC analyses were conducted using + mode ESI and 100 - 500 m / z.

[0475] Initial characterisation of components in the active fraction was performed using ultra-high- performance liquid chromatography-high resolution mass spectrometry (UHPLC-MS). Analyses were done with an Exactive Orbitrap (Thermo Fisher Scientific) mass spectrometer coupled to an Accela Ultra High-Performance Liquid Chromatography (UHPLC) system (Thermo Fisher Scientific).

[0476] Chromatographic separation was done with a reverse (RP) Hypersil Gold C18 1 .9 pm, 2.1 x 150 mm column (Thermo Scientific) using H2O with 0.1 % formic acid (v / v, pH 2.74) as the mobile phase (solvent A), and acetonitrile with isopropanol (10:90) and 10 mM ammonium acetate as the mobile phase (solvent B). Samples (20 ul) were analysed using 0-20% gradient of B from 0.5 to 1 .5 min and then to 100% B in 10.5 min. After 3 min isocratic at 100% B the column was re-equilibrated with 100% A for 7 min. The flow rate was set to 0.34 ml / min, the column temperature to 60° C, and the data were acquired in both positive and negative ESI (4 kV spray) using a heated electrospray ionisation source (HESI). Mass spectra were acquired from 70 to 1400 m / z using a mass resolution of 100,000.

[0477] The final purification and characterisation of the active components in FR10 were conducted using flash column chromatography silica gel with DCM / EtOAc followed by EtOAc / methanol to obtain 5 fractions, the second of these separated using petrol / EtOAc and silica PTLC (preparative thin-layer chromatography). Spot 4 of 6 was active and produced an NMR spectrum (Bruker 600 MHz) that was used with the UHPLC-MS and GCMS data to obtain the structure of the active component that was subsequently synthesized.

[0478] Results. Combining the yields from the successive purification steps in this analysis indicated that the native plant contained at least 0.0019% of the active purified molecule (ULI262-B).

[0479] FIG. 39(a) shows that high resolution UHPLC-MS analysis gave + mode ions equal to 245.11722 (from M - H2O) and 285.10969 (from M + Na+), suggesting that native ULI262-B has the molecular formula C15H1804 (exact mass 262.1205).

[0480] EXAMPLE 11. Synthesis of the ULI262 small molecule

[0481] A summary of the synthesis steps is shown in FIG. 40. Step 1 - 1 ,4-conjugate addition

[0482] •s^MgBr(3°eq)

[0483] This procedure was performed in 10 batches of 4.5 grams. Attempts to scale-up the procedure to 10 and 12 grams failed.

[0484] A suspension of CuBr-DMS (7.64 g, 37.1 mmol, 1 .5 eq) in anhydrous THF (150 ml) was degassed with bubbling nitrogen for 30 min at room temperature. The mixture was then cooled to -78° C and allowed to stir at this temperature for 15 min, before dropwise addition (via syringe pump) of vinyl magnesium bromide (1.0M THF, 74.1 ml, 74.1 mmol, 3.0 eq). The addition took about 1 hour, and gives a dark-brown suspension. After a further 30 min stirring at -78° C, a solution of Hagemann’s ester (4.50 g, 24.7 mmol, 1.0 eq) in anhydrous THF (32 ml) was added dropwise (via syringe pump) over 1 hour. After a further 30 min at -78° C, TLC showed high conversion and the mixture was quenched by the addition of 10% aq. ammonium chloride solution (200 ml). The bath was removed and the mixture diluted with MTBE (200 ml). All 10 batched were combined for the work-up and purification procedure. The organic layer collected, and the aqueous layer back-extracted once with MTBE. Combined organic layers were dried over sodium sulfate and concentrated in vacuo to a brown oil. Purification by silica gel chromatography was performed 2 times (1st: PE / Et2O 0-30%; 2nd: PE / Et2O 0-20%). The product (int-1) was obtained as a light-yellow oil (37.9 g, 73% yield).

[0485] Step 2 - Ketal formation 2, , int-1 int-2

[0486] To a solution of int-1 (17.0 g, 80.95 mmol, 1 .0 eq) in anhydrous diethyl ether (340 ml, 20V) was added trimethyl orthoformate (39.8 ml, 364 mmol, 4.5 eq) then anhydrous ethylene glycol (27.1 ml, 486 mmol, 6.0 eq). PTSA-H2O (615 mg, 3.2 mmol, 0.04 eq) was then added and the mixture allowed to stir at room temperature under a nitrogen atmosphere for 16 hr. The reaction was quenched with 10% aqueous potassium carbonate solution (200 ml), diluted with water (500 ml) and the organic layer collected. The aqueous layer was back-extracted with diethyl ether (2 x 400 ml). The combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo. Purification was achieved by a single subjection to silica gel chromatography (PE / Et2O 0- 20%), to afford the product (int-2) as a light-yellow oil (19.7 g, 96% yield).

