NOVEL INHIBITOR COMPOUNDS

PRIORITY DOCUMENT

The present application claims priority from Chinese Patent Application No. 202210464329.8 titled “Novel Inhibitor Compounds” and filed on 29 April 2022, the content of which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

[0001] The present disclosure relates to a novel compound, which is an analogue of vitamin D. The present disclosure also relates to pharmaceutical compositions including the novel compound, and to the use of the novel compound in treating and/or preventing disorders associated with low vitamin D levels, particularly disorders that respond to the inhibition of the CYP24A1 enzyme.

BACKGROUND

[0002] The CYP24A1 enzyme is a mitochondrial inner membrane cytochrome P450 component that acts naturally to catabolize both 25-hydroxycholecalciferol (25(OH)D ₃ ) and 1,25-dihydroxychole- calciferol (1,25(OH) ₂ D ₃ ) to control vitamin D hormonal actions in different tissues (Anderson, P.H., Curr. Osteoporos. Rep., 2017. 15(5): p. 443-449). This catabolism occurs via hydroxylation at either C23 or C24 of the aliphatic side chain of 25(OH)D ₃ and 1,25(OH) ₂ D ₃ (structures given below), resulting in less-active catabolites and culminating in the formation of calcitroic acid (St-Arnaud, R. and G. Jones, CYP24A1: structure, function, and physiological role, in Vitamin D. 2018, Elsevier, p. 81-95). CYP24A1 gene expression in healthy individuals is regulated by the effects of 1,25(OH) ₂ D ₃ , parathyroid hormone (PTH), and fibroblast growth factor 23 (FGF23) (Jones, G., Prosser, D.E. and Kaufmann, M. Arch. Biochem. Biophys., 2012. 523(1): p. 9-18), with 1,25(OH) ₂ D ₃ being the predominant stimulator of expression (Zierold, C., et al., Arch. Biochem. Biophys., 2000. 381(2): p. 323-327).

[0003] Chronic kidney disease (CKD) is a progressive disease characterized by gradual deterioration of kidney function. CKD is a growing public health issue, with comorbidities such as hypertension and diabetes being major contributors (Webster, A.C., et al., The Lancet, 2017. 389(10075): p. 1238-1252.; Patel, U.D., et al., Am. J. Kidney Dis., 2005. 46(3): p. 406-414). An inevitable event in CKD is the disturbance of the renal vitamin D metabolic pathways, which involve both 25-hydroxy-vitamin D la- hydroxylase (CYP27B1) and CYP24A1. The gradual reduction of renal function translates into a progressive decline in CYP27B 1 activity leading to a decrease in the production of circulating 1,25(OH) ₂ D ₃ (St- Arnaud, R. and G. Jones, ibid). Additionally, elevated serum FGF23 levels inhibit renal CYP27B1 expression and stimulate renal CYP24A1 expression, which contributes to inadequate 1,25(OH) ₂ D ₃ production (Helvig, C.F., et al., Kidney Int., 2010. 78(5): p. 463-472). Circulating levels of 25(OH)D ₃ , as a precursor substrate for 1,25(OH) ₂ D ₃ , have also been reported to decline in CKD due to factors including proteinuria (Caravaca-Fontan, F., et al., Nefrologia (English Edition), 2016. 36(5): p. 510-516). Inappropriately low levels of 1,25(OH) ₂ D ₃ lead to impaired intestinal calcium absorption and hypocalcaemia, which in turn induces secondary hyperparathyroidism (SHPT) (Elder, G., J. Bone Miner. Res., 2002. 17(12): p. 2094-2105; Martinez, I., et al., Am. J. Kidney Dis., 1997. 29(4): p. 496-502).

[0004] 1,25(OH) ₂ D ₃ or VDR-activating vitamin D analogues (such as alfacalcidol, maxacalcitol, doxercalciferol, or paricalcitol) are often prescribed for patients with declining renal function, with the aim being to reduce PTH levels (Driieke, T.B., Curr. Opin. Nephrol. Hypertens., 2005. 14(4): p. 343-349; Frazao, J.M., et al., Am. J. Kidney Dis., 2000. 36(3): p. 550-561). However, some factors limit the usefulness of active vitamin D or vitamin D analogue therapy, such as the resultant hypercalcemia and the inappropriately high levels of CYP24A1 activity, which, besides the deactivation of 1,25(OH) ₂ D ₃ , also have the potential to deactivate vitamin D analogues (Posner, G.H., et al., J. Steroid Biochem. Mol. Biol., 2010. 121(1-2): p. 13-19). [0005] Vitamin D deficiency also directly impacts on systems including immune function, cardiovascular health and reproductive health. The association between vitamin D deficiency and cancer incidence has also been demonstrated in numerous different settings, including breast cancer (Feldman, D. et al., Nat. Rev. Cancer, 2014. 14: p. 342-357; O’Brien, K. M. et al., Environ. Health Perspect. 2017. 125: p. 077004). Further, several reports suggest that vitamin D may play a role in breast cancer therapy (O’Brien, K. M. et al., Environ. Health Perspect. 2017. 125: p. 077004; Swami, S. et al., Endocrinology, 2012. 153: p. 2576-2587) and in predicting disease recurrence and survival. The mechanisms by which vitamin D purportedly exerts breast cancer-protective actions are diverse (Welsh, J. J. Steroid Biochem. Mol. Biol. 2017). Direct interaction and genomic repression of estrogen receptor (ER) activity by VDR has been reported to be a mechanism through which vitamin D exerts growth-suppressive actions in breast cancer cells (Swami, S. et al., Endocrinology, 2012. 153: p. 2576-2587). In addition to VDR, the regulation of vitamin D activity at target tissue level is governed by catabolic actions of CYP24A1. In invasive breast cancer cells and tissues, CYP24A1 gene amplification (Davis, L. M. et al., 2007. 9: p. 327-336; Albertson, D. G. et al., 2000.: p. 144-146; Rennstam, K. and Hedenfalk, I. Breast Cancer Res, 2006. 8(213)), as well as higher CYP24A1 mRNA and protein expression, have been observed when compared to normal or in situ counterparts and independent of 1,25(OH) ₂ D ₃ induction (Lopes, N. et al., BMC Cancer, 2010. 10(483); Townsend, K. et al., Clin. Cancer Res. 2005. 11: p. 3579-3586). While it is uncertain whether CYP24A1 over-activity is causal, or is a consequence, of cancer progression, the therapeutic use of vitamin D or vitamin D analogues may be relatively ineffective in breast cancer where CYP24A1 activity is shown to be aberrantly high.

[0006] 1,25(OH) ₂ D ₃ also exhibits immunomodulatory and anti-inflammatory effects in the intestine and protects against multiple gastrointestinal diseases (Almerighi, C. et al., Cytokine, 2009. 45(3): p. 190- 197; Froicu, M. and Cantorna, M. T., BMC Immunol., 2007. 8(30): p. 5). It protects the intestine via modulation of the gut microbiota and by acting via the VDR, and has demonstrated protective effects in the intestine by improving ulceration, erythema, and pain (Anand, A. et al., Contemp. Oncol. (Pozn), 2017. 21(2): p. 145-151; Meeker, S. et al., Cancer Res., 2014. 74(16): p. 4398-4408). Chemotherapy agents such as 5 -fluorouracil (5-FU) lead to a high rate of gastrointestinal mucositis (GM), posing as a significant clinical problem for cancer patients undergoing chemotherapy. GM is characterised as severe ulcers in the alimentary tract, presenting as symptoms such as pain, diarrhoea, vomiting, and weight loss due to malabsorption in the intestine (Harris, D. J., Ther. Clin. RiskManag., 2006. 2(3): p. 251-258). Therefore, mucositis significantly impacts patients’ quality of life, and poses as a dose-limiting complication as it often results in a reduction or cessation of cancer treatments, severely compromising patients’ survival outcomes (lacovelli, R., et al. Critical Reviews in Oncology /Hematology, 2016.

98(2016/02): p. 24-28). There are currently limited treatment options available for GM.

[0007] Preventing 1,25(OH) ₂ D ₃ breakdown to improve the half-life of 1,25(OH) ₂ D ₃ could offer a strategy that may improve vitamin D activity and improve responsiveness to vitamin D therapies. In work leading to the present disclosure, the inventors investigated a targeted CYP24A1 inhibition approach with prospective therapeutic value in conditions with low vitamin D activity. The inventors synthesized a novel C-24 O-methyl oxime analogue of 1,25(OH) ₂ D ₃ that has specific CYP24A1 inhibitory properties, and therefore may be useful in the treatment and/or prevention of diseases or disorders associated with low physiological vitamin D levels, and, in particular, in diseases or disorders that respond to CYP24A1 inhibition.

SUMMARY

[0008] In a first aspect, the present disclosure provides a compound of formula (I): wherein:

R ₁ is selected from H and OH,

R ₂ , R ₃ and R ₄ are each independently selected from CH ₃ and F, or a pharmaceutically-acceptable salt, solvate or prodrug thereof. [0009] The compound of formula (I) may be a compound of formula (la): or a pharmaceutically-acceptable salt, solvate or prodrug thereof.

[0010] In a second aspect, the present disclosure provides a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, and one or more pharmaceutically-acceptable excipients. The pharmaceutical composition may be adapted for oral administration. The pharmaceutical composition may be adapted for subcutaneous administration.

[0011] In a third aspect, the present disclosure provides a method of treating and/or preventing a disease or disorder associated with low physiological vitamin D levels in a subject, the method comprising administering to said subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, optionally in combination with a pharmaceutically-acceptable excipient.

[0012] The disease or disorder may be a disease or disorder that responds to CYP24A1 inhibition.

[0013] Accordingly, the present disclosure also provides a method of treating and/or preventing a disease or disorder that responds to CYP24A1 inhibition in a subject, the method comprising administering to said subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, optionally in combination with a pharmaceutically-acceptable excipient. [0014] The disease or disorder may be chronic kidney disease (CKD), or a disease or disorder associated with CKD. The disease or disorder associated with CKD may be secondary hyperparathyroidism (SHPT).

[0015] The disease or disorder may be chemotherapy-induced gastrointestinal mucositis. In particular, the disease or disorder may be 5 -fluorouracil-induced gastrointestinal mucositis.

[0016] The disease or disorder may be a gut disorder associated with inflammation and an altered microbiome, such as inflammatory bowel disease, irritable bowel syndrome or diverticulitis.

[0017] The disease or disorder may be cancer. In particular, the disease or disorder may be breast cancer.

[0018] The compound of formula (I) may be administered in combination with another agent, for example, another agent that: treats and/or prevents the disease or disorder; can enhance the activity of the compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, in the treatment and/or prevention of the disease or disorder; and/or an agent whose effects can be potentiated by the compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, in the treatment and/or prevention of the disease or disorder.

[0019] In a fourth aspect, the present disclosure provides the use of a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, for treating and/or preventing a disease or disorder associated with low physiological vitamin D levels.

[0020] The present disclosure also provides the use of a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, for treating and/or preventing a disease or disorder that responds to CYP24A1 inhibition.

[0021] In a fifth aspect, the present disclosure provides a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, for treating and/or preventing a disease or disorder associated with low physiological vitamin D levels.

[0022] The present disclosure also provides a compound of formula (I), or a pharmaceutically- acceptable salt, solvate or prodrug thereof, for treating and/or preventing a disease or disorder that responds to CYP24A1 inhibition. [0023] In a sixth aspect, the present disclosure provides the use of a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, in the manufacture of a medicament (such as a pharmaceutical composition) for treating and/or preventing a disease or disorder associated with low physiological vitamin D levels.

[0024] The present disclosure also provides the use of a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, in the manufacture of a medicament (such as a pharmaceutical composition) for treating and/or preventing a disease or disorder that responds to CYP24A1 inhibition.

[0025] In a seventh aspect, the present disclosure provides a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, and a pharmaceutically acceptable excipient, for use in treating and/or preventing a disease or disorder associated with low physiological vitamin D levels.

[0026] The present disclosure also provides a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, and a pharmaceutically acceptable excipient, for use in treating and/or preventing a disease or disorder that responds to CYP24A1 inhibition.

BRIEF DESCRIPTION OF FIGURES

[0027] Figure 1 shows mean fluorescence polarization (n = 3) measurements in millipolarization (mP) for the bound 1 nM Fluormone™/3.62 nM VDR full-length protein interacting with each of the sixteen tested concentrations (0.0007 to 1 x 10 ⁴ nM represented at Log scale in the figure) for each of the three tested compounds (1,25(OH) ₂ D ₃ , 25(OH)D ₃ and VD1-6). Error bars represent standard deviation from the mean. Solid lines connecting the points represent the curve fitting using sigmoidal dose -response modelling with varying slope.

[0028] Figure 2 shows simulated docking position of 1,25(OH) ₂ D ₃ (left), and VD1-6 (right) in the CYP24A1 active pocket.

[0029] Figure 3 provides results showing the effect of VD1-6 (10 ^-7 M), 1,25(OH) ₂ D ₃ (10 ^-7 M ) and the combined treatment of them each at (10 ^-7 M ) on the mRNA levels of CYP24A1, CYP27B1, and VDR produced in HEK293T cell culture at 24 h. * p < 0.05 vs. vehicle control; * p < 0.05 vs. 1,25(OH) ₂ D ₃ (10 M) treatment. Values are represented as mean ± SD.

[0030] Figure 4 shows levels of 1,25(OH) ₂ D ₃ in HEK293T cell culture at 0, 8 and 24 h in the absence and presence of VD1-6 at 10 ^-8 M and 10 ^-7 M . Mean ± S.D, n=4/group; ^a p < 0.05 vs 0 h; ^b p < 0.05 vs. 1,25(OH) ₂ D ₃ alone at 8 h; ^c p < 0.05 vs. 1,25(OH) ₂ D ₃ alone at 24 h.

[0031] Figure 5 provides 24,25(OH) ₂ D ₃ levels (A) and relative CYP24A1 mRNA levels (B) in HEK293T cell culture at 72 h in the absence and presence of VD1-6 (10 ^-11 - 10 ^-7 M ). Mean ± S.D, n=4/group; ^a p < 0.05 vs. 25(OH)D ₃ only treatment; ^b p < 0.05 vs. 25(OH)D ₃ +10 ^-11 M VD1-6 treatment; ^c p < 0.05 vs. 25(OH)D ₃ + 10 ^-10 M VD1-6 treatment; ^d p < 0.05 vs. 25(OH)D ₃ + 10 ^-9 M VD1-6 treatment; ^e p < 0.05 vs. 25(OH)D ₃ + 10 ^-8 M VD1-6 treatment.

[0032] Figure 6 provides: A) Schematic outline of Cre-mediated excision of exon 5 of CYP24A1 gene and primer placement; B) DNA analysis from intestine and kidney (vitamin D handling tissues) shows CYP24A1 was deleted in IntCYP24A1 ^-/- intestinal tissue (duodenum, jejunum and colon) exclusively, but was present in kidney. CYP24A1 was present in all tissues in CYP24A1 ^fl/fl mice; C) mRNA expression of CYP24A1 relative to RplpO in IntCYP24A1 ^-/- mice was negligible in the small intestine compared to the CYP24A1 ^fl/fl mice (mean ± SEM).

[0033] Figure 7 provides: A) H&E-stained sections of duodenum jejunum, and colon treated with the CYP24Al ^fl/fl saline (A, E, I, respectively), CYP24A1 ^fl/fl 5-FU (B, F, J, respectively), IntCYP24A1 ^-/- saline (C, G, K, respectively) or IntCYP24A1 ^-/- 5-FU (D, H, L, respectively) (photomicrographs taken at 20x objective); B) Histology measures for the duodenal villi (A, B) and crypts (C, D), jejunal villi (E, F) and crypts (G, H) and colonic crypts (I, J, respectively) (each point represents the mean ± SEM of each individual mouse; *p<0.5, **p<0.01 and ***p<0.001 vs. CYP24A1 ^fl/fl saline controls).

[0034] Figure 8 provides: A) H&E stained sections of duodenum and colon treated with saline, VD1-6 (“Polara 1”), 5-FU or VD1-6 + 5-FU (photomicrographs taken at 20x objective); B) Histology measures for the duodenal villi (A, B) and crypts (C, D), jejunal villi (E, F) and crypts (G, H) and colonic crypts (I, J, respectively) (data shown as mean ± SEM. *p<0.5, **p<0.01 and ***p<0.001 vs. untreated controls (saline)).

[0035] Figure 9 provides Alcian blue staining showing the effects of CYP24A1 deletion prior to 5- FU administration on the total number of goblet cell counts in the intestine (data shown as mean ± SEM. *p<0.5, **p<0.01 and ***p<0.001 vs. CYP24A1 ^fl/fl saline controls; photomicrographs taken at 40x objective).

