DIAGNOSTIC METHODS AND KITS

Abstract

The present invention relates to methods for the diagnosis or prognosis of conditions caused by defects in cholesterol biosynthesis, such as Smith-Lemli-Opitz syndrome (SLOS), in particular to early diagnostic methods including in utero methods. In one aspect, the method compries detecting in a biological sample levels of delta-5 bile acid conjugated with 2-(acetylamino)-2-deoxy-D-glucose (GlcNAc) of formula (I)

Claims

1. A method for diagnosing or monitoring the progress of a condition caused by defective cholesterol biosynthesis, said method comprising detecting in a biological sample levels of delta-5 bile acid conjugated with 2-(acetylamino)-2-deoxy-D-glucose (GlcNAc) of formula (I) ##STR00010## or a derivative thereof, or a precursor of any one of formulae (II)-(VH), or (XX)-(XXIII) ##STR00011## ##STR00012## which are higher than those found in a sample from a subject not suffering from said condition.

2. A method according to claim 1 wherein the sample is a blood, plasma, serum, cerebrospinal fluid (CSF) or urine sample.

3. A method according to claim 1 wherein the levels of delta-5 bile acid GlcNAc of formula (I) ##STR00013## or a derivative thereof or a precursor thereof, which are higher than those found in a sample from a subject not suffering from said condition are detected in the biological sample.

4. A method according to claim 3 wherein a precursor of the compound of formula (I) is a compound of formula (I) to (VII), or (XX)-(XXIII) ##STR00014## ##STR00015##

5. A method according to claim 4 wherein the precursor compound of formula (II)-(VII), (XX) or (XXI) is detected in a blood, serum, plasma or CSF sample.

6. A method according to claim 1 which comprises detecting levels of the compound of formula (I) or a derivative thereof, in a urine sample from a subject suspected of or suffering from SLOS or from a urine sample from an expectant mother.

7. A method according to claim 6 wherein the derivative of formula (I) is a compound of formula (X) or (XI) ##STR00016## wherein R is a hydroxyl, glycine or taurine group.

8. A method according to claim 1 which further comprises detecting and/or quantifying a further compound or diagnostic marker which is characteristic of a condition caused by defective cholesterol biosynthesis such as SLOS.

9. A method according to claim 8 wherein the level of 8-dehydocholesterol (8-DHC) of formula (XIII) ##STR00017## or a metabolite thereof, selected from 24-hydroxy-8-DHC (24OH8-DHC) of formula (XIV), 25OH8-DHC of formula (XV) and 26OH8-DHC of formula (XVI) ##STR00018## is detected.

10. A method according to claim 9 wherein the level of the compound of formula (XIV) or (XV) is detected.

11. A method according to claim 8 wherein the level of 7-DHC of formula (IX) ##STR00019## or 7-DHC metabolites are detected and compared with those found in a sample from a subject not suffering from said condition.

12. A method according to claim 11 wherein the 7-DHC metabolites are selected from 4OH7-DHC of formula (XVII), 7-oxocholesterol (Compound VIII), 7,8-epoxycholesterol (XVIII) and 3,5-dihydroxycholest-7-en-6-one (Compound XIX) ##STR00020##

13. A method according to claim 1 wherein the compounds detected are detected and/or quantified using liquid chromatography or mass spectrometry or a combination thereof.

14. A method according to claim 13 wherein a compound is derivatised by reaction with a conjugation agent to facilitate detection.

15. A method according to claim 14 wherein the conjugation agent is a Girard agent

16. A method of modulating Smoothened (Smo) receptor activity comprising administering to a patient in need thereof an amount of Compound VI or Compound XXI, or pharmaceutically acceptable salt thereof.

17. A method of treating cancer comprising administering to a patient in need thereof an amount of Compound VI or Compound XXI, or pharmaceutically acceptable salt thereof.