[0487] Step 3 - Weinreb amide formation

[0488] To a solution of N,0-dimethylhydroxylamine hydrochloride (7.7 g, v.7 mmol, 4.0 eq) in THF (125 ml) at -78° C was added n-BuLi (2.5M, 63 ml, 157.5 mmol, 8.0 eq) dropwise over 30 min. After a further 15 min, the cooling bath was removed and stirred at ambient temperature for 30 min before re-cooling to -78OVC. A solution of int-2 (5.0 g, 19.7 mmol, 1.0 eq) in THF (40 ml) was then added dropwise over 45 min. After a further 15 min at -78° C, the reaction was quenched by addition of aqueous 10% ammonium chloride solution (250 ml), then warmed to room temperature and extracted with diethyl ether (3 x 200 ml). The combined organic layers were dried over sodium sulfate, filtered and concentrated to dryness under reduced pressure. Purification by silica gel chromatography (petroleum ether / diethyl ether 0-40%) afforded int-3 (4.22 g, 80% yield) as a light-yellow oil.

[0489] Step 4 - Allylation

[0490] To a solution of int-3 (10.0 g, 37.2 mmol, 1 .0 eq) in THF (500 ml) at -78° C was added dropwise allyl magnesium bromide (41 ml, 40.9 mmol, 1 .1 eq) over 45 min. After stirring at the same temperature for a further 15 min, the reaction was quenched by the addition of aqueous 10% ammonium chloride solution (400 ml), then warmed to room temperature and extracted with diethyl ether (3 x 300 ml). The combined organic layers were dried over sodium sulfate, filtered and concentrated to dryness under reduced pressure. Purification by silica gel chromatography (petroleum ether / diethyl ether 0-25%) afforded int-4 (8.47 g, 91% yield) as a light-yellow oil.

[0491] Step 5 - Luche reduction

[0492] 96% yield int-4 int-5

[0493] To a solution of int-4 (6.3 g, 25.0 mmol, 1 .0 eq) and cerium trichloride heptahydrate (10.3g, 27.5 mmol, 1.1 eq) in methanol (500 ml) at -78° C was added a freshly-made solution of NaBH4(1 .045 g, 27.5 mmol, 1 .1 eq) in EtOH (55 ml) dropwise over 30 min. The mixture was allowed to warm to -50 oC over 90 min, then quenched by addition of aqueous 10% ammonium chloride solution (600 ml), then warmed to room temperature and most of the methanol was evaporated under reduced pressure. The resulting mixture was extracted with diethyl ether (3 x 300 ml). The combined organic layers were dried over sodium sulfate, filtered and concentrated to dryness under reduced pressure. Purification by silica gel chromatography (petroleum ether / diethyl ether 0-80%) afforded int-5 (6.11 g, 96% yield) as a light-yellow oil.

[0494] Step 6 - Ketal deprotection

[0495] 89% yield int-5 int-6

[0496] To a solution of int-5 (13.0 g, 51 .6 mmol, 1 .0 eq) in acetone (650 ml, 50V) at room temperature was added aqueous 1 M HCI solution (65 ml, 5V). The solution was stirred at room temperature for 16 hr, then quenched with 10% aqueous potassium carbonate solution (250 ml). The mixture was concentrated under vacuum to remove most of the acetone, before diluting with MTBE (500 ml). The organic layer was collected, and the aqueous layer extracted with MTBE (2 x 300 ml). The combined organic layers were dried over sodium sulfate, filtered and concentrated to dryness under reduced pressure. Purification by silica gel chromatography (petroleum ether / MTBE 0-60%) afforded int-6 (9.48 g, 89% yield) as a light-yellow oil.

[0497] Step 7 - Lactone annulation 53% yield brsm To a solution of int-6 (2.5 g, 12.0 mmol, 1 .0 eq) in DCM (50 ml, 20V) at -78° C under nitrogen was added dropwise a solution of TiCI4 (3.87 ml, 36 mmol, 3.0 eq) in DCM (15.5 ml) over 30 min. Then a solution of tributylamine (11 .4 ml, 48 mmol, 4.0 eq) in DCM (46 ml) was added dropwise over 30 min. After a further 30 min at the same temperature, a solution of 2,2- dimethoxyacetone (5.8 ml, 48 mmol, 4.0 eq) in DCM (17.5 ml) was added dropwise over 30 min. The solution was stirred for a further 30 min before the cooling bath was removed and the mixture stirred at room temperature for 20 hr. The dark brown solution was quenched by addition of water (500 ml), followed by extraction with DCM (4 x 300 ml). The combined organic layers were dried over sodium sulfate, filtered and concentrated to dryness. The crude brown oil was subjected to three rounds of silica gel chromatography (1st: PE / EA 0-80%; 2nd: PE / EA 0- 40%; 3rd: DCM / MTBE 0-5%) to afford int-7 as a light-brown oil (1 .01 g, 32% yield, 53% yield brsm). The product was sufficiently pure to continue with the next step. Some int-6 was also recovered (990 mg) as a light-brown oil.