[0036] Figure 10 provides immunostaining of Ki-67 in the intestine following 5-FU administration: Ki-67 antibody-stained sections of duodenum, jejunum, and colon treated with CYP24A1 ^fl/fl saline (A, F, K, respectively), CYP24A1 ^fl/fl 5-FU (B, G, L, respectively), IntCYP24A1 ^-/- saline (C, H, M, respectively) or IntCYP24A1 ^-/- 5-FU (D, I, N, respectively); counts of Ki-67 positive cells per demi-crypt for the duodenum (E), jejunum (J) and colon (O, respectively) (each point represents the mean ± SEM of each individual mouse; *p<0.5, **p<0.01 and ***p<0.001 vs. CYP24A1 ^fl/fl saline controls; photomicrographs taken at 40x objective).

[0037] Figure 11 provides immunostaining of Ki-67 in the intestine following 5-FU administration: Ki-67 antibody-stained sections of duodenum, jejunum, and colon treated with saline (A, F, K, respectively), VD1-6 (“Polara 1”) (B, G, L, respectively), 5-FU (C, H, M, respectively) or VD1-6 + 5-FU (D, I, N, respectively); counts of Ki-67 positive cells per demi-crypt for the duodenum (E), jejunum (J) and colon (O, respectively) (data shown as mean ± SEM. *p<0.5, **p<0.01 and ***p<0.001 vs. untreated controls (saline); photomicrographs taken at 40x objective).

[0038] Figure 12 provides the effect of VD1-6 (“Polara 1”) during 5-FU administration on the expression of vitamin D responsive genes in the colon of treated mice: mean mRNA expression of CYP24A1 (A), CYP27B1 (B), and VDR (C), relative to control gene RPLP0 (data shown as mean ± SEM. *p<0.5, **p<0.01 and ***p<0.001 vs. untreated controls (saline)).

[0039] Figure 13 provides the effect of VD1-6 (“Polara 1”) during 5-FU administration on the expression of inflammatory genes in the colon of treated mice: mean mRNA expression of TNF-α (A) and NF-KB (B) relative to RPEP0 (data shown as mean ± SEM. p<0.5 for all groups).

[0040] Figure 14 provides the effect of VD 1-6 (“Polara 1”) during 5-FU administration on the expression of innate immunity -related genes in the colon of treated mice: average mRNA expression of lipocalin-2 (A), TLR4 (B), and TLR5 (C) relative to RPEP0 (data shown as mean ± SEM. *p<0.5, **p<0.01 and ***p<0.001 vs. untreated controls (saline)).

[0041] Figure 15 provides the relative abundance of family taxonomic level present in colon contents of mice: data shown as A) individual animals in specific treatment groups and B) summary of individuals in each group. [0042] Figure 16 provides alpha diversity of gut microbiota in mice treated with 5-FU and/or VD1-6 (“Polara 1”) (each point represents Shannon index for each animal, expressed as median (solid line), mean (dashed line), interquartile ranges and range (whiskers)).

[0043] Figure 17 provides beta diversity of gut microbiota in mice treated with 5-FU and/or VD1-6 (“Polara 1”): principal component analysis 3-dimensional plot, PCol vs PCo2 vs PCo3 (A) and Principal component analysis 2-dimensional plot, PCol vs PCo2 (B), suggest clustering of Chemotherapy only (stars, 5-FU treated) to right. Pearson’s correlation (C, D) demonstrates shift of PCol driven by Akkermansiaceae family (R ² = 0.764, p < 0.0001) and to a lesser degree Mariniflaceae family (R ² = 0.183, p = 0.0415).

DETAILED DESCRIPTION

[0044] The inventors have found that a compound of formula (I) can selectively inhibit CYP24A1. In particular, the compound of formula (I) does not bind to the vitamin D receptor (VDR), thus avoiding off-target effects that may lead to hypercalcaemia (in the case of VDR activation) or hypocalcaemia (when the VDR is inactivated), which may result in rickets and osteomalacia. Further, by lowering endogenous catabolism of 25(OH)D ₃ and 1,25(OH) ₂ D ₃ , the body’s own vitamin D is used. This allows the body to compensate, or self-regulate, the actions of vitamin D. As endogenous vitamin D activity is elevated, the need for the body to attempt to drive more vitamin D production (via PTH) lowers, leading to a fall in PTH levels. If levels of the compound of formula (I) are stable, the body could reach a new set point of vitamin D activity with a lower level of PTH, which is a safer way to elevate vitamin D activity compared with administering exogenous vitamin D (or any analogues). In addition, the compound of formula (I) has superior binding to CYP24A1 when compared to that of 1,25(OH) ₂ D ₃ , thus making it an effective inhibitor of CYP24A1 even in the presence of endogenous 1,25(OH) ₂ D ₃ . Accordingly, the inventors have found that the compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, shows considerable promise in the treatment and/or prevention of diseases or disorders associated with low physiological vitamin D levels.

[0045] Thus, in a first aspect, the present disclosure provides a compound of formula (I):

wherein:

R ₁ is selected from H and OH,

R ₂ , R ₃ and R ₄ are each independently selected from CH ₃ and F, or a pharmaceutically-acceptable salt, solvate or prodrug thereof. R ₁ may be OH. R ₂ , R ₃ and R ₄ may be CH ₃ . The compound of formula (I) may be a compound of formula (la): or a pharmaceutically-acceptable salt, solvate or prodrug thereof. [0046] The term "pharmaceutically-acceptable salt" as used herein, refers to salts that retain the desired biological activity of the compounds of formula (I), and includes pharmaceutically-acceptable acid addition salts and base addition salts. Suitable pharmaceutically-acceptable base addition salts of the compounds of formula (I) may be prepared from an inorganic base or from an organic base. Examples of such inorganic bases are sodium, potassium, calcium, aluminium and magnesium. Additional information on pharmaceutically-acceptable salts can be found in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Co., Easton, PA 1995.

[0047] "Prodrug" means a compound that undergoes conversion to a compound of formula (I) within a biological system, usually by metabolic means (e.g., by hydrolysis, reduction or oxidation). For example, an ester prodrug of a compound of formula (I) may be convertible by hydrolysis in vivo to the compound of formula (I). Suitable esters of the compounds of formula (I) may be, for example, acetates, citrates, lactates, tartrates, malonates, oxalates, salicylates, propionates, succinates, fumarates, maleates, isethionates, di-p-toluoyltartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, p- toluenesulfonates, cyclohexylsulfamates and quinates. Examples of prodrugs are provided in “Prodrugs: challenges and rewards”, Valentino J Stella (ed), Springer, 2007.

[0048] In the case of compounds of formula (I) that are solid, it will be understood by those skilled in the art that the compounds (or pharmaceutically acceptable salts, solvates or prodrugs thereof) may exist in different crystalline or polymorphic forms, all of which are encompassed within the scope of the present invention.

[0049] The term "solvate" refers to any form of a compound of formula (I) resulting from solvation with an appropriate solvent. Such a form may be, for example, a crystalline solvate or a complex that may be formed between the solvent and the dissolved compound.

[0050] The present invention further provides a method of synthesising a compound of formula (I), or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0051] With regard to the description of the synthetic methods described below and in the referenced synthetic methods that are used to prepare starting materials, it will be understood by those skilled in the art that all proposed reaction conditions, including choice of solvent, reaction atmosphere, reaction temperature, duration of the experiment and workup procedures, can be readily selected. Moreover, it will be understood by those skilled in the art that the functionality present on various portions of the molecule must be compatible with the reagents and reaction conditions utilised. [0052] Necessary starting materials may be obtained by standard procedures of organic chemistry. The preparation of such starting materials is described in conjunction with the following representative process variants and within the examples hereinafter. Alternatively, necessary starting materials may be obtainable by analogous procedures to those illustrated which are within the ordinary skill of those skilled in the art. Further, it will be appreciated that during the synthesis of the compounds, in the processes described below, or during the synthesis of certain starting materials, it may be desirable to protect certain substituent groups to prevent their undesired reaction. Those skilled in the art will readily recognise when such protection is required, and how such protecting groups may be put in place, and later removed. Examples of protecting groups are described in, for example, Protective Groups in Organic Synthesis by Theodora Green (publisher: John Wiley & Sons). Protecting groups may be removed by any convenient method well known to those skilled in the art as appropriate for the removal of the protecting group in question, such methods being chosen so as to effect removal of the protecting group with the minimum disturbance of groups elsewhere in the molecule. Thus, if reactants include, for example, groups such as amino, carboxyl or hydroxyl, it may be desirable to protect the group in some of the reactions mentioned herein.

[0053] An example of a particularly suitable method for synthesising a compound of the present invention is shown in Scheme 1 below. The compound of formula (I) is referred to as VD1-6 in Scheme 1. Iodo VD1-1, which was accessed from vitamin D2 following previously reported methods (Posner, G.H., et al., J. Org. Chem., 1997. 62(10): p. 3299-3314; Kahraman, M., et al., J. Med. Chem., 2004. 47(27): p. 6854-6863), was reacted with an enolate formed in situ from 3,3-diemthyl-2-butanone, to give ketone VD1-2 in 72% yield. The O-methyloxime functionality was installed using methoxyamine hydrochloride in pyridine to give VD1-3 in 69% yield. Desilylation for 1 hour at room temperature using camphor sulphonic acid (CSA) revealed the secondary alcohol which was then oxidised using pyridinium chromate (PDC) to give ketone VD1-4 in a 68% yield over the two steps. Horner-Wads worth-Emmons (HWE) chemistry was then employed to react ketone VD1-4 with commercially-available phosphonate VD1-5, under basic conditions, to conjugate the two fragments by an olefin linker. Removal of the silyl protecting groups, again using CSA, revealed the anti -planar hydroxy groups in VD1-6 in 43% yield over two-steps.

[0054] In a further aspect of the present invention, a method of synthesising a compound of formula

(I), or a pharmaceutically acceptable salt, solvate or prodrug thereof, is provided, wherein the method comprises the steps set out in Scheme 1.

[0055] The resultant compound of formula (I) can be isolated and purified using techniques well known to those skilled in the art.

[0056] A compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, may be formulated into a pharmaceutical composition with a pharmaceutically-acceptable excipient.

Therefore, in a second aspect, the present disclosure provides a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, and one or more pharmaceutically-acceptable excipients.

[0057] Examples of suitable excipients for different forms of pharmaceutical compositions are well known to those skilled in the art and are described in, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 1995, and in the Handbook of Pharmaceutical Excipients, 2 ^nd Edition, (1994), Edited by A Wade and PJ Weller. The choice of excipient may be made with regard to the intended route of administration and standard pharmaceutical practice.

[0058] A pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically- acceptable salt, solvate or prodrug thereof, may comprise any suitable carriers, diluents, binders, lubricants, suspending agents, coating agents and solubilising agents. Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water. Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Preservatives, stabilising agents, dyes and even flavouring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Anti-oxidants and suspending agents may be also used.

[0059] A pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically- acceptable salt, solvate or prodrug thereof, may be adapted for oral, rectal, vaginal, parenteral, intramuscular, intraperitoneal, intraarterial, intrathecal, intrabronchial, subcutaneous, intradermal, intravenous, nasal, buccal or sublingual routes of administration. The pharmaceutical composition may be adapted for oral administration. For oral administration, particular use may be made of compressed tablets, pills, tablets, gellules, drops, and capsules. For other forms of administration, a pharmaceutical composition may comprise solutions or emulsions which may be injected intravenously, intraarterially, intrathecally, subcutaneously, intradermally, intraperitoneally or intramuscularly, and which are prepared from sterile or sterilisable solutions. The pharmaceutical composition may be adapted for subcutaneous administration. A pharmaceutical composition comprising a compound of the present invention may also be in the form of suppositories, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions or dusting powders. A pharmaceutical composition may be formulated in unit dosage form (i.e., in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose).

[0060] As discussed above, the compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, may be useful in the treatment and/or prevention of a disease or disorder associated with low physiological vitamin D levels. Accordingly, in another aspect, the present disclosure provides a method of treating and/or preventing a disease or disorder associated with low physiological vitamin D levels in a subject, the method comprising administering to said subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, optionally in combination with a pharmaceutically-acceptable excipient.

[0061] “Associated with”, in the context of a disease or disorder, means a disease or disorder that is the result of the disease or disorder that it is associated with, or that leads (or contributes) to a disease or disorder that it is associated with. For example, SHPT is associated with CKD as CKD leads to SHPT. In the case of a disease or disorder associated with low physiological vitamin D levels, low physiological vitamin D levels may be the result of a disease or disorder. Examples of disorders that cause or result in low physiological vitamin D levels include cystic fibrosis, Crohn’s disease, celiac disease, obesity, cancer (e.g., breast cancer) and liver and kidney diseases, such as CKD. Conversely, low physiological vitamin D levels can lead to a loss of bone density, and thus may be the cause of diseases and disorders such as osteoporosis, bone fractures, rickets and osteomalacia. Low physiological vitamin D levels can also lead to the depletion or loss of the protective capacity of the gut barrier and the depletion of the mucous lining of the intestine, contributing to the development of gastrointestinal mucositis. Inhibiting vitamin D catabolism may also maintain the composition of the gut microbiota, which contributes to the prevention of gut damage following treatment with 5 -fluorouracil (5-FU), and may also assist is preventing or treating gut disorders associated with inflammation and an altered microbiome, such as inflammatory bowel disease (IBD), irritable bowel syndrome (IBS) or diverticulitis.

[0062] The term “low physiological vitamin D levels”, as used in the art, is correlated with serum 25(OH)D ₃ levels, and is based on bone health parameters. A 25(OH)D ₃ level below 50 nmol/L is considered low, and may lead to osteoporosis. Below 10-20 nmol/L is considered very low and can lead to rickets in children.

[0063] The method may be applied to the prevention or treatment of CKD, or a disease or disorder associated with CKD. CKD is characterised by a slow and progressive loss of kidney function, and occurs when a disease or condition impairs kidney function, causing kidney damage to worsen over several months or years. Some diseases and conditions that cause CKD include: Type 1 and Type 2 diabetes; high blood pressure; glomerulonephritis; interstitial nephritis; polycystic kidney disease or other inherited kidney diseases; prolonged obstruction of the urinary tract (from conditions such as enlarged prostate, kidney stones, and some cancers); vesicoureteral reflux, and pyelonephritis. [0064] CKD is divided into five stages. The stages are based primarily on the estimated glomerular filtration rate (eGFR) test result. As the number of the stage increases, renal function deteriorates. Typically, the stages are designated as follows:

1. Stage 1 - eGFR 90 or greater (mild kidney damage);

2. Stage 2 - eGFR between 60 and 90 (mild kidney damage, but other signs, such as protein in the urine and/or physical damage to the kidneys);

3. Stage 3 - eGFR between 30 and 59 (some damage to kidneys, and symptoms may include swelling in hands and feet, back pain, more frequent or less frequent urination, high blood pressure, anemia, bone disease);

4. Stage 4 - eGFR between 15 and 29 (moderate to severe kidney damage, and similar symptoms to Stage 3);

5. Stage 5 - eGFR less than 15 (very close to kidney failure, or kidney failure has occurred).

Treatment in accordance with the present invention may be useful at Stages 1 and 2, and in some cases at Stage 3.

[0065] As discussed above, the gradual reduction of renal activity observed in CDK leads to the disturbance of the renal vitamin D metabolic pathways, resulting in inappropriately low levels of vitamin D. This causes impaired intestinal calcium absorption and hypocalcaemia, which in turn induces secondary hyperparathyroidism (SHPT). The inventors have found that, by inhibiting one of the enzymes (CYP24A1) involved in the catabolism of 1,25(OH) ₂ D ₃ , it may be possible to restore vitamin D to normal levels (i.e., where the 25(OH)D ₃ level is 50 nmol/L or above).

[0066] Therefore, the present disclosure also provides a method of treating and/or preventing a disease or disorder that responds to CYP24A1 inhibition in a subject, the method comprising administering to said subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, optionally in combination with a pharmaceutically-acceptable excipient. [0067] Accordingly, the disease or disorder may be CKD, or a disease or disorder associated with CKD. The disease or disorder associated with CKD may be SHPT. Therefore, the present disclosure also provides a method of treating and/or preventing CKD, or a disease or disorder associated with CKD, in a subject, the method comprising administering to said subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, optionally in combination with a pharmaceutically-acceptable excipient. The disease or disorder associated with CKD may be SHPT.

[0068] The disease or disorder may be chemotherapy-induced gastrointestinal mucositis (GM). One of the major causes of GM is high-dose cancer therapy. The onset and persistence of GM involves cross- communication between the specific chemotherapeutic drug, the immune system, and gut microbes that results in a loss of mucosal integrity leading to gut-barrier dysfunction. It is defined as inflammatory and/or ulcerative lesions of the gastrointestinal tract, and is characterised by crypt loss, villus atrophy, loss of renewal capacity, and impairment of gut function. In some embodiments, the “gastrointestinal tract”, as defined herein, does not include the mouth or oral cavity. The term “gastrointestinal tract” may also exclude the pharynx, oesophagus and stomach. The term “gastrointestinal tract” may refer to the section of the gastrointestinal tract from the pyloric sphincter to the rectum.

