Patent Application
20230416299
The present invention relates to compounds, methods for the treatment of neurodegenerative conditions and methods of diagnosis of neurodegenerative conditions and in particular but not exclusively to the use of a cholestenoic acid to treat neurodegenerative conditions such as motor neuron disease.
The present invention also relates to deuterated compounds and in particular compounds that are derivatives of cholestenoic acids. The present invention also relates to compositions including said deuterated compounds and their use, in particular but not exclusively, in the treatment of conditions such as motor neurone disease.
The vertebrate central nervous system (CNS) is composed of a wide variety of neurons that are generated following tightly-regulated developmental programs. Characterization of the function and specificity of molecules and regulatory elements working on distinct neuronal populations is thus essential in order to enhance our understanding of how such complexity is achieved in the developing brain and how it is maintained in the adult brain.
One means of developmental and adult regulation is via nuclear receptors. Nuclear receptors expressed in embryonic and adult brain have a developmental role and a function in the adult brain. The liver X receptors (Lxrα and β), which are activated by oxysterols, are required for neurogenesis during ventral midbrain (VM) development. It is believed that Lxrs and their ligands have an effect on adult motor neurons. Enzymes involved in the synthesis of cholesterol and oxysterols, such as 2,3-oxidosqualene-lanosterol cyclase, are localized in Islet1+ oculomotor neurons in the mouse VM at E11.5 and oxysterols and endogenous brain Lxr ligands are sufficient to regulate neurogenesis in the developing ventral midbrain. While endogenous brain Lxr ligands have been identified and found to regulate the development of midbrain dopaminergic neurons and red nucleus neurons, no endogenous ligand capable of regulating the survival of motor neurons in vivo has so far been identified but research has indicated that specific Lxr ligands regulate the balance between motor neuron survival and death.
Cholesterol is present at high levels in the CNS of vertebrates and is metabolized in the brain, predominantly to 24S-hydroxycholesterol (24S-HC). Neurodegenerative conditions that occur are as a result of neurons in the brain being lost. In conditions such as Parkinson's disease, which is a common neurodegenerative disease, the condition is linked to the loss of substantial nigra midbrain dopaminergic neurons. The loss of nigrostriatal neurons results in symptoms such as tremors that are a classic symptom of the illness.
Research has worked on the use of cell replacement therapy (CRT) and regenerative medicine to try and combat the disease, which is becoming more prevalent as populations age. However, an alternative means of developmental and adult regulation is via nuclear receptors. It has been found that the liver X receptor (Lxr) ligand is a specific inducer of midbrain dopaminergic neurons both in embryonic stem cells, neural tissues and even in whole animals. In particular, examples of nuclear receptors expressed in embryonic and adult brain having both a developmental role and a function in the adult brain are the liver X receptors (Lxrα and β). The liver X receptors (Lxrα and β), are activated by oxysterols. Analysis of double Lxrα and Lxrβ knockout mice revealed that Lxrs are required for neurogenesis during ventral midbrain (VM) development. Moreover, adult male Lxrβ knockout mice (Lxrβ−/−) show a progressive accumulation of lipids in the brain and loss of spinal cord motor neurons, suggesting a neuroprotective role of Lxrs and their ligands on adult motor neurons. Similarly, the number of Islet1+ oculomotor neurons is lower in the developing midbrain of Lxrα−/−β−/−mice, indicating a role of Lxrs, not only in the maintenance of adult motor neurons, but also in their development. Enzymes involved in the synthesis of cholesterol and oxysterols, such as 2,3-oxidosqualene-lanosterol cyclase, are localized in Islet1+ oculomotor neurons in the mouse ventral midbrain and it has been found that oxysterols and endogenous brain Lxr ligands are sufficient to regulate neurogenesis in the developing ventral midbrain. While endogenous brain Lxr ligands have been identified and found to regulate the development of midbrain dopamine neurons and red nucleus neurons (Theofilopoulos et al. (2012) Nat. Chem. a Biol. 9, 126-133), to date, no endogenous ligand that is capable of regulating the survival of motor neurons in vivo has so far been identified.
Cholestenoic acids are not mere intermediate metabolites of bile acid biosynthesis, but are rather a diverse family of bioactive compounds, capable of regulating nuclear receptor function. They were found to specifically activate Lxr and elicit an array of functions ranging from the regulation of Islet-1 expression, to the positive and negative regulation of motor neuron survival. It has been shown that 3β,7α-dihydroxycholest-5-en-26-oic acid (3β,7α-diHCA) and 3β,7β-dihydroxycholest-5-en-26-oic acid (3β,7β-diHCA) are Lxr ligands present in human CSF (cerebrospinal fluid). Of these, 3β,7β-diHCA, and the previously identified Lxr ligand, 3β-hydroxycholest-5-en-26-oic acid (3β-HCA), were found to cause neuronal cell death, and the latter was present at high levels in patients with hereditary spastic paresis type SPG5. Also 3β,7α-diHCA, which was found at low levels in SPG5 and was absent from cerebrotendinous xanthomatosis (CTX) patients was found to promote motor neuron survival.