[0498] Step 8 - Ring-closing metathesis

[0499] DCM was freshly degassed before use by vigorous bubbling of nitrogen gas for 30 min. To a solution of int-7 (2.01 g, 8.0 mmol, 1 .0 eq) in DCM (0.02M, 400 ml) at room temperature was added Hoveyda-Grubbs catalyst M720 (100 mg, 0.02 mol%) under a nitrogen atmosphere. The reaction flask was wrapped in foil and the mixture allowed to stir at room temperature for 16 hr. The crude mixture was concentrated to dryness under reduced pressure. The crude mixture was purified by silica gel chromatography (PE / MTBE 0-100%) to afford int-8 as a light-brown solid (1 .55 g, 83%).

[0500] Step 9 - Alcohol oxidation To a solution of int-8 (980 mg, 4.19 mmol, 1 .0 eq) in DCM (0.05M, 85 ml) at room temperature was added Dess-Martin periodane (2.13 g, 5.03 mmol, 1 .2 eq) in 5 portions over 90 min. After a further 90 min at room temperature, the mixture was diluted with saturated aqueous NaHCOs solution (250 ml) and extracted with DCM (2 x 200 ml). The combined organic layers were dried over sodium sulfate, filtered and concentrated to dryness. Purification by silica gel chromatography (PE / MTBE 0-100%) afforded int-9 pale-yellow oil (905 mg, 93% yield).

[0501] Step 10 - Epoxidation crude 100% yield int-9 int-10

[0502] To a solution of int-9 (900 mg, 3.89 mmol, 1 .0 eq) in DCM (56 ml) at room temperature was added m-CPBA (77% purity, 2.58 g, 3.0 eq) in one portion. After stirring for 14 hr at room temperature, the mixture was diluted with saturated aqueous NaHCOs (300 ml) and 15% aqueous sodium thiosulfate solutions (50 ml). The mixture was extracted with DCM (2 x 300 ml) and the combined organic layers dried over sodium sulfate, filtered and concentrated under reduced pressure to afford the crude epoxide int-10 as a white gummy solid (2.5 g, contains a large amount of mCPBA-related residue). This was immediately used in the next step without further purification.

[0503] Step 11 - Epoxide elimination int-11 (Linderolide H)

[0504] The crude int-10 (2.4 g, 40-50% content) was dissolved in DCM / EtOAc (1 :1 , 400 ml) and silica gel (250 g) was added to create a thick slurry. The slurry was stirred at 45 °C for 48 hr, the filtered and washed with DCM / MeOH (95:5, 1000 ml) until no more product eluted. The solution was concentrated to a light-brown crystalline solid, which was re-dissolved in DCM / MeOH (95:5, 15 ml) and subjected to silica gel chromatography (120g silica, DCM / MTBE 0-10%). The pure product int-11 (rac-Linderolide-H) was obtained as off-white crystals (781 mg, 81% yield) after trituration in MTBE (10 ml), [yield based on step-11] Step 12 - Alcohol protection y 83% yield brsm int-12 int-11 (Linderolide H)

[0505] To rac-Linderolide-H int-11 (450 mg, 1 .81 mmol, 1 .0 eq) in DCM (40 ml - not completely dissolved) at 0 °C was added 2,6-lutidine (1 .26 ml, 10.9 mmol, 6.0 eq) followed by dropwise addition of a solution of TBS triflate (830 uL, 3.62 mmol, 2.0 eq) in DCM (12 ml) over 45 min. After 2 hr the solution was quenched with 0.25M aq HCI solution (500 ml) and extracted with DCM (3 x 250 ml). The combined organic layers were dried over sodium sulfate, filtered and concentrated to dryness. Purification by silica gel chromatography (PE / MTBE 0-100%) afforded the product int-12 as a light-yellow oil which crystallised at RT overnight (430 mg, 65% yield, 83% yield brsm) and recovered starting material (95 mg).

[0506] Step 13 - Wittig olefination

[0507] To a solution of methyltriphenylphosponium bromide (740 mg, 2.07 mmol, 3.0 eq) in THF (5.9 ml, 8V) at 0 °C was added n-BuLi (2.5M, 750 uL, 1 .86 mmol, 2.7 eq) over 5 min. The mixture was stirred at this temperature for 30 min to give a yellow solution with some white solids. The solution was then added dropwise over 1 hour to a solution of enone int-12 (250 mg, 0.69 mmol, 1 .0 eq) in THF (5.0 ml, 20V) at 0 °C. After complete addition, the brown suspension was immediately quenched with 10% aqueous ammonium chloride solution (20 ml) and extracted with MTBE (3 x 20ml). The combined organic layers were dried over sodium sulfate, filtered and concentrated to dryness. Purification by silica gel chromatography (PE / MTBE 0-20% then to 100%) afforded the product as a colourless oil (185 mg, 74% yield, 85% yield brsm) and recovered starting material as a light-yellow oil (32 mg).