18. The method according to claim 17 wherein the cancer is selected from the group consisting of an adenocarcinoma of the pancreas, prostate, breast, stomach, esophagus or biliary tract; a medulloblastoma or glioma; a small-cell lung cancer; a basal cell carcinoma; a rhabdomyosarcoma; a urothelial carcinoma; a squamous cell carcinoma of the oral cavity; and a hepatocellular carcinoma.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0049] The invention will now be particularly described by way of example with reference to the accompanying diagrammatic drawings in which:

[0050] FIG. 1 is a graph showing the concentration of the compounds of formula (VIII)(3-Hydroxycholest-5-en-7-one or 7-oxocholesterol), compound (VII) (cholest-5-ene-3,7-diol or 7-hydroxycholesterol), compound (IV) (3,7-dihydroxycholest-5-en-26-oic acid) and compound (II) (3,7-dihydroxychol-5-en-24-oic acid) in plasma from 9 SLOS patients and 50 controls from reference (Theofilopoulos et al JCI 2014). In most control samples the concentration of 3,7-dihydroxychol-5-en-24-oic acid was at or below the limit of quantification of 1 ng/mL.

[0051] FIG. 2 illustrates the novel bile acid biosynthesis starting with 7-DHC and ending with GIcNAC conjugates of 3,7-dihydroxychol-5-en-24-oic acid. The metabolites of elevated abundance found in plasma from SLOS patients are indicated by an upward pointing arrow. Metabolites of elevated abundance found in SLOS urine, and also indicated by an upwards pointing arrow, but are shown in the dashed-box.

[0052] FIG. 3 is a series of graphs showing the concentrations of further compounds that may be used as supplementary diagnostic markers in accordance with an embodiment of the invention, where (A) shows levels of oxysterols enzymatically derived from 7-DHC via oxidation of C-7 and (B) shows levels of dihydroxycholesterols, dihydroxycholestenones and isomers of dihydroxy-8-DHC.

[0053] FIG. 4 is a series of graphs illustrating the concentration of 7-OC (VIII), 7-HC (VII), 26H,7OC (XX), 3H,7OCA (VI), 3,7-diHCA (IV), 3,7,24-triHCA (III), 3,7,25-triHCA (V), 3H,7O-.sup.5-BA (XXI) and 3,7-diH-.sup.5-BA (II) in plasma from 10 SLOS patients and 24 controls determined by LC-MS exploiting charge-tagging utilising the GP reagent [Griffiths FRBM 2013, Crick Olin Chem 2015]. The bottom and top of the box are the first and third quartiles, and the band inside the box represents the median. The whiskers extend to the most extreme data points which are no more than 1.5 times the range between first and third quartile distant from the box. Points beyond that are plotted individually. Non-parametric Mann-Whitney test was used for pair-wise comparison for non-normally distributed data. *, P<0.05; **, P<0.01.

[0054] FIG. 5 is a graph showing proportions (mole %) of bile acids with 7-oxo or 7-hydroxy group conjugated with GlcNAc in urine from 3 SLOS patients and 3 controls determined by LC-MS. Total bile acids include mono-, di-and tri-hydroxylated cholanic acids and their single and doubly unsaturated equivalents singly, doubly or triply conjugated with sulfuric acid, GlcNAc and glycine or taurine. Control data is given on the right hand of each column, SLOS data on the left.

[0055] FIG. 6 is a graph showing mRNA levels of Gli1 in H-3T3 cells at different concentrations of 3-Hydroxy-7-oxocholest-5-enoic acid (Compound XXI).

EXAMPLE 1

[0056] Extraction of Sterols and Oxysterols (II-IX, XIII-XXIII) from Plasma

[0057] The applicants investigated the possibility that patients suffering from SLOS may use 7-DHC as a starting point for bile acid biosynthesis rather than cholesterol. Liquid chromatography (LC)mass spectrometry (MS) was used to determine the nature of bile acid intermediates found in plasma from patients suffering from SLOS as well as from healthy individuals as controls. Specifically compounds (II)-(IX), and (XX) to (XXIII) were identified by a process involving Girard P derivatisation and LC-MS as described by Crick P J et al., J. Olin Chem. 2015 February; 61(2):400-11. Compounds (I) and (X)-(XI) were identified in urine by an LC-MS process as described by Griffiths W J, et al. Mass Spectrometry Handbook, Ed Mike S Lee, 2012 John Wiley & Sons, 2012 p.297-337 and elucidated further below.