[0069] A variety of assessment scales exist for measurement of oral mucositis. Two of the most commonly utilized scales are the WHO and National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) scales (Peterson, D. E. et al., Aim. Oncol., 2011. 22(suppl. 6):p. vi78-vi84):

WHO scale for oral mucositis

Grade 0 = No oral mucositis

Grade 1 = Erythema and soreness

Grade 2 = Ulcers, able to eat solids

Grade 3 = Ulcers, requires liquid diet (due to mucositis)

Grade 4 = Ulcers, alimentation not possible (due to mucositis) NCI Common Terminology Criteria for Adverse Events (CTCAE) version 4.0:

Grade 1 = Asymptomatic or mild symptoms; intervention not indicated.

Grade 2 = Moderate pain; not interfering with oral intake; modified diet indicated

Grade 3 = Severe pain; interfering with oral intake

Grade 4 = Life-threatening consequences; urgent intervention indicated

Grade 5 = Death.

[0070] Most of the scales that are utilized for clinical care incorporate the collective measurement of oral symptoms, signs and functional disturbances (Peterson, D. E. et al., ibid). Prevention in accordance with the present invention will be useful before onset of any symptoms i.e., at Grade 0 of the WHO scale, or Grade 1 of the NCI scale. Prevention may also be useful where some symptoms have developed e.g., at Grade 1 or Grade 2 of the WHO scale, or Grade 2 of the NCI scale. Given the proliferation data (Ki67 antibody) discussed in the Examples, administering a compound of formula (I), even after some damage has occurred, may increase proliferation to restore intestinal villi following damage, and also prevent GM in subsequent rounds of chemotherapy.

[0071] The present inventors have found that the compound of formula (I), or a pharmaceutically- acceptable salt, solvate or prodrug thereof, is effective at preventing chemotherapy-induced GM. Therefore, the present disclosure also provides a method of preventing chemotherapy-induced GM in a subject, the method comprising administering to said subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, optionally in combination with a pharmaceutically-acceptable excipient. Examples of chemotherapies involving 5-FU include chemotherapy for breast cancer (e.g., combination of 5-FU, doxorubicin and cyclophosphamide), and chemotherapy for colon cancer (e.g., combination of 5-FU, leucovorin and oxaliplatin, or combination of 5-FU, leucovorin and irinotecan).

[0072] The compound of formula (I) is effective at preventing GM induced by chemotherapy involving the use of 5 -fluorouracil (5-FU). Therefore, the present disclosure also provides a method of preventing 5-FU-induced GM in a subject, the method comprising administering to said subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, optionally in combination with a pharmaceutically-acceptable excipient. In this method, the compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, may be administered before chemotherapy is administered, may be administered at the same time as chemotherapy is administered, and/or may be administered shortly after chemotherapy is administered, to a subject.

[0073] The present inventors have also found that the compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, is effective in preventing compositional alterations to gut microbiota, including in the context of chemotherapy treatment (e.g., involving 5-FU). DNA damage caused by chemotherapy agents such as 5-FU activates the innate immune system, resulting in the release of damage-associated molecular pattern molecules by damaged cells of the gastrointestinal tract (Cinausero, M. et al., Front. Pharmacol., 2017. 8: p. 354; Sonis, S., Nature Reviews Cancer, 2004. 4: p. 277-284). Over-activation of the innate response damages cell membranes by recruiting macrophages and upregulating transcription factor NF-KB, resulting in the secretion of pro-inflammatory cytokines TNF-a, IL-1 and IL-6; this cascade of events causes apoptosis of the cells of the mucosal epithelia, resulting in crypt ablation in the intestines (Cinausero, M. et al., ibid; Keefe, D. M, K., et al., Seminars in Oncology Nursing, 2004. 20(1): p. 38-47). Crypt hypoplasia in the colon disrupts the barrier function of the gastrointestinal epithelium, allowing invasion and colonisation of intraluminal bacteria into the mucosa where they cause infection and ulceration (Naidu, M. U. et al., Neoplasia, 2004. 6(5): p. 423-431). It is highly desirable that a diverse and symbiotic gut microbiota is maintained, as it plays key roles in modulating host inflammation, homeostatic immune responses, and strengthening gut integrity (Pickard, J. M. et al., Immunol. Rev., 2017. 279(1): p. 70-89). Chemotherapy agents such as 5-FU have detrimental effects on the gut microbiota as they shift the composition of microbes from commensal to pathogenic, causing dysbiosis (Stringer, A. M. et al., J. Support Oncol., 2007. 5(6): p. 259-267). The present inventors have found that the relative abundance of beneficial bacteria, such as Lachnospiraceae and Bifidobacterium, significantly increased, while the abundance of pathogenic bacteria (such as Verrucomicrobia and Faecalibaculum) significantly decreased, when a compound of formula (I) was administered. As discussed previously, 1,25(OH) ₂ D ₃ exhibits immunomodulatory and anti-inflammatory effects in the intestine and protects against multiple gastrointestinal diseases. However, 1,25(OH) ₂ D ₃ is catabolised by CYP24A1. Accordingly, the compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, which acts as a CYP24A1 inhibitor and results in prolonged increased levels of circulating 1,25(OH) ₂ D ₃ in the gut, may prevent or alleviate the intestinal damage caused by chemotherapy agents (such as 5-FU) by maintaining the composition of the gut microbiota. [0074] Accordingly, the present invention also provides a method of treating and/or preventing a gut disorder associated with inflammation and an altered microbiome in a subject, the method comprising administering to said subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, optionally in combination with a pharmaceutically-acceptable excipient. The compound of formula (I) is effective at treating and/or preventing a gut disorder associated with inflammation and an altered microbiome wherein the disorder is induced by chemotherapy. Therefore, the present disclosure also provides a method of treating and/or preventing a gut disorder associated with inflammation and an altered microbiome in a subject, wherein the disorder is induced by chemotherapy, the method comprising administering to said subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, optionally in combination with a pharmaceutically-acceptable excipient. In this method, the compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, may be administered before chemotherapy is administered, may be administered at the same time as chemotherapy is administered, or may be administered shortly after chemotherapy is administered, to a patient. The chemotherapy may involve the use of 5 -fluorouracil (5-FU). Therefore, the present disclosure also provides a method of preventing and/or treating a gut disorder associated with inflammation and an altered microbiome in a subject, wherein the disorder is induced by chemotherapy involving 5-FU, the method comprising administering to said subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, optionally in combination with a pharmaceutically-acceptable excipient. In this method, the compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, may be administered before chemotherapy is administered, may be administered at the same time as chemotherapy is administered, and/or may be administered shortly after chemotherapy is administered, to a subject.

[0075] The gut disorder associated with inflammation and an altered microbiome may be inflammatory bowel disease (IBD). IBD is a general term used to describe a number of disorders that involve chronic inflammation of the digestive tract, and types of IBD include ulcerative colitis and Crohn’s disease. Accordingly, the present invention also provides a method of treating and/or preventing IBD in a subject, the method comprising administering to said subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, optionally in combination with a pharmaceutically-acceptable excipient.

[0076] The gut disorder associated with inflammation and an altered microbiome may be irritable bowel syndrome (IBS). IBS is a common, chronic disorder that affects the large intestine. Signs and symptoms include cramping, abdominal pain, bloating, gas, and diarrhoea or constipation, or both. Accordingly, the present invention also provides a method of treating and/or preventing IBS in a subject, the method comprising administering to said subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, optionally in combination with a pharmaceutically-acceptable excipient.

[0077] The gut disorder associated with inflammation and an altered microbiome may be diverticulitis. Diverticulitis occurs when small, bulging pouches (diverticula) develop in the digestive tract. When one or more of these pouches become inflamed or infected, the condition is called diverticulitis. Signs and symptoms include severe abdominal pain, fever, nausea and a marked change in bowel habits. Accordingly, the present invention also provides a method of treating and/or preventing diverticulitis in a subject, the method comprising administering to said subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, optionally in combination with a pharmaceutically-acceptable excipient.

[0078] The inventors have also found that prevention of vitamin D catabolism by inhibition of CYP24A1 using the compound of formula (I), or a pharmaceutically- acceptable salt, solvate or prodrug thereof, lessens the incidence of breast cancer and lung metastases. In particular, the compound of formula (I) may reduce the aggressiveness of breast cancer, reduce the occurrence of metastases, and reduce the tumour size. The compound of formula (I) may be useful not only when CYP24A1 activity is aberrantly high. If synthesis of 1,25(OH) ₂ D ₃ was low due to low CYP27B1, a compound of formula (I) could assist in preserving what 1,25(OH) ₂ D ₃ is produced. In addition, a compound of formula (I) given systemically may assist in preserving circulating 25(OH)D ₃ levels, contributing to its beneficial effect.

[0079] Accordingly, the present invention also provides a method of treating cancer in a subject, the method comprising administering to said subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, optionally in combination with a pharmaceutically-acceptable excipient. In particular, the cancer may be breast cancer.

[0080] The subject will typically be a human subject, however the method is also applicable in the veterinary context and, as such, the subject may also be selected from, for example, livestock animals (e.g., cows, horses, pigs, sheep and goats), companion animals (e.g., dogs and cats) and exotic animals (e.g., non-human primates, tigers, elephants, etc.). [0081] In a further aspect, the present disclosure provides the use of a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, for treating and/or preventing a disease or disorder associated with low physiological vitamin D levels.

[0082] The present disclosure also provides the use of a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, for treating and/or preventing a disease or disorder that responds to CYP24A1 inhibition.

[0083] In another aspect, the present disclosure provides a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, for treating and/or preventing a disease or disorder associated with low physiological vitamin D levels.

[0084] The present disclosure also provides a compound of formula (I), or a pharmaceutically- acceptable salt, solvate or prodrug thereof, for treating and/or preventing a disease or disorder that responds to CYP24A1 inhibition.

[0085] In a further aspect, the present disclosure provides the use of a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, in the manufacture of a medicament (such as a pharmaceutical composition) for treating and/or preventing a disease or disorder associated with low physiological vitamin D levels.

[0086] The present disclosure also provides the use of a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, in the manufacture of a medicament (such as a pharmaceutical composition) for treating and/or preventing a disease or disorder that responds to CYP24A1 inhibition.

[0087] In another aspect, the present disclosure provides a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, and a pharmaceutically acceptable excipient, for use in treating and/or preventing a disease or disorder associated with low physiological vitamin D levels.

[0088] The present disclosure also provides a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, and a pharmaceutically acceptable excipient, for use in treating and/or preventing a disease or disorder that responds to CYP24A1 inhibition. [0089] As used herein, the phrase "manufacture of a medicament" includes the use of a compound of formula (I) directly as the medicament or in any stage of the manufacture of a medicament (such as a pharmaceutical composition) comprising one or more compounds of formula (I), or a pharmaceutically- acceptable salt, solvate or prodrug thereof.

[0090] In the case of compounds of formula (I) that are solid, it will be understood by those skilled in the art that the compounds (or pharmaceutically acceptable salts, solvates or prodrugs thereof) may exist in different crystalline or polymorphic forms, all of which are encompassed within the scope of the present disclosure.

[0091] The term "therapeutically effective amount" or "effective amount" is an amount sufficient to effect beneficial clinical or desired results. A therapeutically effective amount can be administered in one or more administrations. Typically, a therapeutically effective amount is sufficient for treating and/or preventing a disease or condition or otherwise to palliate, ameliorate, stabilise, reverse, slow or delay the progression of a disease or condition. By way of example only, a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, solvate or prodrug thereof, may comprise between about 10 mcg and about 50 mcg per day, for example between about 10 and about 40 mcg per day. However, notwithstanding the above, it will be understood by those skilled in the art that the therapeutically effective amount may vary and depend upon a variety of factors including the activity of the particular compound (or salt, solvate or prodrug thereof), the metabolic stability and length of action of the particular compound (or salt, solvate or prodrug thereof), the age, body weight, sex, health, route and time of administration, rate of excretion of the particular compound (or salt, solvate or prodrug thereof), and the severity of, for example, the disease or disorder associated with low physiological vitamin D levels.

[0092] In accordance with the present disclosure, a compound of formula (I), or a salt, solvate or prodrug thereof, may be used for both treating and/or preventing a disease or disorder. As such, it is to be appreciated that the scope of the present disclosure includes prophylaxis as well as the alleviation of established symptoms of a disease or disorder. As such, the method and uses of a compound of formula

(1) agonist in accordance with the present disclosure includes: (1) preventing or delaying the appearance of clinical symptoms of a disease or disorder, or a disease or disorder developing in an afflicted subject;

(2) inhibiting a disease or disorder (i.e., arresting, reducing or delaying the development of a disease or disorder or a relapse thereof (in case of a maintenance treatment) or at least one clinical or subclinical symptom thereof; and (3) relieving or attenuating a disease or disorder (i.e., causing regression of a disease or disorder, or at least one of its clinical or subclinical symptoms).

[0093] The compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, may be administered in combination with another agent, for example, another agent that: treats and/or prevents a disease or disorder; which can enhance the activity of the compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, in the treatment and/or prevention of a disease or disorder; and/or whose effects can be potentiated by the compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, in the treatment and/or prevention of a disease or disorder. For example, if a subject’s vitamin D levels are low and there is resistance to elevating vitamin D levels through vitamin D supplementation alone, then a combination of a vitamin D supplement with the compound of formula (I) may be useful. In addition, some subjects are resistant to analog treatment due to excessive catabolism, and therefore a combination of that analog and a compound of formula (I) may potentiate the effects of the analog. With regard to the treatment of cancer (including breast cancer) as discussed above, the combination of chemotherapy and a compound of formula (I) may treat the cancer and limit the side effects (i.e., mucositis).

[0094] Accordingly, in some embodiments, a compound of formula (I), or a pharmaceutically- acceptable salt, solvate or prodrug thereof, will be used in a combination therapy with one or more agents. The agent may be a vitamin D supplement, such as ergocalciferol (vitamin D2) or cholecalciferol (vitamin D ₃ ). The agent may be a vitamin D analog, such as alfacalcidol, maxacalcitol, doxercalciferol, ED-71, or paricalcitol. The agent may be a chemotherapy agent, such as 5-FU, or a combination of 5-FU with other chemotherapeutic agents, such as doxorubicin and cyclophosphamide, leucovorin and oxaliplatin, or leucovorin and irinotecan.

[0095] Where used in combination with another agent, the compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, and the other agent may be administered in the same pharmaceutical composition or in separate pharmaceutical compositions. If administered in separate pharmaceutical compositions, the compound of formula (I), or a pharmaceutically-acceptable salt, solvate or prodrug thereof, and the other agent may be administered simultaneously or sequentially in any order (e.g., within seconds or minutes or even hours (e.g., 1 to 2 hours)).

[0096] The compounds, uses and compositions of the present disclosure are hereinafter further described with reference to the following, non-limiting examples. EXAMPLES

Example 1 - Chemical synthesis and purification

Reagents and chemicals

[0097] Anhydrous tetrahydrofuran (THF) was purchased from Sigma- Aldrich (Shanghai). All other reagents used in the synthesis of a compound of formula (I) (referred to as VD1-6), including VD1-5 and solvents, were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai). ¹ H and ¹³ C NMR spectra were recorded at room temperature on a Bruker 400 MHz NMR or a Bruker 600 MHz NMR spectrometer in CDCI ₃ as the solvent. MS spectral data were acquired by a Sciex® API 4000 mass spectrometer. Column chromatography was performed on silica gel (200-300 mesh). The references to VD1-1, VD1-2, etc. correspond to the references used for the chemical compounds given in Scheme 1.

Synthetic protocol for VD1-6

[0098] As discussed above, iodo-VD1-1 was accessed from vitamin D2 following previously- reported methods.