[0508] Step 14 - Hydroxylation 15% yield 20% yield brsm int-13 int-14

[0509] Anhydrous acetonitrile (4.0 ml) was bubbled with oxygen gas for 20 min at room temperature before addition of int-13 (125 mg, 0.35 mmol, 1.0 eq) followed by DBU (260 uL, 1.73 mmol, 5.0 eq). The solution was warmed to 40 °C and stirred under an oxygen balloon for 16 hr. The orange solution was then poured into 0.5M aq. HCI (400 ml) and extracted with EtOAc (3 x 250 ml). The combined organic layers were dried over sodium sulfate, filtered and concentrated to a light-brown oil. Purification by silica gel chromatography (see below) afforded int-14 (20 mg, 15% yield, 20% yield brsm) as a colourless oil, along with recovered starting material int-13 (29 mg) as a colourless oil.

[0510] The crude oil was first subjected to silica gel chromatography (12 g silica, 0-25% PE / MTBE over 30 min, then 100% MTBE for 15 min).

[0511] Peaks 1 , 2, and 3 were individually resubjected to silica FCC (4g silica, PE / MTBE) to further remove minor impurities. Peak 4 was re-purified by prep-TLC (PE / MTBE 80 / 20). After repurification:

[0512] Peak 1 - 9 mg, unknown product, mass isomer of desired product

[0513] Peak 2 - mixture of 3 unknown products

[0514] Peak 3 - 20 mg, desired product

[0515] Peak 4 - mixture of 2 or 3 unknown dihydroxylated (or peroxide) products, mass = SM+32

[0516] Step 15 - Alcohol deprotection int-14

[0517] To a solution of int-14 (13 mg, 0.035 mmol) In THF (2 ml) was added TBAF (100 uL, 1 M in THF). The reaction was stirred at room temperature and monitored by TLC until full conversion of the starting material. The reaction was diluted with sat. NaHCC>3 (100 ml) and extracted with TBME (3 x 100 ml). The combined organic layers were washed with brine, dried over sodium sulfate and concentrated in vacuo. The crude product was purified by FCC (30-50% EA / PE) followed by preparative HPLC (C18, gradient 0-30% MeCN / water) to give ULI262 as an off- white solid, 3.26 mg, 36% yield.

[0518] EXAMPLE 11.1. structural equivalence of isolated native (ULI262-B) and synthesised ULI262

[0519] NMR analysis and structure. GCMS analysis of synthesised ULI262 (FIG. 41(a)) showed the same fragmentation pattern as the isolated native ULI262-B (FIG. 41(b)). The structure of ULI262-B shown in FIG. 41(c) was further confirmed by the1H NMR shifts in native ULI262-B and synthesised ULI262 (TABLE 6).

[0520] TABLE 6 of ULI262 in establishment, differentiation, and rescue of

[0521] EXAMPLE 12.1 Confirmation of synthesised ULI262 activity in human crypt organoids

[0522] The native ULI262-B (FIG.42(b)) and synthesised ULI262 (FIG.42(c)) both activated the production of more development crypt organoids at day 5 from healthy human ATZ crypts than culture standard intestinal crypt organoids cultures alone ((FIG.42(a)).

[0523] The initial in vitro organoid assays testing synthesised ULI262 used 10 pM as the starting treatment concentration. A dose-response study spanning a 16-fold concentration range centred on 10 pM was then performed. At day 5, ULI262 produced a greater number of established organoids than vehicle (0.1% DMSO / water) across all concentrations (FIG. 42(d)). The response was biphasic, with more organoids at lower ULI262 concentrations and fewer at higher concentrations. EXAMPLE 12.2 Establishment of organoids from cryopreserved FAP pig polyp-derived organoids with synthesised ULI262

[0524] Organoids were established from crypts originally isolated from a section of the single progressive rectal polyp remaining in the APC1311 / + (FAP) pig after 3.5 months of daily oral ULI262-A treatment (see EXAMPLE 4.2). A total of 1 ,000 crypts cryopreserved in CS10 (STEMCELL Technologies) were thawed and cultured under three culture conditions in standard human organoid growth medium (IntestiCult: (1 ) no addition, (2) 10 pM Y27 (STEMCELL Technologies), or (3) 5 pM synthesised ULI262. Y27 (Y-27632), a ROCK inhibitor, was included as it is routinely used to enhance survival and establishment of organoids from cryopreserved or dissociated intestinal crypts by preventing anoikis (Sato et al., Nature 459:262-265, 2009).

[0525] FIG. 43(a) shows that both Y27 and ULI262 were required to establish organoids; however, only a single organoid cultured in ULI262 ultimately survived. This organoid was subsequently dissociated and passaged under two conditions: (1 ) standard medium without additions or (2) medium supplemented with 5 pM synthesised ULI262. The left panel of FIG. 43(b) shows that passage without additions generated multiple small ring-shaped organoids, suggesting that the residual FAP polyp may have acquired additional mutation(s). The right panel of FIG. 43(b) shows that continued culture in 5 pM ULI262 produced more differentiated organoids with normal morphology and tissue-like structures.