[0058] Plasma (100 L) was added dropwise to a solution of absolute ethanol (1.05 mL) containing 24R/S-[25,26,26,26,27,27,27-.sup.2H.sub.7]hydroxycholesterol ([.sup.2H.sub.7]24OHC) and 22R-[25,26,26,26,27,27,27-.sup.2H.sub.7]hydroxycholest-4-en-3-one ([.sup.2H.sub.7]22ROHCO]) (20 ng of each in 1.05 mL of absolute ethanol) in an ultrasonic bath. After 5 min the solution was diluted to 70% ethanol by addition of 0.35 mL of water, ultrasonicated for a further 5 min and centrifuged at 14,000g at 4 C. for 30 min. The supernatant was loaded onto a 200 mg Certified Sep-Pak C.sub.18 cartridge (pre-conditioned with 4 mL of absolute ethanol followed by 6 mL 70% ethanol) and allowed to flow at 0.25 mL/min. The flow-through was combined with a column wash of 70% ethanol (5.5 mL) to give SPE1-Fr1 containing the oxysterols. A second fraction (SPE1-Fr2) was collected by eluting with a further 4 mL of 70% ethanol before elution 5 of cholesterol, 7-dehydrocholesterol and similarly hydrophobic sterols using 2 mL of absolute ethanol (SPE1-Fr3). Each fraction was divided into two portions (A) and (B) and concentrated under reduced pressure using a vacuum concentrator (ScanLaf, Denmark).

[0059] Charge Tagging of Sterols and Oxysterols from Plasma

[0060] The sterol and oxysterol fractions (A) from above were re-constituted in 100 L of propan-2-ol then treated with KH.sub.2PO.sub.4 buffer (1 mL 50mM, pH 7) containing 3 L of cholesterol oxidase (2 mg/mL in H.sub.2O, 44 units/mg protein). The reaction mixture was incubated at 37 C. for 1 hr then quenched with 2.0 mL of methanol. Glacial acetic acid (150 L) was added followed by Girard P (GP) reagent (190 mg bromide salt or 150 mg chloride salt, 0.80 mmol). The mixture was vortexed then incubated at room temperature overnight in the dark. To remove excess reagent from the reaction mixture a recycling method was used. A 200 mg Certified Sep-Pak C.sub.18 cartridge was preconditioned with methanol (6 mL), 10% methanol (6 mL) and finally 70% methanol (4 mL). The derivatization mixture from above (3.25 mL in -70% organic) was applied to the column and allowed to flow through at -0.25 mL,/min. The column was washed with 70% methanol (1 mL) followed by 35% methanol (1 mL) and the combined eluent diluted with water (4 mL) to give a solution in 9 mL of 35% methanol. This solution was applied to the column, collected, and combined with a column wash of 17.5% methanol (1 mL). Water (9 mL) was added to give a solution in 19 mL of 17.5% methanol which was again applied to the column. The flow-through was discarded and the column washed with 10% methanol (6 mL). Derivatized sterols/oxysterols were then eluted from the column with methanol (31 mL, SPE2-Fr1, Fr2, Fr3) followed by absolute ethanol (1 mL, SPE2-Fr4). Cholesterol and 7-dehydrocholesterol were found to be almost exclusively present in SPE2-Fr3 while oxysterols elute in SPE2-Fr1 and Fr2. The fractions (B) were treated in an identical fashion to the (A) fractions but in the absence of cholesterol oxidase. This allows differentiation of sterols oxidised to contain an oxo group from those naturally possessing one. In later studies the 200 mg Certified Sep-Pak C18 cartridge has been replaced by an Oasis HLB 60-mg column [Crick An Bio Chem 2015].

[0061] LC-MS(MS.sup.n) on 5 the LTQ-Orbitrap

[0062] To analyse GP-tagged oxysterols, SPE2-Fr1 and -Fr2 were combined and diluted to give a final solution of 60% methanol. For each experiment, 20 L was injected onto the LC column and MS, MS.sup.2 and MS.sup.3 spectra recorded as described below. For the analysis of the more non-polar sterols SPE2-FR1, -Fr2 and -Fr3 were combined prior to dilution to 60% methanol.