[0099] (6R)-2,2-Dimethyl-6-((1R,4S,7aR)-7a-methyl-4-((triethyysilyl )oxy)octahydro-1H -inden-1- yl)heptan-3-one (“VD1-2”)

An n -BuLi (1.09 mL, 2.5 M in n-hexane) solution was added slowly into diisopropylamine (0.27 mL, 2.22 mmol) at 0 °C under a nitrogen atmosphere. The mixture was diluted using THF (5 mL) at -78 °C and stirred for 20 min. 3,3-Dimethyl-2-butanone (0.37 mL, 2.10 mmol) was then added and stirred for 30 min, before HMPA (1.71 mL, 7.74 mmol) was added and stirred for a further 15 min. To the stirring mixture was added iodo-VDl-1 (130 mg, 0.30 mmol) stirring was maintained for 2 h before being warmed to room temperature. The reaction was quenched with H ₂ O (1.0 mL) and extracted with EtOAc (3 x 20 mL). The organic portions were combined and washed with brine (10 mL), dried over anhydrous Na ₂ SO ₄ , and concentrated. The crude material was purified using column chromatography ( n-hexane) to give VD1-2 (85 mg, 72%) as a colourless oil. ¹ H NMR (400 MHz, CDCI ₃ ) 54.03 (s, 1H), 2.54—2.33 (m, 2H), 1.99-1.90 (m, 1H), 1.87-1.76 (m, 2H), 1.75-1.63 (m, 2H), 1.59-1.50 (m, 1H), 1.43-1.18 (m, 9H), 1.13 (s, 9H), 0.99-0.84 (m, 15H), 0.54 (q, J = 8.0 Hz, 6H); ¹³ C NMR (100 MHz, CDCI ₃ ) 5 216.8, 56.8, 53.2, 44.4, 42.3, 40.9, 35.2, 34.8, 33.5, 30.2, 27.4, 26.6, 23.2, 18.6, 17.8, 13.7, 7.1, 5.1. MS (ESI) calc, for C ₂₅ H ₄₈ NaO ₂ Si [M+Na] ⁺ : 431.33, found: 431.43, 839.21 [2M+Na] ⁺ . [00100] (6R,E)-2,2-Dimethyl-6-((1R,4S,7aR)-7a-methyl-4-((triethylsil yl)oxy)octahydro-1H -inden-1- yl)heptan-3-on-O-methyl oxime (“VD1-3”)

To a solution of VD1-2 (300 mg, 0.74 mmol) in pyridine (6 mL) was added NH ₂ OCH ₃ ·HCl (1.23 g, 14.70 mmol). The mixture was stirred at room temperature for 4 h before being quenched with H2O (10 mL) and extracted with EtOAc (3 x 30 mL). The organic portions were combined and washed with brine (20 mL), dried over anhydrous Na ₂ SO ₄ , and concentrated. The crude material was purified using column chromatography (2-10% CH ₂ Cl ₂ in n-hexane ) to give VD1-3 (220 mg, 69%) as a colourless oil. ¹ H NMR (400 MHz, CDCI ₃ ) 5 4.03 (s, 1H), 3.78 (s, 3H), 2.38-2.25 (m, 1H), 2.08-1.89 (m, 2H), 1.91-1.74 (m, 2H), 1.72-1.18 (m, 12H), 1.10 (s, 9H), 1.00-0.90 (m, 15H), 0.55 (q, J = 8.0 Hz, 6H); ¹³ C NMR (100 MHz, CDCI ₃ ) 5 167.6, 69.6, 61.1, 56.5, 53.5, 42.3, 41.0, 37.4, 36.5, 34.8, 32.6, 28.0, 27.4, 23.4, 23.3, 18.5, 17.9, 13.7, 7.1, 5.1. MS (ESI) calc, for C ₂₆ H ₅₂ O ₂ Si [M+H] ⁺ : 438.38, found: 438.62.

[00101] (1R,7aR)-1-((R,E)-5-(Methoxyimino)-6,6-dimethylheptan-2-yl)- 7a-methyloctahydro-4H - inden-4-one (“VD1-4”)

To a solution of VD1-3 (220 mg, 0.50 mmol) in MeOH (4 mL) was added camphor sulfonic acid (CSA) (151 mg, 0.65 mmol). The mixture was stirred at room temperature for 1 h before the reaction was quenched with sat. NaHCO ₃ (5 mL). The mixture was extracted with EtOAc (3 x 20 mL) and the organic portions were combined and washed with brine (20 mL), dried over anhydrous Na ₂ SO ₄ , and concentrated. The crude material was dissolved in CH ₂ Cl ₂ (5 mL) and PDC (415 mg, 0.32 mmol) was added. The mixture was stirred at room temperature for 40 min before being diluted with CH ₂ Cl ₂ (20 mL) and filtered through celite. The filtrate was collected and washed with sat, NaHCO ₃ (10 mL) and brine (10 mL), dried over anhydrous Na ₂ SO ₄ , and concentrated. The crude material was purified using column chromatography (5% EtOAc in n-hexane) to give VD1-4 (110 mg, 68%) as a colourless oil. ¹ H NMR (400 MHz, CDCI ₃ ) 5 3.78 (s, 3H), 2.47-2.42 (m, 1H), 2.34-2.20 (m, 3H), 2.12-2.09 (m, 1H), 2.06-1.98 (m, 2H), 1.96-1.87 (m, 2H), 1.77-1.69 (m, 1H), 1.57-1.49 (m, 3H), 1.47-1.40 (m, 2H), 1.36-1.29 (m, 2H), 1.10 (s, 9H), 1.01 (d, J = 6.0 Hz, 3H), 0.64 (s, 3H); ¹³ C NMR (100 MHz, CDCI ₃ ) 5 212.1, 167.1, 62.1, 61.1, 56.3, 50.0, 41.1, 39.1, 37.4, 36.6, 32.5, 28.0, 27.6, 24.2, 23.2, 19.3, 18.6, 12.6. MS (ESI) calc, for C20H ₃ 6NO2 [M+H] ⁺ : 322.27, found: 322.76.

[00102] (6R,E)-6-((1R,7aR,E)-4-((Z)-2-((3S,5R)-3,5-dihydroxy-2-methy lenecyclohexylidene)- ethylidene )-7a-methyloctahydro - 1 H-inden- 1 -yl )-2,2-di methylheptan-3-on-O-methyl oxime (“VD 1 -6”)

To a stirring solution of VD1-5 (286 mg, 0.49 mmol) in anhydrous THF (6 mL) at -78 °C under nitrogen atmosphere was added n-BuLi (0.2 mL, 0.50 mmol, 2.5 M in n-hexane ) dropwise. The mixture was stirred at -78 °C for 15 min before a solution of VD1-4 (110 mg, 0.35 mmol) in anhydrous THE (3 mL) was added. The stirring was continued at -78 °C for 4 h and then 12 h at 0 °C. The reaction was quenched with sat. NH ₄ CI (1 mL) and extracted with EtOAc (3 x 20 mL). The organic portions were combined and washed with brine (10 mL), dried over anhydrous Na ₂ SO ₄ , and concentrated. The crude material was purified using column chromatography (1% EtOAc in n-hexane ) to give a colourless oil (60 mg) that was dissolved in MeOH (3 mL). To this solution, CSA (60 mg, 0.32 mmol) was added, and the mixture was stirred at room temperature for 3 h before being quenched with sat. NaHCO ₃ (2 mL). The mixture was extracted with EtOAc (3 x 10 mL) and the organic portions were combined and washed with brine (5 mL), dried over anhydrous Na ₂ SO ₄ , and concentrated. The crude material was purified using column chromatography (50% EtOAc in n-hexane ) to give VD1-6 (30 mg, 43%, over two steps) as a colourless oil. ¹ H NMR (600 MHz, CDCI ₃ ) δ 6.39 (d, J = 11.2 Hz, 1H), 6.03 (d, J = 11.2 Hz, 1H), 5.33 (s, 1H), 5.00 (s, 1H), 4.46-4.40 (m, 1H), 4.27-4.19 (m, 1H), 3.79 (s, 1H), 2.84-2.81 (m, 1H), 2.61-2.59 (m, 3H), 2.33- 2.28 (m, 2H), 2.05-1.97 (m, 4H), 1.94-1.88 (m, 2H), 1.72-1.67 (m, 2H), 1.51-1.40 (m, 6H), 1.33-1.25 (m, 5H), 1.11 (s, 9H), 0.99 (d, J = 6.5 Hz, 3H), 0.55 (s, 3H); ¹³ C NMR (150 MHz, CDCI ₃ ) δ 167.4, 147.6, 143.3, 132.8, 125.04, 117.01, 111.8, 70.8, 66.9, 61.0, 56.3, 55.9, 45.9, 45.3, 42.9, 40.4, 37.2, 37.1, 32.5,

29.1, 27.9, 27.5, 23.6, 23.2, 18.6, 12.0. MS (ESI) calc, for C ₂₉ H ₄₈ NO ₃ [M+H] ⁺ : 458.36, found: 458.65.

Example 2 - Biological assessment of VD1-6

VDR competitor binding assay

[00103] Protocol

PolarScreen™ VDR Competitor Assay Kit, Red (Life Technologies Australia Pty Ltd, Thermo Fischer Scientific, Scoresby, Victoria, Australia) was used for the assay. VDR recombinant human protein (32,100 nM), Fluormone™ VDR Red tracer (100 nM), and dithiothreitol (DTT) solutions provided by the kit manufacturer were thawed for 30 min on ice before use. A sufficient volume of complete VDR Red screening buffer “complete buffer” (5 μL of DTT for each 1 mL of the VDR Red screening buffer) was prepared and kept on ice until use. Primary stock solutions (106 nM) of 1,25(OH) ₂ D ₃ , 25(OH)D ₃ (Sigma Aldrich, Castle Hill, NSW, Australia), and VD1-6 were prepared separately in DMSO. From these solutions, fifteen further three-fold serial dilutions were prepared for each of the three compounds in DMSO with the lowest concentration in the series of 0.07 nM. For conducting the assay, each of the sixteen DMSO dilutions for each compound was further diluted 50-fold using the complete buffer for the resultant highest and lowest concentrations being 2 x 104 nM and 0.0014 nM, respectively and for a post- dilutional DMSO concentration of 2%. To prepare VDR/Fluormone™ complex, separate 4 nM Fluormone™ and 14.48 nM VDR full-length protein solutions were prepared by appropriate dilution of the manufacturer’s stock solutions using complete buffer followed by vortex mixing of the Fluormone™ solution for 10 sec and inversion mixing only of the VDR solution. Sufficient equal volumes of the Fluormone™ and VDR dilutions were combined and mixed by inversion and the final solution (2 nM Fluormone™/7.24 nM VDR) was kept on ice until use.

[00104] In a 384 microwell plate (Corning® 4511, low volume, black, polystyrene, round bottom, non-sterile, non-treated), 10 μL aliquots from each of the sixteen (0.0014 - 2 x 104 nM) serial dilutions for each of the three tested compounds were pipetted into triplicate adjacent wells. To those wells, 10 μL of the (2 nM Fluormone™/7.24 nM VDR) solution was then added for final concentrations of test compounds ranging from 0.0007 to 1 x 104 nM, final Fluormone™ concentration of 1 nM, and a final VDR full-length concentration of 3.62 nM (the manufacturer’s recommended lot-specific concentration). Maximum and minimum polarization controls were prepared for monitoring assay performance similarly by the addition of the Fluormone™/VDR complex to a column of wells with 10 μL blank 2% DMSO in complete buffer and a column of wells with 10 μL 2 x 104 nM 1,25(OH) ₂ D ₃ , respectively. The final DMSO concentration in each well was 1% to match the manufacturer’s recommended solvent tolerance for the assay. The plate was lid covered and shaken horizontally on an orbital shaker at 60 rpm for 5 min then incubated for 3 h at room temperature. Fluorescence polarization values (mP) were then measured on the plate reader Cytation™ 5 multi-mode (Biotek, Winooski, VT, United States) equipped with a red fluorescence polarization filter (Excitation/Emission, 530/590 nm) at 25 °C. Modelling of the mean fluorescence polarization readings versus corresponding concentrations for each compound using sigmoidal dose-response curve fitting with a variable slope as recommended by the kit manufacturer [36] was performed using GraphPad Prism® 8.3.0 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com.

[00105] Results and discussion

It was important to establish whether VD1-6 has affinity for VDR binding at concentrations used in biological assessment. Results of VDR competitor assay experiments for VD1-6, 1,25(OH) ₂ D ₃ and 25(OH)D ₃ are represented in Figure 1. Curve fitting (solid connecting lines) using sigmoidal dose- response (variable slope) curve (Carpenter, V.T., et al., Bone, 2012. 50(3): p. 688-94) yielded modelled binding IC ₅₀ values of 0.93, 56.2 and 438 nM for 1,25(OH) ₂ D ₃ , 25(OH)D ₃ and VD1-6, respectively. These concentrations are those required to displace half of the high affinity fluorescent tracer from its VDR binding, which results in a corresponding drop in the mean fluorescence polarization by 50%. The results illustrate that VD1-6 has approximately 470-fold (438/0.93) lower binding affinity than 1,25(OH) ₂ D ₃ to the active site of VDR. Further, VD1-6 binding to VDR was only evident at concentrations greater than 10 ^-7 M (Figure 1). 25(OH)D ₃ , known to bind VDR with markedly less affinity than 1,25(OH) ₂ D ₃ , had 60-fold reduced binding affinity than 1,25(OH) ₂ D ₃ for VDR, consistent with previous reports and indicated that VD1-6 has lower affinity for VDR than 25(OH)D ₃ .

Comparative in silico docking of 1,25(OH) ₂ D ₃ and VD1-6 into CYP24A1

[00106] The computer-aided simulated measure of the change of molecular free energy that occurs due to binding (relative binding free energy) is considered a potentially reliable measure of binding affinities of small ligand molecules to protein molecular targets (Cournia, Z., B. Allen, and W. Sherman, J. Chem. Inf. Model., 2017. 57(12): p. 2911-2937; Wan, S., et al., Interface Focus, 2020. 10(6): p. 20200007). In-silico docking of VD1-6 into CYP24A1 binding pocket was conducted as an assessment of the affinity to CYP24A1 binding site in comparison to 1,25(OH) ₂ D ₃ . In the absence of a crystal structure of the human CYP24A1 in the PDB, the available rat CYP24A1 protein was utilized for docking studies. The reported similarity between human and rat CYP24A1 is 85% with the rat variant matching 11 of 13 key amino acids for substrate binding and catalysis (Annalora, A.J., et al., J. Mol. Biol., 2010. 396(2): p. 441-451; Jayaraj, J.M., et al., J Biomol Struct Dyn, 2019. 37(7): p. 1700-1714).

[00107] Protocol

The crystal structure of a rat mitochondrial cytochrome P450 24A1 co-crystallized with a zwitterionic surfactant was used: protein data bank (PDB) ID: 3K9V (Resolution = 2.5A and receptor pocket-size = 2512 A) (Annalora, A.J., et al., ibid). Ligand structures for docking were acquired by first determining the appropriate SMILES string for 1,25(OH) ₂ D ₃ and VD1-6. Enumeration of 3D conformers was undertaken via OMEGA 3.3.0.3 with the default maximum number of conformers (200) (Hawkins, P.C.D., et al., J. Chem. Inf. Model., 2010. 50(4): p. 572-584; OMEGA 3.3.0.3. 2019, OpenEye Scientific Software: Santa Fe, NM, USA). VD1-6 and 1,25(OH) ₂ D ₃ produced 60 and 62 conformations, respectively. With the selected crystal structure for CYP24A1, binding pockets were derived using Make Receptor 3.3.0.3 (Make Receptor 3.3.0.3. 2019, OpenEye Scientific Software: Santa Fe, NM, USA). Appropriate sections of the protein and the co-crystallized ligand were chosen, and auto-generated constraints were not included as per the default settings. No alterations to the site shape potential were made. Docking studies to the receptor were performed using FRED 3.3.0.3 as part of the OF Docking suite (FRED 3.3.0.3. 2019, OpenEye Scientific Software: Santa Fe, NM, USA; McGann, M., J. Chem. Inf. Model., 2011. 51(3): p. 578-596; McGann, M., J. Comput. Aided Mol. Des., 2012. 26(8): p. 897-906). Docking resolution was set to ‘High’ and the number of poses limited to 1. The two ligands docked successfully and the predicted orientations were examined in 3D space using VIDA 3.3.0.3 (VIDA 3.3.0.3. 2019, OpenEye Scientific Software: Santa Fe, NM, USA). Generated chemgauss4 scores ranked the docked 1,25(OH) ₂ D ₃ and VD1- 6 according to their goodness of fit into the receptor pocket, and the interactions with surrounding residues.

[00108] Results and discussion

The simulated docking positions of 1,25(OH) ₂ D ₃ and VD1-6 in the CYP24A1 binding pocket are shown in Figure 2. The ligand binding site demonstrates similar amino acid residues to that indicated by Jayaraj et al. (ibid) i.e., GLU 329 and THR 330 for the CYP24A1 active binding tunnel. 1,25(OH) ₂ D ₃ has one hydrogen bond between its terminal C-25 hydroxyl group and THR 330, while VD1-6 displays a single hydrogen bond to MET 246 from the C-l hydroxyl group. The docking pose of 1,25(OH) ₂ D ₃ is consistent with the findings of Annalora et al. (ibid), where the C-l and C-3 hydroxyl groups are located near the THR 395 residue.