[0526] As shown in FIG. 43(c), FAP pig polyp-derived organoids were cultured in the presence of synthesised ULI262 at a concentration of 1 nM. This observation is consistent with the biphasic response described in EXAMPLE 12.1 , where low pM concentrations of ULI262 supported greater organoid establishment compared to higher concentrations.

[0527] EXAMPLE 12 demonstrates that synthesised ULI262 is biologically active in both healthy human crypt organoids and FAP pig polyp-derived organoids. At 10 pM, ULI262 supported establishment of human crypt organoids and more fully differentiated aberrant FAP polyp- derived organoids into structures with normal morphology. At low nanomolar concentrations, ULI262 expanded the number of established FAP polyp organoids, consistent with a biphasic dose-response observed with human organoids.

[0528] Importantly, ULI262 acted to rescue cryopreserved crypts and organoids, enabling survival under conditions where cryodamage typically induces anoikis and apoptosis. These effects provide protection against the consequences of cryoinjury, including membrane and organelle disruption from ice crystal formation, osmotic shock, and ROS, thereby supporting crypt and organoid viability during recovery after cryopreservation. As described in EXAMPLE 13, downstream activation of PPAR pathways by ULI262 includes upregulation of lipid metabolic and membrane-stabilising genes (e.g., FABP1 , FABP2, SCP2), mitochondrial -oxidation genes (e.g., CPT1A, ACAA1 , ACOX1 ) that sustain energy balance, and antioxidant and stress-response genes (e.g., ANGPTL4, HMGCS2). Collectively, these PPAR-regulated mechanisms account for the observed protective effect by maintaining membrane integrity, reducing oxidative stress, and preventing anoikis in the post-thaw recovery phase.

[0529] Balanced pan-PPAR activation. Taken together, these results demonstrate that ULI262 produces balanced activation of PPARA (or PPARa), PPARD (or PPAR-p / b), PPARG (or PPAR-y), and RXRA, supporting organoid establishment, differentiation, and rescue from cryopreservation injury. Balanced pan-PPAR activation enables efficacy at nanomolar-low micromolar concentrations while mitigating liabilities associated with single-isoform agonists such as PPARG-selective fluid retention, PPARA-related liver enzyme elevations, or PPARD- linked tumorigenicity (Kersten, Nat Rev Endocrinol 2014; Barish et al., J Clin Invest 2006;

[0530] Chandra et al., Nat Rev Drug Discov 2008). By distributing transcriptional load across isoforms, ULI262 engages complementary pathways for lipid metabolism, epithelial renewal, and antiinflammatory signalling (Shearer & Billin, J Lipid Res 2007; Dubois et al., Nat Common 2017), thereby enhancing safety and efficacy relative to selective agonists. These findings establish the basis for the downstream mechanistic studies presented in EXAMPLE 13.

[0531] EXAMPLE 13. ULI262 is a balanced pan-PPAR agonist

[0532] Overview. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors of the nuclear receptor superfamily that regulate lipid metabolism, glucose balance, inflammation, and cellular differentiation. The three isoforms — PPARa, PPARS (p / 6), and PPARy — have distinct but overlapping tissue distributions and functions. Upon ligand binding, PPARs heterodimerize with retinoid X receptors (RXRs) and modulate gene expression through peroxisome proliferator response elements (PPREs), thereby influencing key metabolic and proliferative pathways.

[0533] Limitations of prior PPAR agonists for colorectal cancer prevention. Previous attempts to harness PPAR agonism for colorectal cancer prevention have been hindered by a trade-off between efficacy and safety.

[0534] • Synthetic agonists. Bezafibrate (pan-PPAR a / 6 / y) showed anti-inflammatory activity in preclinical models, though direct evidence in colorectal cancer remains limited (Botta M, Audano M, Sahebkar A, Mitro N, Ruscica M. Pharmacol Res. 2018;132:49-56). Elafibranor (PPAR-a / b) and Fenofibrate (PPAR-a) did not demonstrate significant antitumour efficacy in clinical studies (Westerouen van Meeteren MJ, et al. Sci Rep. 2020;10:18581 ; Lange L, et al. Cancers (Basel). 2022;14:5236). GW501516 (PPAR-5) enhanced fatty acid oxidation and suppressed inflammation in preclinical studies but was linked to carcinogenicity, halting development (Sahebkar A, Chew GT, Watts GF. Expert Opin Drug Saf. 2014;13:373-387). Muraglitazar, Pioglitazone, and Rosiglitazone (PPAR-y agonists) have shown mixed effects in colorectal tumour models, with pioglitazone showing reduced polyp burden in some studies, though cardiovascular risks limit their clinical use (Pramanik KC, et al. Mol Carcinog. 2024;63:310-322; Ishii H, et al. IntJ Mol Sci. 2023;24:10255). Overall, six of seven major synthetic agents reported adverse outcomes including cardiovascular events, fluid retention, carcinogenicity, and organ toxicity.