[0063] LC was performed on a Ultimate 3000 HPLC system (Dionex, Surrey, UK) using a Hypersil GOLD revered phase column (1.9 pm particle size, 502.1 mm, Thermo Fisher). Mobile phase A consisted of 33.3% methanol, 16.7% acetonitrile and 0.1% formic acid. Mobile phase B consisted of 63.3% methanol, 31.7% acetonitrile and 0.1% formic acid. The chromatographic run started at 20% B for 1 min before increasing the proportion of B to 80% over 7 minutes and maintaining this for a further 5 min. The proportion of B was returned to 20% over 6 s and re-equilibration was for 3 min, 54 s to give a total run time of 17 min. The flow rate was 200 L/min and the eluent was directed to the atmospheric pressure ionization (API) source of an LTQ-Orbitrap. The Orbitrap was calibrated externally before each analytical session and the mass accuracy was better than 5 ppm.

[0064] The method consisted of a Fourier Transform (FT)-MS scan in the Orbitrap at 30,000 resolution (full width at half-maximum height; FWHM), simultaneous to which sequential MS.sup.2 or MS.sup.3 scans were carried out in the linear ion trap (LIT) with normalised collision energies of for MS.sup.2 and for MS.sup.3 (instrument settings).

[0065] Elevated levels of the 7-DHC metabolites, 7-oxocholesterol (Compound VIII), 7-hydroxycholesterol (Compound VII), 3, 7-dihydroxycholest-5-en-26-oic (Compound IV) and 3, 7-dihydroxychol-5-en-24-oic (Compound II) acids in SLOS plasma were detected (FIG. 1, FIG. 4). Also, elevated levels of 313-hydroxy-7-oxocholest-5-en-26-oic acid (Compound VI), 3,26-dihydroxycholest-5-en-7-one (26-Hydroxy-7-oxocholesterol) (Compound XX), 3,7,24-trihydroxycholest-5-en-26-oic acid (Compound Ill), 3,7,25-trihydroxycholest-5-en-26-oic acid (Compound V) and 3-hydroxy-7-oxochol-5-en-24-oic acid (Compound XXI) were detected (FIG. 4).

[0066] In patient samples where the 7-DHC to cholesterol ratio is high, 3-hydroxy-7-oxocholest-5-en-26-oic acid (Compound VI) was also observed. Low levels of metabolites with retention time and fragmentation patterns consistent with 3, 7, 24-trihydroxycholest-5-en-26-oic (Compound III) and 3, 7,25-trihydroxycholest-5-en-26-oic structures (Compound V) were also presumptively identified in these patient samples by comparison to the 7-epimers which were available as authentic standards.

[0067] Sterols with a 3, 7-dihydroxy-5-ene function are not substrates for HSD3B7, the oxidoreductase required to initiate A/B ring transformation to the 3-hydroxy-5-hydrogen configuration found in primary bile acids [Russell ARB 2003], so the 3, 7-dihydroxy-5-ene structure is maintained in the products of this bile acid biosynthesis pathway.

EXAMPLE 2

[0068] Extraction and Analysis of Bile Acids (I, X, XI) from Urine

[0069] Sterols possessing a 7-hydroxy group are known to be conjugated with N-acetylglucosamine (GlcNAc) and excreted in urine, and so the applicants investigated the urine of SLOS patients for GlcNAc conjugated bile acids using LC-MS methods.

[0070] Working solutions of [2,2,4,4-.sup.2H.sub.4]cholic acid (20 ng/L), [2,2,4,4-.sup.2H.sub.4]glycochenodeoxycholic acid (20 ng/L) and [2,2,4,4-.sup.2H.sub.4]taurochenodeoxycholic (20 ng/L) were prepared in absolute ethanol. 2 L (40 ng) of each working solution was added to 994 L of water in a 2 mL Eppendorf tube.

[0071] Urine (100 L, pH 6-7) was added drop-wise to the 1 mL of water containing deuterated standards (above). After 10 min ultrasonication the solution was centrifuged at 14,000 rpm, 4 C. for 30 min and the supernatant 5 retained. An Oasis HLB (60 mg, Waters) column was washed with absolute ethanol (4 mL), methanol (4 mL) and conditioned with water (4 mL). The supernatant from above was loaded onto the column and allowed to flow at 0.25 mL/min. After a 3 mL wash with water bile acids were eluted in 41 mL of methanol. The first two 1 mL fractions were combined, diluted to 60% methanol and analysed by LC-MS(MS).sup.n in an identical fashion to derivatised oxysterols as described in Example 1 with the exception that bile acid urine analysis was performed in the negative ion mode.