[00109] Simulated docking into the active binding pocket of CYP24A1 shows that the binding of VD1-6 involves a change in the hydrogen bonding pattern relative to 1,25(OH) ₂ D ₃ . This may be attributed to the lack of a terminal hydroxyl moiety at the C-25 end of VD1-6 which may also explain the predicted inversion of orientation inside the binding pocket as seen in Figure 2. Docking scores revealed VD1-6 to rank higher than the endogenous ligand 1,25(OH) ₂ D ₃ (-12.30 and -11.64 relative chemgauss4 docking scores) in their docking to the CYP24A1 active binding site, which indicates a higher relative binding affinity of VD1-6 to CYP24A1.

[00110] It is noteworthy that for 1,25(OH)2D3 to be catabolically hydroxylated at either the C-23 or C-24 in the active site of CYP24A1, the terminal side chain (C-21 to C-25) needs to be positioned towards the heme moiety of the enzyme (Schuster, I., Biochim Biophys Acta, 2011. 1814(1): p. 186-99). The docking experiments conducted herein reveal that simulated poses for 1,25(OH) ₂ D ₃ and VD1-6 appear to be consistent with this concept with aliphatic side chains being pitched toward the heme moiety and having appropriate flexibility to interact. From a metabolism perspective, the C-24 O-methyl oxime moiety of VD1-6 may block C-24 hydroxylation leaving it only possible to proceed down the C-23 pathway. On the other hand, the endogenous ligand 1,25(OH) ₂ D ₃ can proceed through both the C-23 and C-24 catabolic routes. The greater potential of interaction of VD1-6 with CYP24A1 besides its occupied C-24 suggest that VD1-6 is a more efficient CYP24A1 binder with partial catabolic immunity i.e., a likely inhibitor of vitamin D catabolism with a presumably longer half-life than the endogenous ligand. HEK293T cell culture experiments

[00111] HEK293T cells were maintained at 37 °C in a growth media comprised minimum essential medium alpha (alpha-MEM) (Thermo-Fisher Scientific, Scoresby, VIC, Australia), 10% v/v fetal bovine serum (FBS) (Thermo-Fisher) and a standard usage of tissue culture additives including 25 nM HEPES, 2 mM L-glutamine, and antibiotics: 100 U/mL penicillin G/streptomycin. For experimentation, cells were seeded in 24-well tissue culture plates with 30% confluency and were allowed to attach for 24 h before treatment application.

[00112] Experiment 1 - Genetic response to VD1-6

[00113] Protocol

Upon cell attachment, culture wells (n = 5) were exposed for 24 h to a vehicle control (0.2% v/v ethanol (Sigma Aldrich, Castle Hill, NSW, Australia)) or three separate treatments of VD1-6 (10 ^-7 M ), 1,25(OH) ₂ D ₃ (10 ^-7 M ) and a mixture of VD1-6 and 1,25(OH) ₂ D ₃ both at 10 ^-7 M. The cells from replicate wells for each of the three treatment groups were then treated for RNA extraction using TRIzol® reagent (Thermo-Fisher Scientific, Scoresby, VIC, Australia) at room temperature following the manufacturer’s protocol (ThermoFisherScientific. TRIzol Reagent User Guide. [Accessed July 2021]; Available from: https://tools.thermofisher.com/content/sfs/manuals/trizol_re agent.pdf). Briefly, after media removal, 200μL TRIzol® was added to unwashed cells in each well to lyse them. Eysate of each well was then transferred to 1.5 mL Eppendorf tubes, then 40 μL of chloroform (Sigma Aldrich, Castle Hill, NSW, Australia) was added then the tube content was mixed by inversion and centrifuged for 15 min at 12,000 rpm at 4 °C. The upper aqueous layer was transferred to a new tube to which 100 μL of isopropanol (Sigma Aldrich, Castle Hill, NSW, Australia) was added and the tube content was incubated for 10 min followed by centrifugation for 10 min at 12,000 rpm at 4 °C, then the supernatant was discarded. For washing, 200 μL of 70% ethanol (Sigma Aldrich, Castle Hill, NSW, Australia) was added to the RNA pellet, vortexed for 5 sec then centrifuged for 5 min at 7,500 rpm at 4 °C, then the supernatant was discarded, and the RNA pellet was air-dried for 10 min. The RNA pellet was resuspended in 25 μL RNase-free water (Sigma Aldrich, Castle Hill, NSW, Australia) then the tubes were incubated at 60 °C water bath for 10 min before storage at -80 °C for real-time PCR analysis.

[00114] Results and discussion

A comparative study of the effects of VD1-6 versus 1,25(OH) ₂ D ₃ , alone, and in combination on the mRNA levels of CYP24A1, CYP27B1, and VDR in HEK293T cells at 24 h is illustrated in Figure 3. At 10 ^-7 M, VD1-6 did not affect CYP24A1 mRNA levels compared to vehicle control (Error! Reference source not found. A). However, 1,25(OH) ₂ D ₃ at an equivalent concentration was associated with a significant (p < 0.05) elevation in CYP24A1 mRNA levels, at 3.5-fold that seen with vehicle control. The combination of VD1-6 and 1,25(OH) ₂ D ₃ resulted in greater stimulation of CYP24A1 expression at 6-fold (p < 0.05) the baseline mRNA levels seen with vehicle control while also being significantly greater than the mRNA levels corresponding to 1,25(OH) ₂ D ₃ treatment at 1.7-fold (p < 0.05) (Figure 3 A). On the other hand, mRNA levels of CYP27B1 (Figure 3B) and VDR (Figure 3C) did not show any significant change either with VD1-6, 1,25(OH) ₂ D ₃ or their combined treatment, when compared to mRNA levels seen with vehicle control.

[00115] The combined in vitro cell free VDR binding assay and in silico docking results of VD1-6 in comparison to the endogenous ligand (as discussed above) provide a clear indication that VD1-6 has the potential to be an inhibitor of CYP24A1 without significant VDR binding, at least up to concentrations of 10 ^-7 M. Consistent with this, VD1-6 at 10 ^-7 M over 24 h did not induce CYP24A1 mRNA, unlike

1,25(OH) ₂ D ₃ , indicating the absence of VDR binding by VD1-6. However, the inclusion of VD1-6 with 1,25(OH) ₂ D ₃ resulted in a more pronounced increase in CYP24A1 mRNA levels, suggesting that VD1-6 potentiated the transcriptional effects of 1,25(OH) ₂ D ₃ , likely through binding to and inhibiting CYP24A1 and thus increasing 1,25(OH) ₂ D ₃ half-life. Furthermore, VD1-6 did not impact CYP27B1 and VDR mRNA levels suggesting that the effects of VD1-6 are specific to 1,25(OH) ₂ D ₃ activity.

[00116] Experiment 2 - 1,25(OH) ₂ D ₃ preservation by VD1-6

[00117] To establish whether reduced 1,25(OH) ₂ D ₃ catabolism occurred with VD1-6 treatment, the preservation of 1 ,25(OH) ₂ D ₃ in HEK293T cell culture up to 24 h was tested.

[00118] Protocol

Cultured wells (n = 4) were exposed for 8 and 24 h to a vehicle (0.2% v/v ethanol) and VD1-6 at 10 ^-7 M as controls and to separate treatments of 1,25(OH) ₂ D ₃ at 5 x 10 ^-10 M without and with VD1-6 at 10 ^-8 M and 10 ^-7 M . Supernatants from control and treated wells were collected at 8 and 24 h post-treatment and stored with time zero samples at -80 °C for 1,25(OH) ₂ D ₃ LC-MS/MS analysis.

[00119] Results and discussion

Results of LC-MS/MS analysis of the concentration of the added 1,25(OH) ₂ D ₃ to HEK293T cell culture, in absence and presence of VD 1 -6 at concentrations of 10 ⁸ and 10 ^-7 M at time zero, and at 8 h and 24 h post-treatment, are illustrated in Figure 4. In the absence of VD1-6, 1,25(OH) ₂ D ₃ concentrations exhibited a rapid mean decline of 83 pM (p < 0.05) over 8 h, to reach a mean concentration of 451 ± 27.7 pM. This decline rate was found to slow beyond 8 h (i.e., 1 ^st order rate) with a further but non-significant mean decline of 29 pM over the following 16 h. This resulted in a mean 1,25(OH) ₂ D ₃ levels of 422 ± 11.9 pM at 24 h which was significantly different from the time zero mean levels (p < 0.05).

[00120] The inclusion of VD1-6 resulted in dose-dependent preservation of the added 1,25(OH) ₂ D ₃ levels. VD1-6 at 10 ^-8 M did not exert a significant inhibition of catabolism at 8 h in terms of mean 1,25(OH) ₂ D ₃ levels compared to levels in the absence of VD1-6 at a similar time. However, with that VD1-6 concentration, the level of catabolism inhibition seen at 8 h seemed to be sustained up to 24 h, which resulted in a mean 1,25(OH) ₂ D ₃ concentration of 480 ± 28.3 pM which was significantly different (p < 0.05) when compared to that at 24 h without VD1-6. However, with a higher VD1-6 concentration, i.e., 10 ^-7 M , a significant catabolism inhibition was evident by 8 h with mean 1,25(OH) ₂ D ₃ concentrations of 534 ± 25.9, which was significantly different (p < 0.05) from the mean concentration in the absence of VD1-6 at 8 h and non-significantly different from the mean concentrations at time zero. This inhibition of catabolism effect was persistent up to 24 h (i.e., non-significantly different from that at 8 h and zero time), with a mean 1,25(OH) ₂ D ₃ concentration of 506 ± 28.9 pM, which was significantly different (p < 0.05) from mean concentrations at 24 h in the absence of VD1-6. At 24 h, mean 1,25(OH) ₂ D ₃ concentrations with both 10 ^-8 M and 10 ^-7 M VD1-6 were non-significantly different, indicating possibly comparable longer-term preservation effects of these two concentrations.

[00121] In summary, while 1,25(OH) ₂ D ₃ levels declined by 16% by 8 h and 22% by 24 h, at 10 ^-7 M , VD1-6 prevented this decline in 1,25(OH) ₂ D ₃ over the 24 h period. While, at 10 ^-8 M, VD1-6 was less preventive of 1,25(OH) ₂ D ₃ decline at 8 h, the protective effects of this VD1-6 concentration on 1,25(OH) ₂ D ₃ levels at 24 h were comparable to that of the effects of 10 ^-7 M VD1-6.

[00122] Experiment 3 - Effect of VD1-6 on 24,25(OH) ₂ D ₃ production in HEK293T cell culture

[00123] To assess the effects of VD1-6 on 25(OH)D ₃ catabolism, HEK293T cells were treated with

25(OH)D ₃ over 72 hours, and media levels of 24,25(OH) ₂ D ₃ and mRNA levels for CYP24A1 were measured in absence and presence of VD1-6.

[00124] Protocol

72 h exposure of cultured wells (n = 4) was done to similar controls as under Experiment 2 above and to 25(OH)D ₃ at 10 ^-6 M without and with VD1-6 at 10 ^-11 , 10 ^-10 , 10 ^-9 , 10 ^-8 and 10 ^-7 M , each as a separate treatment. At the end of treatment, supernatants from quadruplicate wells for each group were collected and stored at -80 °C for 24,25(OH) ₂ D ₃ LC-MS/MS analysis. Cells were then treated for total RNA isolation as described under Experiment 1.

[00125] Results and discussion

In the presence of 25(OH)D ₃ (10 ⁶ M), HEK293T cells produced 24,25(OH) ₂ D ₃ which was approximately 170-fold higher than levels in vehicle treated cells (Figure 5 A). The inclusion of VD1-6 resulted in a biphasic concentration-dependent effect on the mean accumulated 24,25(OH) ₂ D ₃ concentrations. At 10 ^-10 M VD1-6, a 14% increase in mean 24,25(OH) ₂ D ₃ concentration was observed when compared to levels with 25(OH)D ₃ treatment alone (p < 0.05). However, at higher concentrations, VD1-6 treatment did not increase 24,25(OH) ₂ D ₃ levels, with 10 ⁸ M VD1-6 treatment resulting in no change in levels, and at 10 ^-7 M VD1-6, a marked decrease in 24,25(OH) ₂ D ₃ levels was seen when compared to levels in 25(OH)D ₃ treatments alone (16%, p < 0.05), with VD1-610 ^-11 M (20%, p < 0.05), with 10 ^-10 M VD1-6 (27% p < 0.0001), with VD1-6 10 ⁹ M (24%, p < 0.0001) or VD1-610 ^-8 M (15%, p < 0.05). Although 25(OH)D ₃ treatment increased CYP24A1 mRNA levels in HEK293T cells, VD1-6 inclusion did not further elevate CYP24A1 mRNA levels (Figure 5B). Although the elevated 24,25(OH) ₂ D ₃ levels at 10 ^-10 M VD1-6, appeared to correlate with elevated CYP24A1 mRNA levels, this was not statistically significant. The decline in 24,25(OH) ₂ D ₃ levels in 25(OH)D ₃ treatments with 10 ^-7 M VD1-6 did not correspond to a change in CYP24A1 mRNA levels when compared to mRNA levels in 25(OH)D ₃ only treatments, suggesting that 24,25(OH) ₂ D ₃ decline was not due to reduced CYP24A1 expression.

[00126] Thus, VD1-6 at 10 ^-7 M was also capable of reducing 25(OH)D ₃ catabolism, as measured by the formation of 24,25(OH) ₂ D ₃ . the first product of 25(OH)D ₃ catabolism, most likely through significant competition and occupation of the CYP24A1 active site. Interestingly, the elevation in 24,25(OH) ₂ D ₃ levels with VD1-6 at 10 ^-10 M may have occurred due to counter-elevation of CYP24A1 activity by the preservation of 1,25(OH) ₂ D ₃ . While, in this experiment, the discrimination of the effects of 25(OH)D ₃ , the synthesis of 1,25(OH) ₂ D ₃ , and the anti-catabolic effects of VD1-6 on the expression of CYP24A1 mRNA is not possible, the exacerbated CYP24A1 induction by the anti-catabolic effects of VD1-6 is plausible, given VD1-6 treatment, when combined with 1,25(OH) ₂ D ₃ enhances CYP24A1 expression (Figure 3A).

Other general experimental protocols

[00127] Real-time PCR analysis

Replicate total RNA samples obtained from experimental cultured wells described under 2.4.1 were analyzed for CYP24A1, CYP27B1, and VDR mRNA by quantitative PCR while quadruplicate RNA extractions from 2.4.3 were quantified only for CYP24A1 mRNA. 2000 ng of total RNA from each sample were input for reverse transcription (RT) using iScript™ cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) following the manufacturer’s protocol [46] with genomic DNA cleaning incorporated. RT product of 12.5 ng total RNA equivalent was then input to each of the real-time PGR reactions for determining the target genes mRNA levels, using Forget-Me-Not™ EvaGreen® qPCR Master Mix (Biotium, Fremont, CA, USA). Real-time PCR was performed using a CFX Connect Real-Time PCR Detection System (Bio-Rad, California, USA). Sequences of the primer set used were: CYP24A1 gene, forward- 5'-CCTGCTGCCAGATTCTCTGGAA-3', reverse- 5'-TTGCCATACTTCTTGTGGTACTCC- 3', CYP27B1 gene, forward- 5'-CAGACAAAGACATTCATGTGGG-3', reverse- 5'- GTTGATGCTCCTTTCAGGTAC-3', VDR gene, forward- 5'-CCAGTTCGTGTGAATGATGG-3', reverse- 5'-GTCGTCCATGGTGAAGGA-3' and Actin B (ACTB) gene (the housekeeping gene for relative quantification of each of the three monitored genes), forward- 5'-AAGAGATGGCCACGGCT-3', reverse- 5 '-CAATGATCTTGATCTTC ATTGTGC-3 ' .

[00128] EC-MS/MS analysis

24,25(OH) ₂ D ₃ concentrations were analyzed in cell culture samples from Experiment 3 using D APT AD derivatisation and EC-MS/MS as previously described (Alshabrawy, A.K., et al., J. Chromatogr. B, 2021. 1173: p. 122654). The calibrators were prepared in phosphate -buffered saline (PBS) and the samples were appropriately diluted with PBS to bring them into the linearity range of the assay. The same EC-MS/MS method was modified for 1,25(OH) ₂ D ₃ analysis to enhance the sensitivity through using higher initial sample volume (200 μL instead of 50 μL) and reconstituting the final derivatized 1,25(OH) ₂ D ₃ in 20 μL and injecting 15 μL (rather than reconstituting in 25 μL and injecting 5 μL) onto the EC-MS/MS column. Multiple reaction monitoring (MRM) transitions were used for quantifying the 1,25(OH) ₂ D ₃ -DAPTAD derivative and the derivative of its deuterated isotope (1,25(OH) ₂ D ₃ -d ₆ ) as previously reported (Ishige, T., et al., Clin. Chim. Acta, 2017. 473: p. 173-179). Similar twin chromatographic peaks for R and S isomers of the derivatives (Ishige, T. et al. , ibid) were also obtained and used similarly for quantification.