[0535] • Natural agonists. Several naturally occurring compounds, such as fatty acids, flavonoids, polyphenols, triterpenoids, and dietary metabolites, have been reported to activate PPAR isoforms and evaluated in both preclinical models and, in some cases, clinical trials for colorectal cancer prevention. Aspirin and other NSAIDs reduced adenoma recurrence in randomized controlled trials (Baron JA, et al. N Engl J Med. 2003;348:891 -899), while omega-3 fatty acids such as eicosapentaenoic acid (EPA) reduced polyp number and size in familial adenomatous polyposis (FAP) patients (Hull MA, et al. Gut. 2010;59:918-925). Resveratrol modulated colorectal cancer biomarkers in patients (Patel KR, et al. Cancer Prev Res. 2010;3:1452-1460). Other compounds such as curcumin, genistein, selenium, and ursolic acid have demonstrated PPAR- linked activity in intestinal models (Carroll RE, et al. Cancer Prev Res. 2011 ;4:354-364; Shimizu M, et al. Cancer Sci. 2008;99:1573-1581 ; Clark LC, et al. JAMA. 1996;276:1957-1963; Ikeda Y, et al. Biochem Biophys Res Commun. 2011 ;404:493- 499). While these natural agonists have not been associated with toxicity, their clinical utility has been constrained by low potency, poor selectivity, and limited bioavailability.

[0536] Taken together, these findings highlight the unmet need for PPAR-targeted agents that deliver efficacy without unacceptable safety risks.

[0537] Insights from knockout models. Genetic studies further underscore the essential role of PPAR isoforms in intestinal homeostasis, immune-epithelial balance, and cancer susceptibility.

[0538] • PPARa knockout mice (PPARa- / -). These animals show defective fatty acid metabolism and heightened inflammatory responses, leading to abnormal epithelial proliferation and increased susceptibility to chemically induced colitis (Lee SS, et al. Mol Cell Biol. 1995;15:3012-3022; Peters JM, et al. J Biol Chem. 1997;272:27307-27312).

[0539] • PPARy knockout mice (PPARy- / -). Loss of PPARy results in spontaneous colitis or heightened disease severity after chemical induction, with disruption of epithelial barrier integrity and impaired differentiation. Knockout studies also demonstrated increased tumour susceptibility under inflammatory conditions, confirming the tumour-suppressive role of PPARy in the intestine (Adachi M, et al. J Exp Med. 2006;203:2459-2465;

[0540] Dubois V, et al. Nat Commun. 2017;8:15108).

[0541] • PPAR5 knockout mice (PPAR5- / -). These animals display impaired epithelial regeneration and abnormal stem / progenitor cell dynamics. While PPAR5 is required for effective repair following injury, constitutive activation of PPAR5 has been associated with accelerated tumour formation, demonstrating the need for balanced signalling (Peters JM, et al. Mol Cell Biol. 2000;20:5119-5128; Harman FS, et al. Gastroenterology. 2004;127:1674-1685).

[0542] Collectively, these knockout studies confirm that PPARa, PPARy, and PPAR5 are non- redundant regulators of epithelial production and mucosal defence, and that their disruption increases susceptibility to conditions such as inflammatory bowel disease (IBD) and cancers, such as colorectal cancer.

[0543] Rationale for ULI262. This combined pharmacological and genetic evidence suggests PPARs as critical modulators of intestinal integrity and colorectal cancer prevention. These findings provide the rationale for developing ULI262 as a balanced pan-PPAR agonist designed to deliver potent receptor activation while maintaining a favourable safety profile suitable for longterm use in prevention.

[0544] EXAMPLE 13.1. ULI262-A treatment of ApcMin / + mice increased expression of canonical PPAR downstream targets.

[0545] An in vivo ApcMin / + mouse study was conducted to identify early transcriptional changes in intestinal tissues induced by daily oral administration of ULI262-A using materials and methods described in EXAMPLE 3.1 . A total of 20 female ApcMin / + mice aged 14-15 weeks were allocated into 5 groups. Either PBS vehicle control (PBS) or ULI262-A was administered by gavage at 10 ml / kg daily in a time course over 5 days. Duodenal, ileal, and colonic tissue was isolated and prepared for transcriptional profiling at days 0, 2, 3, 4, and 5.

[0546] FIG. 44(a) shows principal component analysis (PCA) clustering of ileal samples from ApcMin / + mice following initiation of ULI262-A treatment at day 0. Samples collected on days 0, 2, and 3 clustered separately from those collected on days 4 and 5, indicating a time-dependent transcriptional shift induced by ULI262-A, with early (days 2+3) and late (days 4+5) treatment groups displaying distinct gene expression profiles. FIG. 44(b) shows that daily ULI262-A treatment of ApcMin / + mice resulted in increased expression of canonical PPAR targets in the ileum in the late treatment group (days 4+5). Specifically, ULI262-A treatment resulted in increased expression of Apoa4, Fabp2, 1118, and Reg3. Induction of Apoa4 and Fabp2 is consistent with enhanced differentiation of enterocytes and activation of fatty acid transport and metabolic pathways. Increased expression of 1118 and Reg3 indicates early engagement of innate immune and barrier defence mechanisms.