This indicates that compounds such as cholestenoic acids may be Lxr ligands and as such are key regulators of motor neuron function in development and disease. However, the neuroprotective 3β,7α-diHCA is metabolised by enzymes such as HSD3B7 to its 3-oxo metabolite 7α-hydroxy-3-oxocholest-4-en-26-oic acid (7αH,3O-CA) or to its neurotoxic metabolite 3β,7β-diHCA. Alternatively, metabolism may lead to side-chain shortening through beta oxidation to C24 acids.
It has also been reported that cholesterol metabolites that had the capacity to activate Lxrs can be identified in human cerebrospinal fluid (CSF) (Ogundare M, et al. J Biol Chem 2010; 285(7):4666-79).
In order to identify novel Lxr ligands that regulate motor neuron function the applicants delved deeper into the human CSF sterolome and examined plasma of patients with different human diseases associated with motor neuron degeneration, hereditary spastic paresis (HSP) type 5 (SPG5) and cerebrotendinous xanthomatosis (CTX) as well as infants with oxysterol 7α-hydoxylase deficiency (07αHD). These diseases result from mutations in the cytochrome P450 CYP7B1 (SPG5 and 07αHD) and CYV27α1 genes (CTX). The enzymes coded by these genes are responsible for 7α-hydroxylation of oxysterols and (25R),26-hydroxylation of sterols, respectively, reactions that generate further oxysterols and ultimately cholestenoic acids (
In addition, the applicants showed that patients with CTX, SPG5 and 07αHD are unable to synthesize normal amounts of the Lxr ligand 3β,7α-dihydroxycholest-5-en-26-oic acid (3β,7α-diHCA), a cholestenoic acid that the applicants found promotes neuronal survival. This is of interest in relation to the fact that patients with SPG5 present with motor neuron degeneration and spastic paraplegia. Patients with CTX may sometimes also present with spasticity, possibly due to upper motor neuron degeneration. These results have important implications for the treatment neurological diseases leading to motor neuron degeneration.
They indicate that cholestenoic acid acting as Lxr ligands, as well as inhibitors of specific biosynthetic enzymes in the cholestenoic acid biosynthetic and metabolic pathways, are useful pharmaceuticals for the treatment of motor neuron disorders.
Furthermore, the applicants showed that whilst patients with CTX had abnormally low levels of all of cholest-(25R)-5-en-3β,26-diol 26-HC (26-HC), 3β-hydroxycholest-5-en-26-oic acid (3β-HCA) and 3β,7β-dihydroxycholest-5-en-26-oic (3β,7β-diHCA), patients with SPG5 and 07αHD had elevated levels of some or all of these (
All of these results have important implications for the diagnosis of neurological diseases leading to motor neuron degeneration. They indicate that the levels of certain cholestenoic acids (3β-HCA, 3β,7β-diHCA and 3β,7β-diHCA) and a precursor (26-HC) alone or in combination are diagnostic and/or prognostic for motor neuron degenerative disease and/or the level of neurodegeneration.
According to a first aspect of the invention there is provided a reagent selected from a cholestenoic acid, a cholestenoic acid precursor or an inhibitor of an enzyme in the cholestenoic acid biosynthetic or metabolic pathway or a pharmaceutically acceptable salt thereof for use in the treatment of neurodegenerative conditions.
The applicants have found that cholestenoic acids, and in particular certain forms including some forms not previously identified as being present in the CSF have a direct effect on neuron generation and survival. Some forms of cholestenoic acid appear to show neuroprotective effects, whilst others may be harmful or neurotoxic. As a result, modifying the relative amounts of the specific forms of cholestenoic acid, for example by administering favourable cholestenoic acids or by inhibiting the production of undesirable cholestenoic acids will constitute a useful method for treating or preventing neurodegenerative conditions.
In a particular embodiment of the first aspect of the invention, a cholestenoic acid or a pharmaceutically acceptable salt thereof, used in the treatment of neurodegenerative conditions. In particular the cholestenoic acid is an acid which has a neuroprotective effect, such as 3β,7α-dihydroxycholest-5-en-26-oic (3β,7α-diHCA), or a precursor of this form such as cholest-(25R)-5-en-3β,26-diol 26-HC (26-HC).