[0072] It was found that, in urine from SLOS patients, there was elevated levels of 3, 7-dihydroxychol-5-en-24-oic conjugated with GlcNAc (compound of formula (I)) presumably at position 7, and also the double conjugate as the 3-sulphate (FIG. 5).

EXAMPLE 3

[0073] Identification of Additional Sterols and Oxysterols in Plasma

[0074] Historical residual clinical plasma samples from SLOS patientswere analysed along with samples from newly diagnosed patients and a range of healthy controls.

[0075] Sterols and oxysterols were analysed by LC-ESI-MS.sup.n using a chargetagging approach (enzyme-assisted derivatisation for sterolanalysis, EADSA) as described in Example 1 above. In brief, nonpolar sterols including cholesterol, 7-DHC and 8-DHC were separated from more-polar oxysterols by reversed-phase solid phase extraction (RP-SPE). The separated fractions were individually treated with cholesterol oxidase to convert 3-hydroxy-5-ene and 3-hydroxy-5,7(or 8)-diene to their 3-oxo-4-ene and 3-oxo-4,7(or 8)-diene equivalents, then derivatised with Girard P (GP) reagent to add a charged quaternary nitrogen group to the analytes which greatly improve their LC-ESI-MS and MS.sup.n response. When fragmented by MS.sup.2 GP-tagged analytes give an intense 5 [M-Py]' ion, corresponding to the loss of the pyridine (Py) ring, which can be fragmented further by MS.sup.3 to give a structurally informative pattern. Some sterols and oxysterols naturally contain an oxo group and can be differentiated from those oxidised to contain one by omitting the cholesterol oxidase enzyme from the sample work-up procedure.

[0076] Representative results from these studies are illustrated in FIG. 3. These show that in SLOS patients, compounds of formulae (VII) (VIII), (XVIII), (XIV), (XV), (XVI) and (XIX) are significantly elevated in plasma as compared to those of the normal control samples and that thus these compounds may also give rise to a diagnostic application.

EXAMPLE 4

[0077] Hedgehog Signalling Assays Using Quantitative RT-PCR

[0078] NIH-3T3 cells were grown to confluency in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% Fetal Bovine Serum (FBS, Optima Grade, Atlanta Biologicals). Confluent cells were exchanged into 0.5% FBS DMEM for 24 hours to allow ciliogenesis prior to treatment with sterols in DMEM containing 0.5% FBS for 16 hours. SHH protein carrying a C-terminal hexa-histidine tag was expressed in bacteria and purified as described previously [Bishop Nat Struct Mol Biol 2009]. The mRNA levels of Gli1, a direct Hh target gene commonly used as a metric for signalling strength, were measured using the Power SYBR Green Cells-To-CT kit (Thermo Fisher Scientific). The primers used are Gli1 (forward primer: 5-ccaagccaactttatgtcaggg-3 and reverse primer: 5-agcccgcttctttgttaatttga-3), Gapdh (forward primer: 5-agtggcaaagtggagatt-3 and reverse primer: 5-gtggagtcatactggaaca-3). Transcript levels relative to Gapdh were calculated using the elta-Ct method. Each qRT-PCR experiment, which was repeated twice, included two biological replicates, each with two technical replicates.

[0079] FIG. 6 shows results using compound 3-Hydroxy-7-oxocholest-5-enoic acid (XXI), indicating that this compound is an inhibitor of Hedgehog signalling.

[0080] FIG. 7 shows the results using 27-hydroxy-7-oxocholesterol (compound XX, alternatively known as 26-hydroxy-7-oxocholesterol), indicating that this compound is an activator of Hedgehog signalling.

REFERENCES

[0081] Defects in bile acid biosynthesis--diagnosis and treatment. Setchell K D, Heubi J E. J Pediatr Gastroenterol Nutr. 2006 July; 43 Suppl 1:S17-22.