[0547] Together, these transcriptional changes demonstrate rapid epithelial re-programming that supports a differentiated and protective intestinal phenotype.

[0548] EXAMPLE 13.2. Binding of ULI262 to human recombinant PPAR isoforms.

[0549] Biotinylated, synthesised ULI262 was evaluated for direct binding to human recombinant PPARa, PPARy, and PPAR5 proteins using a pull-down assay. Recombinant proteins (Abnova Ltd, Cheshire, UK or MedChem Express, Monmouth Junction, US) were incubated with biotinylated ULI262 in binding buffer and subsequently captured with streptavidin-coated magnetic beads (Dynabeads™ M-280 Streptavidin, Thermo Fisher). Following extensive washing to remove unbound protein, bound complexes were analysed by protein gels (4-15% Mini-PROTEAN TGX Stain-FreeTM Protein Gels, Bio-Rad Laboratories, Hercules, US or NovexTM Tricine Mini Protein Gels, 10 to 20%, Thermo Fisher) and visualised by SimplyBlueTM SafeStain (Thermo Fisher).

[0550] Control studies for the pull-down experiments included incubations with biotin only, beads only, or ULI262 only, and confirmed that biotinylation of ULI262 did not abolish or alter its ability to interact with PPAR proteins, as activity remained comparable to non-biotinylated ULI262 in crypt organoid assays.

[0551] Bound PPARa, PPARy, and PPAR5 proteins were retained on ULI262-coated beads and could only be effectively released under high-stringency conditions (FIG. 45). Specifically, elution required addition of 350 mM or 500 mM NaCI, whereas lower ionic strength buffers failed to disrupt the interaction. These results demonstrate that ULI262 binds directly and specifically to PPAR family proteins with high affinity, and that such binding is preserved following biotinylation.

[0552] EXAMPLE 13.3. Mechanistic validation of ULI262-A activity through PPAR isoform engagement and downstream targets

[0553] RNA-Seq analysis of FAP crypt-derived organoids (EXAMPLE 7; 48 h; ULI262-A vs PBS) was performed to assess expression of PPAR isoforms and establish downstream targets. Gene expression changes were plotted if they exceeded a 2-fold difference and 1 ,000 read counts in either treatment group. ULI262-A significantly upregulated transcripts for PPARA, PPARD, PPARG, and RXRA relative to PBS controls at 48 h, confirming engagement of the nuclear receptor signalling complex (FIG. 46(a)). Among these, PPARD showed the greatest induction (p = 0.0002), followed by PPARG (p = 0.0015), PPARA (p = 0.0142), and RXRA (p = 0.0184).

[0554] Analysis of canonical downstream targets further demonstrated coordinated activation of pathways linked to metabolic support, antioxidant defence, and barrier conditioning (FIG. 46(b)):

[0555] Cell production / metabolic support

[0556] • ACOX1 — peroxisomal -oxidation to meet energetic demand. Lambe KG et al. Toxicol Lett. 1999;110:119-127.

[0557] • ACSL1 — long-chain fatty acid activation for membrane / lipid synthesis during proliferation. Lefterova Ml et al. Genes Dev. 2008;22:2941-2952.

[0558] • ACSL5 — intestinal fatty acid activation supporting renewal dynamics. Gassier N et al. Gastroenterology. 2007;133:587-598. Klaus C et al. World J Gastroenterol. 2013;19:7369-7373.

[0559] • HMGCS1 — cholesterol pathway supporting membrane biosynthesis. Meertens L et al. EMBO J. 1998;17:6972-6980.

[0560] Anti-oxidant / redox support

[0561] • HMGCS2 — ketogenesis; shifts fuel use to lower mitochondrial ROS burden. Meertens L et al. EMBO J. 1998;17:6972-6980.

[0562] • ME1 — NADPH generation for glutathione recycling and biosynthesis. IJpenberg A et al. J Biol Chem. 1997;272:20108-20117.

[0563] Barrier / anti-inflammatory conditioning (in vitro)

[0564] • ANGPTL4 — PPAR-responsive mediator that supports barrier function and moderates inflammatory influx. Mandard S et al. J Biol Chem. 2004;279:34411-34420. Staiger H et al. Diabetes. 2009;58:369-377. Robciuc MR et al. PLoS One. 2012;7:e46212.

[0565] Balanced PPAR activation in EXAMPLE 13 provides the mechanistic basis for ULI262’s dual actions observed in EXAMPLE 7 and EXAMPLE 9.

[0566] These RNA-Seq data confirm that ULI262-A engages multiple PPAR isoforms and induces a balanced downstream gene program that supports cell production, antioxidant defence, and barrier conditioning. The induction of metabolic genes (e.g., ACOX1 , ACSL1 , ACSL5, HMGCS1 ) provides a mechanistic explanation for the transcriptomic validation of morphologic redirection described in EXAMPLE 7.1 . Similarly, the upregulation of antioxidant and stress- response genes (e.g., HMGCS2, ME1 , ANGPTL4) accounts for the protective and antiinflammatory effects observed in EXAMPLE 7.2.