[00129] Statistical analysis

Statistical analysis was performed using one-way analysis of variance (ANOVA) for non-parametric data with secondary Tukey's multiple comparisons test to determine the difference between treatment groups, using GraphPad Prism® 8.3.0 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com. Example 3 - Effect of VD1-6 in 5-FU-induced gastrointestinal mucositis

Protocols

[00130] CYP24A1 knock-out mice

To selectively delete Cyp24a1 from intestinal epithelial cells, Villin-Cre transgenic mice (kindly donated by Prof Joanne Bowen, University of Adelaide) were bred with homozygous Cyp24a1 floxed (Cyp24a1fl/fl) mice on a C57B1/6 background. Homozygous Cyp24a1fl/fl and Villin-Cyp24fl/fl offspring expressing the Cre -recombinase transgene were subsequently bred to generate mice containing the Cre- recombinase transgene and homozygous for the floxed Cyp24a1 allele (IntCyp24a1-/-).

[00131] The recombinant Cyp24a1 knockout allele was confirmed by PGR using primer pl (5 ' -

GCGCATAACGATACCACGAT-3 ') in combination with primer p2

(5 '-CCAGCCCCAGGTTTTAATGT-3 '). The location of both primers is illustrated in Fig. 6A, and the resulting PGR amplicon is 369bp after deletion of exon 5 of the CYP24A1 gene.

[00132] Male Villin-Cre-CYP24A1 homozygous knockout (IntCyp24a1-/-) and CYP24A1 floxed control (Cyp24a1fl/fl) mice (8 weeks of age, n = 10 and 8 per genotype, respectively) were used for the deletion study. Wild type C57Black6 (n=36) were obtained (Animal Resource Centre, Perth, Australia) for the therapeutic inhibition study. All mice were fed standard AIN-93 semi-synthetic diet containing standard vitamin D (1000 lU/kg) and calcium (1%), with free access to water and group-housed in an animal facility regulated at 22 ± 1°C and subject to a 14:10 hour light-dark cycle.

[00133] All experimental mice were anaesthetised with inhaled isoflurane (2% in 100% oxygen) prior to injection with a single sub-lethal dose of 450 mg/kg 5-FU intraperitoneally (i.p.) in a volume of 200 μL, to induce mucositis without mortality. Control mice were injected i.p. with equivalent volume sterile saline.

[00134] Mice were weighed daily and monitored twice daily following 5-FU administration for toxicity criteria: dull/ruffled coat, dull/sunken eyes, lack of movement when stimulated, hunching and diarrhoea. At 48 hours following 5-FU administration, mice were humanely killed via deep inhalation anaesthesia and cardiac puncture, followed by cervical dislocation. The gastrointestinal tract was dissected from pyloric sphincter to rectum and separated into small intestine (pyloric sphincter to ileocecal sphincter) and colon (ascending colon to rectum). The small intestine was flushed with chilled, sterile PBS, and weighed. Samples (1 cm) were collected at the proximal end (duodenum) and 25% (jejunum) of the length for histology, with subsequent 2cm samples collected for DNA and RNA analysis. The colon was also flushed with chilled sterile PBS and weighed. Samples (1 cm) were collected at the proximal end for histology, with subsequent 2cm samples collected for RNA analysis. Samples for histology were fixed in 10% neutral buffered formalin, processed and embedded in paraffin. Samples for RNA analysis were snap frozen in liquid nitrogen and stored at -80°C. Blood collected from cardiac puncture was centrifuged at 11,000 rpm and serum stored at -20° C.

[00135] DNA isolation and analysis

To demonstrate CYP24a1 had been selectively deleted in the intestine, tissue samples were analysed from Cyp24a1fl/fl and IntCyp24-/- mice including intestine (duodenum, jejunum and colon), kidney and liver. Each tissue (0.5 g) was diced and then then placed in 500 μL digestion mix containing lx TES (2-[(2- by droxy-l,l-bis(hydroxymethyl)ethyl)amino] ethanesulfonic acid) buffer, 30 μg/μL proteinase K and 20% sodium dodecyl sulfate (SDS), then incubated overnight at 55°C.

[00136] Digested organs were subsequently placed on ice for 10 minutes to allow for precipitation and then centrifuged at 13,000 rpm for 10 minutes at 4°C. Supernatant was collected, and ImL isopropanol and 3uL glycogen were added to pellet the DNA. Samples were centrifuged at 13,000 rpm for 1 minute at 4°C, and supernatant discarded. The DNA pellet was washed in ImL 70% ethanol and centrifuged at 13,000 rpm for 1 minute at 4°C. The supernatant was removed, and the pellet was air-dried for 30 minutes at room temperature. The DNA was then resuspended in WOμL TE buffer (pH 8) and stored at 4°C overnight. DNA was analysed with a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, Del., USA) to determine purity and concentration. DNA was stored at -20°C in aliquots of working solution containing 20ng of DNA using TE buffer (pH 8).

[00137] RNA isolation

RNA was isolated and purified from jejunum and colon with NucleoSpin™ RNA isolation Kit (Machery- Nagel, Duren, Germany), according to manufacturer’s instructions. Briefly, tissue (40 mg) was homogenised and added to lysis solution. The lysate was vortexed and filtered by column centrifugation for 1 min at 11,000 rpm. The supernatant was mixed with 350 μL of 70% ethanol to adjust RNA binding conditions. The homogenised solution was transferred into an RNA binding column and centrifuged. The membrane was desalted to improve effectiveness of DNA digestion. DNase solution was added to binding column for DNA digestion and residual DNA removed with high and low stringency washing buffers, respectively, with centrifugation between washes. RNA was eluted from the membrane with RNase-free H2O. RNA was analysed with a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, Del., USA) to determine purity and concentration. RNA integrity number (RIN) values were determined as an external service from SA Genomics Centre, South Australian Health and Medical Research Institute

(SAHMRI). Only samples with a RIN value greater than 7 were utilised for downstream analyses.

[00138] cDNA conversion

RNA was converted to cDNA using the Transcriptor™ First Strand cDNA Synthesis Kit (Roche Molecular Systems Inc., Basel, Switzerland). Briefly, 4 μL Transcriptor™ deoxynucleotide mix, 1 μL Transcriptor™ reverse transcriptase and 1 μL of RNA template were added to nuclease free water to make a reaction volume of 20 μL. Samples were denatured for 10 min at 25 °C for denaturing, annealing for 60 min at 50°C and elongation for 5 min at 85°C.Target cDNA was amplified by PCR for 39 cycles. Transcribed samples were analysed using a NanoDrop (NanoDrop Technologies, Wilmington, Del., USA).

[00139] Real-time PCR analysis

Real-time PCR was performed using a CFX Connect Real-Time PCR Detection System (Bio Rad, California, USA). Amplification mixes contained 1 μL of cDNA, 5 μL of 2X SYBR green (Bio-Rad Laboratories Inc. USA), 3 μL PCR-grade water and 0.5 μL of forward and reverse primers (GeneWorks, Adelaide, Australia), making a final volume of 10 pl.

[00140] Thermal cycling conditions were individually optimised for each primer set (Table 1). RlplO was used as a housekeeping gene for relative quantification for each gene of interest in intestinal and renal samples, respectively. All samples were repeated in duplicate, with experimental thresholds (Ct) calculated by CFX Connect program.

Table 1. Intestinal primer sequences

[00141] Wang, F. et al., Anal. Biochem., 2010. 399(2): p. 211-217; Bai, X., et al., J. Clin. Invest., 2016. 126(2): p. 667-680.

[00142] Histology

Structural integrity of the intestine was analysed using routine H&E staining (as previously described, ref). Sections were scanned on a Nanozoomer (details) using 20x objective, and images analysed using NDP.view2 software (Hamamatsu, Japan). Villous height (base of villus to apical tip of villus) was measured for a minimum of 15 complete villi per section. Crypt depth (base of crypt to crypt-villus junction) was also measured for a minimum of 15 complete crypts per section.

[00143] Immunohistochemistry

To determine whether CYP24A1 deletion or inhibition affected proliferating cell number, immunohistochemistry with anti-ki-67 antibody (Sigma-Aldrich) was performed. Sections (4 μm) were placed onto silane-coated slides (HD Scientific, Sydney, New South Wales, Australia) and heated at 60°C for 2 hours. Sections were dewaxed in xylene, and rehydrated through graded ethanols and distilled water, followed by PBS. Antigen retrieval was performed via enzymatic retrieval using proteinase K digestion at 37°C for 20 min. Sections were then washed in PBS with 1% Tween-20. Endogenous peroxidases were blocked with 3% hydrogen peroxide in methanol for 1 min. Non-specific antibody binding was blocked with Blocking solution (BioLegend, Dedham, USA) for 30 min. After washing in PBS, endogenous avidin and biotin were blocked using the Avidin and Biotin kit (Vector Laboratories, California, USA). Sections were incubated overnight at 4°C with 0.1 mg/mL anti-Ki-67 antibody diluted in PBS with 2% normal goat serum.

[00144] Sections were washed in PBS (3 x 5 min), incubated with Linking reagent (BioLegend, Dedham, USA) for 30 min, then washed in PBS (3 x 5 min). Labelling reagent was then applied for 30 min then washed in PBS (3 x 5 min). Staining was visualised using 3,3 ’-diaminobenzidine (DAB) and counterstained using haematoxylin, before dehydrating with increasing concentrations of ethanol, clearing with xylene and cover slipping with DePex mounting medium. Sections were scanned using 20x objective on the Nanozoomer. Positive (brown) cells were counted per demicrypt in a minimum of 15 complete crypts per section, using NDP.view2 viewing software for NanoZoomer. [00145] Alcian blue staining for goblet cell analysis

To determine the effect of 5-FU, intestine-specific CYP24A1 gene deletion, and VD1-6 on goblet cells, we performed goblet cell specific alcian blue staining. Duodenum, jejunum and colon sections from all mice were analysed.

[00146] Sections were dewaxed in xylene and rehydrated through graded ethanol solutions and distilled water. Sections were stained in fresh Alcian Blue solution for 5 min and were washed with distilled water until clear. Sections underwent oxidation in 1% aqueous periodic acid for 10 min and were washed in running water. Sections were then dehydrated through increasing ethanol solutions, before clearing with xylene and mounting with DPX. NDP.view2 viewing software for NanoZoomer (Hamamatsu, Japan) was used to capture images. We counted stained goblet cells in a minimum of 15 complete villi and crypts for each section, and normalised counts against the perimeters of respective villi and crypts.

[00147] Statistical analysis

Statistical analyses were performed using One-way analysis of variance (ANOVA) for non-parametric data with secondary Tukey's range test to determine the difference between mean ranks, using GraphPad Prism 7.03 (Graphpad software, California, U.S.A). Differences between mean ranks were determined to be significant at p <0.05.

Results and discussion

[00148] CYP24A1 deletion in intestinal epithelial cells does not affect survival or growth The recombinant CYP24A1 knockout allele was confirmed by PGR (Figure 6B). CYP24A1 was successfully deleted from the mouse genome and Cre-facilitated recombination was validated. No difference in pup survival, growth or serum 1,25(OH) ₂ D ₃ levels were observed between Cyp24 ^fl/fl and IntCyp24a1 mice for the duration of the study.

[00149] CYP24A1 expression was also measured in jejunum of Cyp24a1 ^fl/fl and IntCyp24a1 ^-/- . Real time RT-PCR analysis confirmed that CYP24A1 expression was low in Cyp24a1 ^fl/fl mice and absent in IntCyp24a1 ^-/- mice, irrespective of treatment with 5-FU (Figure 6C).

[00150] Response to inhibition of CYP24A1 with VD1-6

Administration of VD1-6 for a total of 7 days to wt mice resulted in no change to body weight, serum 1,25(OH) ₂ D ₃ levels, or intestinal histological parameters when compared with saline (vehicle control). [00151] Response to 5-FU treatment

All mice (wild type, Cyp24a1 ^fl/fl and IntCyp24a1 ^-/- ) tolerated 5-FU with no mortality within 48 h following administration. Clinical indications of deterioration were not observed in the experimental groups of mice. Diarrhoea was not observed in any mice for the duration of the study. 5-FU administration resulted in significant reduction in body weight 48 h following administration. Neither intestinal deletion of CYP24A1, nor inhibition of CYP24A1 significantly improved body weight following administration of 5-FU. Organ wet weights (relative to % body weight of animal) of small intestine, colon, liver and kidney did not significantly vary between groups.

[00152] Intestinal CYP24A1 deletion reduces 5-FU-induced intestinal damage

5-FU results in significant damage to intestine structure. In Cyp24a1 ^fl/fl mice, duodenum villous area significantly decreased 48 h following 5-FU administration compared to saline controls (p = 0.0334) (Figure 7). Villous area did not change in the jejunum, and was not affected by 5-FU in IntCyp24 ^-/- mice. Villous height, crypt depth and crypt area were not affected by genotype or treatment. Jejunum and colon parameters were not significantly affected by 5-FU administration.

[00153] Therapeutic inhibition of CYP24A1 with VD1-6 reduces 5-FU-induced intestinal damage In wild type mice, duodenum villous height and area significantly decreased 48 h following 5-FU administration compared to saline controls (p = 0.0002 and p = 0.0006, respectively) (Figure 8). VD1-6 administration in combination with 5-FU significantly increased villous height compared to 5-FU alone (p = 0.043) and reduced the effect of 5-FU on villous area (no significant difference between 5-FU/VD1- 6 combined compared to saline controls). Crypt depth and area were not affected by 5-FU or VD1-6.

[00154] Jejunum villous height and area also significantly decreased 48 h following 5-FU treatment compared to saline controls (p = 0.0451 and p = 0.0180, respectively). VD1-6 administration in combination with 5-FU resulted in some minor variation in villous height and crypt depth compared with other groups, but did not significantly reduce the effects of 5-FU on villous height and area. Crypt depth and area were not affected by 5-FU in the jejunum.

[00155] In the large intestine, colon crypt depth and area significantly decreased 48 h following 5-FU administration compared to saline controls (p = 0.0034 and p = 0.0136, respectively). VD1-6 administration in combination with 5-FU significantly increased crypt depth compared to 5-FU alone (p = 0.0068) and reduced the effect of 5-FU on crypt area, so that there was no significant difference between 5-FU and VD1-6 crypt area combined compared to saline control crypt area (Figure 8). [00156] 5-FU depletes goblet cells in the small intestine

In wild type and Cyp24a1fl/fl mice, villus goblet cell counts significantly decreased 48 h following 5-FU administration in the small intestine. In wild type mice, goblet cell counts were significantly lower in the duodenum of 5-FU treated mice compared with saline control (p = 0.0002). When the same comparison was made with goblet cell counts normalised against villus perimeter there was no significant difference. Similarly, in the jejunum, goblet cell counts were significantly lower in the 5-FU treated mice compared to saline control (p = 0.003). However, there was no significant difference when goblet cell counts were normalised against villus perimeter. Colon goblet cell counts did not differ significantly between treatment groups.

[00157] In Cyp24a1fl/fl mice villus goblet cell counts significantly decreased 48 h following 5-FU administration compared to saline control in the duodenum and jejunum (p = 0.0403 and p = 0.0107, respectively) (Figure 9). When normalised against villus perimeter, goblet cells counts of 5-FU treated mice were still significantly different to saline control in the duodenum (p = 0.0377), and trending towards significance in the jejunum (p = 0.0581) (Figure 9). Colon goblet cells counts did not differ between genotype or treatment.

[00158] Intestinal CYP24A1 deletion maintains goblet cells following 5-FU administration

In IntCyp24a1 ^-/- mice, there was no significant difference between goblet cell counts in the duodenum or jejunum between 5-FU treated mice and saline controls for both actual counts and those normalised against villus perimeter (Figure 9).

[00159] Therapeutic inhibition of CYP24A1 with VD1-6 has no effect on goblet cells with mice treated with 5-FU

Goblet cell counts in wild type mice treated with both 5-FU and VD1-6 were not significantly different compared to mice treated with 5-FU alone, but were significantly different compared to saline control mice in duodenum and jejunum (p = 0.0063 and p < 0.0001, respectively). When normalised against villus perimeter, goblet cell counts were not significantly different between any groups. However, when goblet cell counts were normalised against villus perimeter, there was no significant difference when mice treated with both 5-FU and VD1-6 were compared with saline controls. There was no significant difference in goblet cell counts between treatment groups in the colon.