[0082] Cerebrotendinous xanthomatosis: an inborn error in bile acid synthesis with defined mutations but still a challenge. Bjrkhem I, Hansson M. Biochem Biophys Res Commun. 2010 May 21; 396(1):46-9.

[0083] Role of a disordered steroid metabolome in the elucidation of sterol and steroid biosynthesis. Shackleton C H. Lipids. 201230 January; 47(1):1-12.

[0084] Identification of unusual 7-oxygenated bile acid sulfates in a patient with Niemann-Pick disease, type C. Alvelius G, Hjalmarson 0, Griffiths W J, Bjrkhem I, Sjvall J. J

[0085] Lipid Res. 2001 October; 42(10):1571-7.

[0086] Cholestenoic acids regulate motor neuron survival via liver X receptors. Theofilopoulos S, Griffiths W J, 5 Crick P J, Yang S, Meljon A, Ogundare M, Kitambi S S, Lockhart A, Tuschl K, Clayton P T, Morris A A, Martinez A, Reddy M A, Martinuzzi A, Bassi M T, Honda A, Mizuochi T, Kimura A, Nittono H, De Michele G, Carbone R, Criscuolo C, Yau J L, Seckl J R, Schle R, Schls L, Sailer A W, Kuhle J, Fraidakis M J, Gustafsson J , Steffensen K R, Bjorkhem I, Ernfors P, Sjovall J, Arenas E, Wang Y. J Olin Invest. 2014 November; 124(11):4829-42.

[0087] The enzymes, regulation, and genetics of bile acid synthesis. Russell D W. Annu Rev Biochem. 2003; 72:137-74.

[0088] Analytical strategies for characterization of oxysterol lipidomes: liver X receptor ligands in plasma. Griffiths W J, Crick P J, Wang Y, Ogundare M, Tuschl K, Morris A A, Bigger B W, Clayton P T, Wang Y. Free Radic Biol Med. 2013 June; 59:69-84.

TABLE-US-00001 From Theo- NIST From filopoulos PLASMA SLOS SLOS SRM(1) Griffiths (2) (3) Sterol Systematic Name (Common name) CODE Mean SEM Mean SD Mean SEM Mean SEM 3,7-Dihydroxychol-5-en-24-oic acid (II) 27.22 11.71 1.49 NM NM Cholesta-5,8-diene-3,24(or25)-diol (XIV + XV) 3.42 0.79 ND NM NM Cholesta-5,8-diene-3,26-diol (XVI) 5.21 1.61 0.05 NM NM 7,8-Epoxycholest-5-en-3-ol (XVIII) 19.51 13.59 ND NM NM Cholest-5-ene-3,7-diol (7- (VII) 17.10 4.40 0.48 0.28 0.00 0.32 1.02 0.58 Hydroxycholesterol) 3-Hydroxycholest-5-en-7-one (7- (VIII) 44.55 17.82 0.59 0.33 3.77 1.29 4.98 2.25 Oxocholesterol) 3,5-Dihydroxycholest-7-en-6-one (XIX) 0.72 0.35 ND NM NM 3,7-Dihydroxycholest-5-en-26-oic acid (IV) 78.86 38.16 2.74 0.18 5.36 0.80 1.67 0.32 3-Hydroxy-7-oxocholest-5-en-26-oic acid (VI) 11.87 7.86 0.04 NM NM 3,7,24-Trihydroxycholest-5-en-26-oic acid (III) 0.50 ND NM NM 3,7,25-Trihydroxycholest-5-en-26-oic acid (V) 0.63 ND NM NM ND not detected. NM not measured (1)NIST standard reference material 1950. Pooled sample, representative of the USA population (2) Analytical strategies for characterization of oxysterol lipidomes: liver X receptor ligands in plasma. Griffiths W J, Crick P J, Wang Y, Ogundare M, Tuschl K, Morris A A, Bigger B W, Clayton P T, Wang Y. Free Radic Biol Med. 2013 Jun; 59: 69-84. (3) Cholestenoic acids regulate motor neuron survival via liver X receptors. Theofilopoulos 5, et al. J Clin Invest. 2014 Nov; 124(11): 4829-42