[0567] Consistent with these findings, EXAMPLE 9 demonstrates that ULI262 promotes the growth and differentiation of liver, pancreatic, lung, kidney, cardiac muscle, striated muscle, brain, and skin organoids, extending the dual cell-production and antioxidant activities across multiple tissue types.

[0568] PPAR activation induces downstream targets across multiple tissues. In the liver, muscle, and heart, PPARa / b regulate energy metabolism via ACOX, HMGCS2, CPT1 , PDK4, and CD36 (Lambe KG, Toxicol Lett 1999; Gilde AJ, Circ Res 2003). In adipose tissue, PPARy directs adipogenesis and insulin sensitivity through aP2, adiponectin, and PEPCK (Tontonoz P, Genes Dev 1994). In the lung and fibrotic liver, PPARy represses NF-KB / AP-1 signalling and downregulates a-SMA and collagen I, reducing inflammation and fibrosis (Staiger H, Diabetes 2009; Zhang Y, Front Pharmacol 2021 ).

[0569] This activity is consistent with pancreatic organoid differentiation in FIG. 36 (EXAMPLE 9).

[0570] ULI262A may thus achieve these actions through balanced activation of PPARA, PPARD, and PPARG, which enables efficacy at nanomolar to low micromolar concentrations while mitigating the safety liabilities associated with selective single-isoform agonists. This distributed activation provides greater therapeutic efficacy at lower concentrations with an improved safety margin, thereby explaining the dual cell-production and antioxidant actions of ULI262.

Claims

Claims1 . A compound of Formula (I) or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof:

2. The compound of claim 1 , wherein Formula (I) is in the form of Formula (la):

3. An isolated compound of formula (la):(la)4. A pharmaceutical composition comprising the compound of any preceding claim, and a pharmaceutically acceptable excipient.

5. A composition comprising the compound of any of claims 1 to 4, in an amount of at least 0.5% by weight of the composition.

6. The composition according to claim 5, wherein the compound is present in the composition in an amount of at least 1%, at least 2% at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, or at least 50%, by weight.

7. The composition according to claim 5 or claim 6, comprising a pharmaceutically acceptable excipient.

8. The composition according to any of claims 4 to 7, which is sterile.

9. The composition according to any of claims 4 to 8, in the form of a solution, suspension, powder, tablet, capsule, lozenge, suppository, buccal product, cream, ointment, gel, film or patch.

10. The composition according to any of claims 4 to 9, which is substantially free of flavonoids, alkaloids, essential oils, phenolics and / or tannins.

11. The composition according to any of claims 4 to 10, which, aside from the compound Formula (I) or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof, comprises substantially no other lactone, or comprises substantially no other sesquiterpene lactone.

12. The composition according to any of claims 4 to 11 , comprising cell growth factors, optionally a lineage-specific medium (LSM).

13. The composition according to any of claims 4 to 12, comprising isolated cells, optionally wherein the isolated cells are stem cells.

14. The composition according to claim 13, wherein the isolated cells comprise pluripotent stem cells, multipotent stem cells and / or progenitor cells.

15. The composition according to any of claim 13 or claim 14, wherein the isolated cells are anorectal transition zone (ATZ) cells.

16. The compound or composition of any preceding claim, for use as a medicament.

17. An agent which is an agonist of PPAR-a, PPAR-p / 5 and PPAR-y, for use as a medicament, optionally wherein the agent is a compound as defined in any of claims 1 to 3.

18. The compound, agent or composition for the use of claim 16 or claim 17, for oral, subcutaneous, intradermal, intravenous, intra-arterial, intramuscular, intrathecal, epidural,intracistemal, intraperitoneal, transdermal, topical, transmucosal, buccal, sublingual, transmucosal, inhalation, intranasal, intra-atricular, intranasal, rectal or ocular administration.

19. The compound, agent or composition for the use according to any of claims 16 to 18, for use in treating cancer, optionally wherein: i) the cancer is colorectal cancer; and / or ii) the cancer is characterised by at least one mutation in the adenomatous polyposis coli (APC) gene.

20. The compound, agent or composition for the use according to any of claims 16 to 18, for use in treating Familial adenomatous polyposis (FAP).21 . The compound, agent or composition for the use according to any of claims 16 to 18, for use in treating inflammation.

22. The compound, agent or composition for the use according to any of claims 16 to 18, for use in treating oxidative stress.

23. The compound, agent or composition for the use according to any of claims 16 to 18, for use in treating damaged, defective or dysfunctional tissue, optionally wherein the damaged, defective or dysfunctional tissue is endodermal tissue, mesodermal tissue, or ectodermal tissue.

24. The compound, agent or composition for the use according to any of claims 16 to 18, for use in treating a disease or disorder of the gastrointestinal tract, optionally wherein the disease or disorder is cancer, Crohn’s disease, chronic gut inflammation or inflammatory bowel disease.

Images