[00160] Intestinal CYP24A1 deletion maintains crypt cell proliferation following 5-FU administration Duodenum, jejunum and colon were immunostained with Ki-67 antibody to identify proliferating cells. In duodenum crypts, Cyp24a1 ^fl/fl mice treated with 5-FU had a significantly lower number of proliferating cells per demicrypt than Cyp24a1 ^fl/fl mice treated with saline (p = 0.0004), and IntCyp24a1 ^-/- mice treated with 5-FU (p = 0.0130). There was no significant difference in Ki-67 positive cells in crypts between IntCyp24a1 ^-/- mice treated with 5-FU compared to saline (Figure 10). In jejunum crypts Cyp24a1 ^fl/fl mice treated with 5-FU demonstrated the lowest number of Ki-67 positive cells per demicrypt, significantly lower than Cyp24a1 ^11/(1 mice treated with saline (p = 0.0015), and IntCyp24a1 ^-/- mice treated with 5-FU (p = 0.0255) (Figure 10). In the colon crypts, Cyp24a1 ^11/(1 mice treated with 5-FU had significantly lower Ki- 67 positive cells per demicrypt compared with saline Cyp24a1 ^11/(1 mice treated with saline (p = 0.0104), and IntCyp24a1 ^-/- mice treated with 5-FU (p = 0.0127).

[00161] Therapeutic inhibition of CYP24A1 maintains crypt cell proliferation in the small intestine in mice treated with 5-FU

Duodenum, jejunum and colon were immunostained with Ki-67 antibody to determine the effect of Cyp24a1 inhibition on proliferating crypt cells. In the duodenum, the highest number of Ki-67 positive cells per demicrypt was recorded in saline control mice. Mice treated with VD1-6 had a significantly lower number of Ki-67 positive cells per demicrypt than the saline control group (p = 0.0059). Mice treated with 5-FU demonstrated the lowest number of Ki-67 positive cells per demicrypt, significantly lower than saline controls (p < 0.0001). However, mice treated with both VD1-6 and 5-FU demonstrated significantly increased Ki-67 positive cells per demicrypt compared to mice treated with 5-FU (p < 0.0001) (Figure 11). Mice treated with both VD1-6 and 5-FU demonstrated lower Ki-67 positive cells per demicrypt than saline control mice (p<0.0001) and mice treated with VD1-6 alone (p=0.0267).

[00162] In jejunum, mice treated with 5-FU demonstrated the lowest Ki-67 positive cell count, significantly lower than saline controls (p < 0.0001). Mice treated with both VD1-6 and 5-FU recorded lower Ki-67 counts than the saline control (p<0.0001) and the VD1-6 alone (p = 0.0001). However, mice treated with both VD1-6 and 5-FU demonstrated a significantly higher number of Ki-67 positive cells than mice treated with 5-FU (p<0.0001) (Figure 11).

[00163] In the colon, mice treated with 5-FU demonstrated significantly lower Ki-67 positive cells per demicrypt than saline controls (p = 0.0006) (Figure 11). Despite a trend towards an increase in the number of Ki-67 positive cells in mice treated with both VD1-6 and 5-FU compared to mice treated with 5-FU, there was no significant difference. There was also no significant different between saline control mice and mice treated with both VD1-6 and 5-FU.

[00164] The present inventors have developed a novel competitive inhibitor of CYP24A1 (VD1-6), which was demonstrated as being effective at preventing the gastrointestinal damage caused by 5-FU. [00165] Intestine specific deletion of CYP24A1 did not affect growth or survival of pups, nor did it affect serum 1,25(OH) ₂ D ₃ levels, similar to inhibition of CYP24A1 in wt mice. As discussed above, VD1-6 has low affinity for VDR, suggesting it is not acting directly through activation of VDR responsive pathways.

[00166] The findings related to 5-FU-induced mucositis align with previous findings (Inomata, A., Horii, I. and Suzuki, K., Toxicol. Lett., 2002. 133(2-3): p. 231-240; Logan, M. et al., Cancer Chemother. Pharmacol., 2009. 63(2): p. 239-251), where damage is present at 48 h following bolus administration of i.p. 450 mg/kg 5-FU. 5-FU was tolerated, irrespective of genotype, and no mortality was recorded in this study. However, in wild type and Cyp24a1fl/fl mice, 5-FU administration resulted in a significant decrease in villus height in the duodenum, and significant decrease in crypt depth in the colon, with loss of function likely to accompany this loss of structure, leading to significant gastrointestinal symptoms such as severe diarrhoea observed in cancer patients receiving 5-FU treatment (Stein, A., Voigt, W. and Jordan, K., Ther. Adv. Med. Oncol., 2010. 2(1): p. 51-63), and animal studies of longer duration (Zang, S. et al., Biomed. Pharmacother. , 2018. 106: p. 910-916).

[00167] When 5-FU was administered to IntCyp24a1-/- mice, there was no significant difference in villus height in the duodenum compared to Cyp24a1fl/fl mice that were administered 5-FU, or IntCyp24a1-/- mice that were administered saline, suggesting intestinal Cyp24a1 expression may play a role in the development of 5-FU-induced gastrointestinal mucositis. Further, competitive inhibition of CYP24A1 with VD1-6 in wt mice also resulted in no significant difference in villus height in the duodenum when 5-FU was administered when compared with saline. Colon crypt depth was also not significantly different in mice administered both VD1-6 and 5-FU compared with saline control mice, demonstrating effects throughout the small and large intestine and suggesting widespread intestinal protection of CYP24A1 inhibition against 5-FU-induced GM.

[00168] 5-FU also depleted goblet cells in wt and floxed mice, with the effect driven by decreased villous height in wt mice (no difference when normalised against villous perimeter), but this was not the case in CYP24A1, with goblet cells remaining significantly depleted in 5-FU treated compared with saline treated Cyp24a1fl/fl mice. Deletion of CYP24A1 in the intestine maintained goblet cells following 5-FU, with no significant difference observed between saline and 5-FU treated mice for duodenum, jejunum or colon, suggesting that CYP24A1 deletion contributes to protection of the intestine from 5-FU- induced GM through preservation of goblet cells and mucin stores. Previous studies have shown vitamin D depletion to decrease muc2 expression (Zeng, Y. et al., Am. J. Physiol. Gastrointest. Liver Physiol., 2020. 318(3): p. G542-G553), therefore CYP24A1 deletion may be maintaining muc2 expression, maintaining the composition and integrity of the mucus layer. However, inhibition of CYP24A1 with VD1-6 had no significant effect on goblet cells and mucin stores, suggesting preservation of goblet cells and mucin stores in the absence of CYP24A1 may be a longer-term effect requiring a longer treatment time.

[00169] Chemotherapy agents, including 5-FU, are known to diminish cell proliferation caused by targeting specific phases of the cell cycle. The decreases in Ki-67 positive cells in the crypts of duodenum, jejunum and colon in mice treated with 5-FU implies decreased proliferating cells migrating up the crypt towards the villus-crypt axis, subsequently reducing the number of cells available to populate the epithelial surface of the villus, reducing villous height. The maintenance of proliferating cells migrating up the crypt in mice with CYP24A1 deleted from the intestine and mice treated with VD1-6 following 5-FU suggests that protection of the intestine structure observed with deletion or inhibition of CYP24A1 is likely driven by protecting the proliferating cells against 5-FU damage. The exact mechanism for this phenomenon is unknown, but the link between improved villous and crypt structure associated with Ki-67 positive (proliferating) cells has been established (Wu et al., J Radiat Res, 2019. 60(6):740-746). A study by Riehl and colleagues suggests TLR4 regulates proliferation in the intestine (Riehl, T. T. et al., American Journal of Physiology - Gastrointestinal and Liver Physiology, 2015. 309(11): p. G874-887), and the driver for TLR4 activation may be hyaluronic acid (Riehl, T. T. et al., ibid; Riehl, T. T. et al., American Journal of Physiology-Gastrointestinal and Liver Physiology, 2020. 319(1): p. G63-G73) as opposed to microbial activation through lipopolysaccharide (EPS). Vitamin D and VDR signalling is essential for Lgr5+ stem cell function (Peregrina, K. et al., Carcinogenesis, 2015. 36(1): p. 25-31), and directly or indirectly regulates proteins involved in intestinal epithelial cell proliferation and migration (Kuhne, H. et al., Lipids in Health and Disease, 2014. 13(1): p. 1-9), suggesting that inhibiting the catabolism of vitamin D and extending its presence in the intestine may be contributing to the maintenance, or restoration, of Ki67 positive cells and crypt cell proliferation.

[00170] Extended presence of 1,25(OH) ₂ D ₃ is likely to promote intestinal epithelial cell differentiation by regulating caudal -related homeobox transcription factor 2 (CDX-2) activity. CDX-2 plays a vital role in intestinal development by mediating intestinal cell differentiation (Gao, N. et al., Developmental Cell, 2009. 16(4): p. 588-599), suggesting that cell differentiation may also have a role in maintaining the villous and crypt structure. [00171] The present results suggest that inhibition of CYP24A1 may be an alternative intervention approach in chemotherapy-induced mucositis, enhancing the immunomodulatory and intestinal health effects of vitamin D through prolonging the half-life of 1,25(OH) ₂ D ₃ in the intestine, and may overcome the aberrantly increased CYP24A1 levels associated with many cancers (Anderson, M. G. et al., Cancer Chemotherapy and Pharmacology, 2006. 57(2): p. 234-240; Sun, H. et al., Human Pathology, 2016. 50: p. 101-108). Recent investigations demonstrate many signalling pathways of 1,25(OH) ₂ D ₃ exist in normal intestine, suggesting that maintaining adequate intestinal 1,25(OH) ₂ D ₃ would interfere with the pathogenesis of gastrointestinal mucositis (Hassanshahi, M. et al., Experimental Biology and Medicine, 2019. 244(12): p. 1040-1052).

[00172] The present findings indicate that CYP24A1 may be contributing to development of gastrointestinal damage by excessively catabolising 1,25(OH) ₂ D ₃ , removing the homeostatic function of 1,25(OH) ₂ D ₃ in the intestine subsequently decreasing crypt cell proliferation and migration.

Example 4 - Effect of VD1-6 on gut microbiota

Protocols

[00173] Animals

Eight-week-old, wild-type male C57BL/6 mice (6 per treatment group) weighing between 20-25 grams received subcutaneous (s.c.) injections of VD1-6 (500 ng/kgBW/day) or vehicle control (0.9% saline, equivalent volume, i.p.) for five days prior, and two days after the administration of a single dose of 5- fluorouracil (5-FU).

[00174] Experimental mice were anaesthetised with inhaled isoflurane (3% in 100% oxygen) daily prior to injection with a single sub-lethal dose of 450 mg/kgBW 5-FU (DBL 5 -Fluorouracil Injection BP, Hospira, 500 mg in 100 mL), i.p. in a volume of 200 μL in sterile 0.9% saline, to induce mucositis without mortality. Control mice were then injected with 200 μL sterile saline. Mice were fed standard AIN-93 semi-synthetic diet containing standard vitamin D (1000 lU/kg) and calcium (1%), with free access to water. The mice were group-housed in an animal facility regulated at 22 ± 1°C and subject to a 14:10 hour light-dark cycle.

[00175] Mice were humanely killed at 48 hours following 5-FU administration via deep inhaled anaesthesia and cardiac puncture, followed by cervical dislocation. The gastrointestinal tract (GIT) was dissected from the pyloric sphincter to the rectum and separated into the small intestine (pyloric sphincter to ileocecal sphincter) and colon (ascending colon to rectum). Contents were carefully removed from the large intestine (colon) and aseptically collected into sterile 1.5 mL tubes. The colon was flushed with chilled, sterile PBS, and 1 cm samples were collected at the proximal end, 25% and 75% for histology, with subsequent 2 cm samples collected for RNA analysis. The colon contents of all mice were stored for further analysis. Samples for RNA analysis were submerged in RNAlater and stored at -80 °C for later RNA extraction using NucleoSpin™ RNA isolation Kit (MACHEREY-NAGEL, Duren,

Germany).

[00176] RNA isolation

RNA was extracted from intestinal and renal tissue with NucleoSpin™ RNA isolation Kit (MACHEREY- NAGEL, Duren, Germany), according to manufacturer’s instructions. Briefly, intestinal tissue (approx. 40 mg) was homogenised and added to a 1.5 mL RNase-free tube containing lysis solution. The lysate mixture was then vortexed and filtered via column centrifugation for 1 min at 11 ,000 rpm. The supernatant was mixed with 350 μL of 70% ethanol to adjust RNA binding conditions. The homogenised solution was then decanted into an RNA binding column/wash tube, and centrifuged for 30 sec. After binding the RNA to the silica membrane, the membrane was desalted using 350 μL of membrane desalting buffer and was centrifuged for 1 min at 11 ,000 rpm. This was done in order to improve the effectiveness of the DNA digestion step.

[00177] The binding column was treated with rDNAse for DNA digestion at room temperature for 15 min. Residual DNA was removed from the column from the silica membrane via washing/drying with 200 μL high stringency washing buffer and 600 μL low stringency washing buffer, respectively.

Centrifugation took place after each wash for 30 sec at 11 ,000 rpm. RNA was eluted from the silica membrane by adding RNAse-free H2O to the binding column in a 1.5 mL capped microcentrifuge tube, followed by centrifugation for 1 min at 11 ,000 rpm.

[00178] RNA was analysed with a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, Del., USA) to determine concentration. Sample purity was determined by RIN analysis conducted at SAMHRI (North Terrance, Adelaide, SA) by an independent research group. Sample purity was averaged at around 7.5-8, with any samples scoring below 7 discarded from further analysis.

[00179] cDNA conversion

RNA was converted to cDNA using the Transcriptor™ First Strand cDNA Synthesis Kit (Roche

Molecular Systems Inc., Basel, Switzerland). Briefly, 4 μL Transcriptor™ deoxynucleotide mix, 1 μL Transcriptor™ reverse transcriptase and 1 μL of RNA template were added. Samples were made up to 20 μL with nuclease free water. Samples were then incubated for 10 min at 25 °C for denaturing, annealing for 60 min at 50°C and elongation for 5 min at 85 °C. Target cDNA was amplified by PCR for 39 cycles.

Transcribed samples were analysed using a NanoDrop (NanoDrop Technologies, Wilmington, Del.,

USA).

[00180] Real-time PCR analysis

Real-time PCR was performed using a CFX Connect Real-Time PCR Detection System (Bio Rad,

California, USA). Amplification mixes contained 1 μL of cDNA, 5 μL of 2X SYBR green (Bio-Rad

Laboratories Inc. USA), 3 μL PCR-grade water and 0.5 μL of forward and reverse primers (GeneWorks,

Adelaide, Australia), making a final volume of 10 pl. Thermal cycling conditions were individually optimised for each primer set (Table 2).

Table 2. Intestinal and renal primer sequences

[00181] Wang, F. et al., Anal. Biochem., 2010. 399(2): p. 211-217; Bai, X. et al., J. Clin. Invest., 2016.

126(2): p. 667-680; Ranch, D. et al., J. Bone Miner. Res., 2011. 26(8): p. 1883-1890; Reyes-Fernandez,

P.C. and Fleet, J.C., J. Bone Miner. Res., 2016. 31(1): p. 143-151; Liu, Y. et al., Int. J. Biol. Macromol., 2020. 162: p. 935-945; Kang, Z. et al. Syst. Biol. Reprod. Med., 2017. 63(6): p. 364-369; Yamakawa, I. et al., Am. J. Physiol. Endocrinol. Metab., 2011. 301(5): p. E844-852; Shen, Y. et al., J.

Ethnopharmacol., 2020. 259: p. 112919; Romano, K. A. et al., Cell Host & Microbe, 2017. 22(3): p. 279- 290.

[00182] All samples were repeated in duplicate, with experimental thresholds (Ct) calculated by CFX Connect program. RT-PCR analysis was used to investigate the expression of lipocalin-2, NF-kB, TLR4 and TLR5 as they are all vitamin D dependent involved with calcium regulation. TNF-a is a cell signalling protein involved in systemic inflammation and is a probable target of 5-FU action in the intestine. RPLP0 was used as the house-keeping gene (HKG).

[00183] Microbiome analysis

Colon contents collected from 23 mice were sent to the Australian Genome Research Facility (AGRF) for DNA extraction and 16S rRNA Illumina sequencing, specifically targeting the V3-V4 region of 16S rRNA, allowing the microbial composition to be sequenced to the genus level. Sequencing data was processed by AGRF and provided as fastq files for each sample and resulting operational taxonomic unit (OTU) relative abundance data in a spreadsheet. The data received from AGRF was compared for relative abundance between treatment groups, looking at both individual samples and combined group data.

[00184] Sequencing data was then analysed for alpha diversity (diversity within individual samples) using Shannon’s Entropy calculation, beta diversity (diversity between samples in a population) using Bray-Curtis analysis and visualised using Principle Coordinate Analysis (PCoA) plot with multiple axes (PCol, PCo2 and PCo3).

[00185] Statistical analysis

All data was analysed using GraphPad Prism version 8.0 with the exception of microbiota (16S rRNA) data. Continuous data were analysed for normality using the D’Agostino and Pearson test and Kolmogorov-Smirnov test. When normality was confirmed, a two-way analysis of variance (ANOVA) or mixed model (when data points were missing) was used to identify statistically significant differences. When normality was not confirmed, a Kruskal-Wallis with Dunn’s correction for multiple comparisons was used. Where possible, paired or repeated measures were prioritized and indicated using line graphs. In cases where this was not possible, biospecimens collected at termination were used and indicated by grouped data (column/bar graphs). In all cases, P<0.05 was considered statistically significant. Results and discussion

[00186] VD1-6 administration does not upregulate the expression of vitamin D-responsive or inflammatory genes in the colon

VD1-6 alone and/or in combination with 5-FU did not increase CYP24A1 and CYP27B1 expression in the large intestine (i.e., colon) (Figure 12A-B). However, VDR mRNA expression in the colon was significantly higher in the saline control group when compared to all the other groups, including VD1-6 alone (p = 0.0472), 5-FU alone (p = 0.0141) and the 5-FU and VD1-6 combination group (p = 0.0063) (Figure 12C). There was no significant difference between the remaining groups. NF-KB expression was unaffected by the presence of either VD1-6 or 5-FU (Figure 13B). There was a trend towards a higher NF-KB expression in the VD1-6 groups, but this did not reach significance. TNF-a mRNA expression was unaffected by the presence of either VD1-6 or 5-FU (Figure 13 A).

[00187] VD1-6 stimulates expression of innate immune gene lipocalin-2 (LCN2) in the colon

There was a significant increase in LCN2 expression in the VDl-6-treated group compared to the 5-FU group (p = 0.0324) and the saline controls (p = 0.0139) (Figure 14A). There were no significant differences in TLR4 or TLR5 expression between groups (Figure 14B-C).

[00188] The relative abundance of family taxonomic level in the colon microbiota is altered by 5-FU and VD1-6

The relative abundance of bacteria at the family taxonomic level comprising the gut microbiota of mice differed between 5-FU-treated mice and VDl-6-treated mice compared with control mice. Despite the changes induced by each individual treatment, when 5-FU was combined with VD1-6 the relative abundance at the family taxonomic level remained similar to the control group (Figure 15). Compositional differences in colon microbiota were observed across individual mice within groups (Figure 15 A).

[00189] Microbial diversity is not significantly altered following 5-FU and/or VD1-6

Shannon entropy was used to evaluate alpha diversity (species diversity within samples). 5-FU and/or VD1-6 did not significantly affect Shannon entropy, indicative of no change in alpha diversity 48 hours after 5-FU administration. A high range between the Shannon entropy values of each sample in the vehicle control and 5-FU groups was observed. Less range in the VD1-6 and 5-FU + VD1-6 groups (Figure 16) was observed. Principle component analyses showed no significant change in beta diversity. However, when PCol, PCo2 and PCo3 values was analysed as most likely to account for any shifts in microbiota, samples from 5-FU treated mice clustered towards higher PCol (Figure 17A, 17B). To identify whether specific microbiota were contributing to this shift, Pearson’s correlation was conducted, revealing a direct correlation between PCol and Akkermansiaceae family (R2 = 0.761; p < 0.0001), and a weak correlation between PCol and Marinifilaceae family (R2 = 0.18; p = 0.041) (Figure 17C, 17D).

[00190] Whilst there were minimal disruptions to microbial diversity 48 hours following 5-FU treatment, changes to some bacteria species when microbial taxa were compared across groups were observed. 5-FU treatment resulted in a 12.2-fold increase in the abundance of Clostridium sp. K4410.MGS-306 (p = 0.003), and 4.4- and 10.1-fold decreases in the abundances of Lactobacillus murinus (p = 0.039) and Faecalibaculum rodentium (p = 0.047), respectively, compared to vehicle control. VD1-6 treatment alone resulted in a 6.3-fold increase in the abundance of Bacteroides caecimuris

(p = 0.021), and 16.1- and 12.4-fold decreases in the abundances of Lachnospiraceae bacterium 28-4 (p = 0.002) and Faecalibaculum rodentium (p = 0.031), respectively, compared to vehicle control. VD1-6 used with 5-FU resulted in an 8.9-fold increase in the abundance of Clostridium sp. K4410.MGS-306 (p = 0.013), and 14.2-fold increase in the abundance of Lachnospiraceae bacterium A4 (p = 0.044) compared to vehicle control (Figure 15). VD1-6 used with 5-FU resulted in a 16.2-fold decrease in the abundance of Lachnospiraceae bacterium A4 (p = 0.035) compared with VD1-6 alone. When corrected for false discovery rate, values were no longer deemed significant (<0.05). However, these species are of interest due to the inherent interindividual variability of microbiota and low sample size.

[00191] These data demonstrate that whilst diversity is not significantly altered 48 hours following administration of 5-FU, there are many microbial shifts occurring that may have downstream immune signalling effects. 5-FU administration did not significantly change the mRNA expression for CYP24A1 or CYP27B1 relative to RPLP0 in the colon 48 hours after administration (Figure 12). VDR expression in the colon, however, was significantly upregulated in the saline control group relative to all other groups. 5-FU’s effect on VDR mRNA expression has been previously shown to be minor. Milczarek, M., et al. (Oncol. Rep., 2014. 32(2): p. 491-504) found that VDR expression was only raised in 5-FU groups when combined with a vitamin D analogue in both tumour models and in vitro cell culture.

[00192] A possible explanation for this upregulation is the action of normal homeostatic mechanisms suppressing VDR expression in VDl-6-treated groups where CYP24A1 expression is being suppressed, slowing or preventing 1,25(OH) ₂ D ₃ catabolism and causing vitamin D levels to remain high. The lack of CYP24A1 and CYP27B1 up- or down-regulation from saline controls does not seem to support this, however prior work does confirm that CYP24A1 mRNA levels do not change following 5-FU and/or VD1-6 administration in the duodenum or jejunum. Alternatively, this upregulation does appear to be due to an individual outlier and may not be representative of the whole group. One value in particular appears as an outlier from the remaining samples (at 0.04), which are closely clustered together (0.015) and may be more representative of the true average value. Arguably, this high value in one individual mouse accounts for the statistically significant difference displayed in saline control mice against all other groups, which would otherwise not reach statistical significance. The work discussed above with VD1-6 VDR binding and in silico docking investigations implies that VD1-6 is not binding to VDR, therefore the upregulation is not due to VD1-61 binding to and potentially blocking VDR activity.

[00193] The lack of VDR upregulation in the VD1-6 groups further supports that VD1-6 does not induce the vitamin D responsive pathway as many vitamin analogues do. Of the pro -inflammatory markers investigated, NF-KB and the proceeding TNF-a, there was no difference between all groups in terms of both RNA expression in the colon. There was a trend for the VD1-6 groups with or without 5-FU treatment to have increased NF-KB mRNA expression, but this did not reach significance. This indicates that pro-inflammatory cytokines may not be the main driver of inflammation following 5-FU administration. This lack of NF-KB elevation with 5-FU treatment is consistent with current evidence from Logan, R. M. et al. (Cancer Biol. Ther., 2008. 7(7): p. 1139-1145), where 5-FU administration did not result in statistically significant elevation of NF-KB mRNA in the colon.

[00194] Inhibiting vitamin D catabolism may improve the composition of the gut microbiota, possibly through increasing the expression of lipocalin-2. The data demonstrate that lipocalin-2, an antimicrobial protein, is significantly upregulated in mice treated with VD1-6 and thus may be modulating the microbiota present. Lipocalin-2 binds to siderophores, restricting their ability to facilitate bacterial uptake of iron, restricting bacterial proliferation and survival (Lim, D. et al., 2020. 8(5)). Circulating 1,25(OH) ₂ D ₃ levels are positively correlated with levels of lipocalin-2, therefore, inhibiting 1,25(OH) ₂ D ₃ catabolism may be responsible for the upregulation of lipocalin-2 seen in the VD1-6 group (Figure 14) (Metheniti, D. et al., Hormones (Athens), 2013. 12(3): p. 397-404).

[00195] Lipocalin-2 is stimulated by recognition of bacterial lipopolysaccharides (LPS) and flagellin by toll-like receptors (TLRs) 4 and 5, respectively (Mori, K. et al. Crit. Care, 2013. 17(Suppl 2): p. P14;

Toyonaga, T. et al., Set. Rep., 2016. 6: p. 35014). TLRs’ role in detecting pathogens and activating pro- inflammatory pathways make them a crucial component of the innate immune system; their activation leads to stimulation of the adaptive immune response, inducing the production of IgA antibodies by intestinal epithelial cells (Bahadur, T. et al., J Family Med. Prim. Care, 2019. 8(5): p. 1567-1570). Therefore, VD1-6 may be protecting the intestine against mucositis via the upregulation of lipocalin-2 (an antimicrobial peptide), leading to the generation of other antimicrobial actions, such as immune memory and downregulation of pro-inflammatory pathways.

[00196] VD1-6 had no effect on TLR4 or TLR5 gene expression patterns in the colon. TLR4 upregulation is correlated with increased mucosal epithelium permeability and pro-inflammatory actions in the small intestine during the acute phase of GM (Figure 14) (Hamada, K. et al., Cancer Chemother. Pharmacol., 2013. 72(4): p. 757-765). TLR4 mRNA expression has been found to be increased in the gut during peak mucosal injury and undetectable during the healing phase (Wardill, H. R. et al., Mol. Cancer Ther., 2016. 15(6): p. 1376-1386). Extensive work with TLR4-deficient mice shows that TLR4 reduction is correlated with significant reductions in diarrhoea, weight loss and intestinal apoptosis as compared to wild-type mice after irinotecan administration (Coller, J. K. et al., Cancer Chemother. Pharmacol., 2017. 79(2): p. 431-4; Wardill et al. ibid). The lack of upregulation from 5-FU is most likely explained as peak damage during GM occurs at approximately 24 hours after 5-FU administration before decreasing back to basal levels as the (Hamada, K. et al., ibid).

[00197] The current evidence indicates that TlR5 upregulation has a protective role in the gut, as TER5 agonists have been found to offer significant protection to the mucosal epithelia from injury resulting from irradiation and chemotherapy (Burdelya, L. G. et al., Int. J. Radiat. Oncol. Biol. Phys., 2012. 83(1): p. 228-234; Burdelya, L. G. et al., Science, 2008. 320(5873): p. 226-230). The lack of upregulation in the VD1-6 groups suggests that TLR5 -meditated epithelium protection is likely not the main driver of VD1-6 action. Although TLR4 and TLR5 expression is not significantly increased in the VD1-6 group, this may be due to the delay between TLR activation and the collection of colonic tissue, as innate immunity is short-lived (Boraschi, D. & Italiani, P., Front. Immunol., 2018. 9(799)).

[00198] 1,25(OH) ₂ D ₃ has been shown to modulate the composition of the gut microbiota, while 5-FU has shown to shift the microbial composition of the gut towards a more pathogenic profile (Figures 15, 16 and 17) (Hong, B. Y. et al. Microbiome, 2019. 7(1): p. 662019; Ooi, J. H. et al., The Journal of nutrition, 2013. 143(10), p. 1679-1686). 5-FU significantly decreased the abundance of Firmicutes and increased the abundance of Parabacteroides, which is consistent with previous reports in the literature (Carvalho, R. et al. Sci. Rep., 2018. 8(1): p. 15072 2018; Li, H. L. et al., Front Cell Infect. Microbiol., 2017. 7: p. 455). Firmicutes are beneficial, anti-inflammatory microbes and are associated with healthy conditions, while Parabacteroides are associated with inflamed, pathological conditions (Carvalho, R. et al., ibid). Low abundance of Firmicutes in the gut increases the intestines sensitivity to inflammation (Natividad, J. M. et al., Inflammatory bowel diseases, 2015. 21(8): p. 1883-1893). VDR knockout mice have shown to have lower abundance of bacteria from the Firmicutes phyla, including bacteria from the Lachnospiraceae genus, highlighting the importance vitamin D in maintaining healthy gut microbiota (Ooi, J. H. et al., ibid). Higher abundance of Lachnospiraceae in the gut has proven to have anti- inflammatory effects and strengthens the barrier of the colon via increasing tight junction production (Geirnaert, A. et al., Sci. Rep., 2017. 7(1): p. 11450). Low abundances of Lachnospiraceae species are also correlated with inflammatory states (Natividad, J. M. et al., ibid), highlighting the role they may play in protecting the intestine in BVDl-6-treated mice.

[00199] VD1-6 significantly decreases the abundance of Verrucomicrobia at the phylum level, which is mostly comprised of mucin-degrading bacteria (Hamouda, N. et al., Basic Clin. Pharmacol. Toxicol., 2017. 121(3): p. 159-168). Verrucomicrobia have been found to be significantly increased in inflamed animals with 5-FU-induced mucositis (Carvalho, R. et al., ibid); bacteria belonging to this phylum are known to advance gut inflammation by degrading the protective mucous layer of the colon, suggesting they play a significant role in 5-FU-induced mucositis pathogenesis (Derrien, M. et al., Frontiers in Microbiology, 2011. 2(166)). Vitamin D deficiency in mice causes a thinner mucous layer in the colon, while supplementing with vitamin D has found to significantly reduce the abundance of Verrucomicrobia species in faecal samples and maintain the colonic mucosal barrier (Zhu, W. et al., Gut pathogens, 2019. l l:p. 8). However, there were no significant differences in abundance of the Akkermansia genus from the Verrucomicrobia phylum between the four groups, thus the significant decrease in this phylum seen in the VD1-6 group may be within a higher-ranking taxon.

[00200] Upregulated signalling of NF-KB and amplification of TNF-a, IL-1 and IL-6 are hallmarks of chemotherapy-induced GM pathogenesis (Cario, E., Curr. Opin. Support Palliat. Care, 2016. 10(2): p. 157-164; Sonis, S. Nature Reviews Cancer, 2004. 4: p. 277-284). Commensal bacteria such as Bifidiobacterium from the Actinobacteria phyla have been shown to inhibit NF-KB activation resulting in lower levels of pro-inflammatory cytokines (Ewaschuk, J. B. et al., Am. J. Physiol. Gastrointest. Liver Physiol., 2008. 295(5): p. G1025-1034; Riedel, C. U. et al., World J. Gastroenterol., 2006. 12(23): p. 3729-3735). The present data reveal that VD1-6- treated mice have significantly increased abundance of Bifidobacterium which may be inhibiting the initial NF-KB inflammatory pathway involved in chemotherapy-induced mucositis pathogenesis. Faecalibaculum species, belonging to the Firmicutes phyla, have also been shown to decrease activation of NF-KB via the secretion of substances that stimulate anti-inflammatory cytokine IL-10 production, resulting in reduced inflammation (Sokol, H. et al., Proc. Natl. Acad. Sci. USA, 2008. 105(43): p. 16731-16736). [00201] Faecalibaculum is significantly reduced in both the 5-FU and VDl-6-treated groups, however, appears to be restored in the combination treatment group as the abundance did not significantly differ from the control group. It has been previously shown that NF-KB and TNF-a peak in concentration 12 hours, and 90 minutes post 5-FU treatment respectively, returning to baseline by 48 hours (Logan, R. M. et al., Cancer Biol. Ther., 2008. 7(7): p. 1139-1145). Thus, no significant differences in expression were observed between the four groups as concentrations of NF-KB and TNF-a had returned to baseline before mice were euthanised at 48 hours post chemotherapy.

[00202] These data demonstrate that VD1-6 prevents damage/restores villus structure in a wild-type mouse line after treatment with 5-FU. There are no changes to VDR, TNF-a and NF-KB expression in the colon, indicating that VD1-6 administration does not alter calcium absorption. Inhibiting 1,25(OH) ₂ D ₃ catabolism has proven to maintain a beneficial composition of gut microbiota, which may be achieved via upregulated expression of lipocalin-2. VD1-6 increases the abundance of anti-inflammatory microbes and decreases the abundance of mucin-degrading, inflammation-inducing bacteria. 5-FU increases the abundance of pathogenic bacteria and decreases the abundance of beneficial bacteria, however when administered with VD1-6, this is mitigated.

[00203] Throughout the specification and the claims that follow, unless the context requires otherwise, the words "comprise" and "include" and variations such as "comprising" and "including" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[00204] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgement of any form of suggestion that such prior art forms part of the common general knowledge.

[00205] It will be appreciated by those skilled in the art that the compounds, uses and the compositions disclosed herein are not restricted by the particular application(s) described. Neither are the compounds, uses and compositions restricted in their preferred embodiment(s) with regard to the particular elements and/or features described or depicted herein. It will also be appreciated that the compounds, uses and compositions disclosed herein are not limited to the embodiment or embodiments disclosed, but are capable of numerous rearrangements, modifications and substitutions without departing from the scope of the disclosure as set forth and defined by the following claims.