Alternatively, inhibition of the production of undesirable cholestenoic acids, such as of 3β,7β-dihydroxycholest-5-en-26-oic (3β,7β-diHCA) or 3β-hydroxycholest-5-en-26-oic (3β-HCA), may be achieved by interfering with the biosynthetic pathway involved in the production of such cholestenoic acids, in particular by administration of an inhibitor of an enzyme that produces such a form of cholestenoic acid. For example, an inhibitor of an epimerase enzyme, that prevents the production of 3β,7β-dihydroxycholest-5-en-26-oic (3β,7β-diHCA), a cholestenoic acid which has been identified by the applicants as having a degree of toxicity to neurons from the desirable epimeric form, 3β,7α-diHCA would provide a beneficial effect. Enzymes that produce this effect and inhibitors therefore may be determined using conventional screening methods. For example, HSD1 enzymes such as 11β-hydroxy steroid dehydrogenase type 1 (11β-HSD1-EC 1.1.1.146) may show epimerase activity (Hennebert 0, et al. J Steroid Biochem Mol Biol. 2009 114 (1-2) 57-63). The use of inhibitors of HSD1 enzymes has been suggested in the treatment of certain neurological disorders previously (see WO2005/060694), but applications in the control of cholestenoic acid biosynthesis and the effects on conditions such as motor neuron disease has not previously been evaluated.
Methods for screening for compounds useful in the treatment of neurodegenerative conditions, which comprises identifying suitable enzyme inhibitors, in particular, for inhibitors of an epimerase enzyme that converts 3β,7α-diHCA to 3β,7β-diHCA form a further aspect of the invention. The precise form of these methods will be determinable by the skilled person with reference. For instance, these methods may involve incubation of the enzyme with the enzyme substrate (such as 3β,7α-diHCA) and the proposed inhibitor in vitro, after which the presence or amount of the enzymatic reaction product (such as 3β,7β-diHCA) is determined and the results compared with similar results obtained in the absence of the proposed inhibitor. Alternatively, the binding site of the enzyme to the substrate such as 3β,7α-diHCA is identified and isolated peptides or proteins comprising said binding site may be used in binding assays, to detect possible inhibitor compounds that bind specifically to that site.
As used herein, the expression ‘pharmaceutically acceptable salt’ includes both pharmaceutically acceptable base and acid addition salts. For example a base-addition salt of a cholestenoic acid may be an alkali or alkaline earth metal salt such as a sodium, calcium or magnesium salt, or an ammonium salt, or a salt with an organic base such as methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine. Such salts may be prepared by methods known to those skilled in the art.
According to a second aspect of the invention there is provided the use of a reagent selected from cholestenoic acid, a cholestenoic acid precursor or an inhibitor of an enzyme in the cholestenoic acid biosynthetic or metabolic pathway, or a pharmaceutically acceptable salt in the preparation of an agent for the treatment of neurodegenerative conditions.
In a particular embodiment of the second aspect, a cholestenoic acid such as 3β,7α-dihydroxycholest-5-en-26-oic (3β,7α-diHCA), or a precursor of this form such as cholest-(25R)-5-en-3β,26-diol (26-HC) is used in the preparation.
The cholestenoic acid or inhibitor of an enzyme in the cholestenoic acid biosynthetic or metabolic pathway will generally be administered in the form of a pharmaceutical composition, in which it is combined with a pharmaceutically acceptable carrier.
According to a third aspect of the invention there is provided a pharmaceutical or veterinary composition comprising a cholestenoic acid, a cholestenoic acid precursor or inhibitor of an enzyme in the cholestenoic acid biosynthetic or metabolic pathway, or a pharmaceutically acceptable salt for use in the treatment of neurodegenerative conditions. In particular, the pharmaceutical or veterinary composition will comprise a cholestenoic acid.
Suitable pharmaceutical compositions will be in either solid or liquid form including pharmaceutically acceptable salts, crystalline polymorphs, solvates, hydrates, co-crystals and amorphous forms. They may be adapted for administration by any convenient peripheral route, such as parenteral, oral, vaginal or topical administration or for administration by inhalation or insufflation. The pharmaceutical acceptable carrier may include diluents or excipients which are physiologically tolerable and compatible with the active ingredient. These include those described for example in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
Parenteral compositions are prepared for injection or infusion, for example either subcutaneously, intramuscularly, intradermally, intravenously, intraspinally, intrathecally, epidurally or via needle-free injection systems. They may be liquid solutions or suspensions, or they may be in the form of a solid that is suitable for solution in, or suspension in, liquid prior to injection. Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof. In addition, if desired the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing or pH-buffering agents, and the like.
Oral formulations will be in the form of solids or liquids, and may be solutions, syrups, suspensions, tablets, pills, capsules, sustained-release formulations, or powders. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.
Topical formulations will generally take the form of suppositories, pessaries, intranasals sprays or aerosols, buccal or sublingual tablets or lozenges. For suppositories or pessaries, traditional binders and excipients may include, for example, polyalkylene glycols or triglycerides; such suppositories or pessaries may be formed from mixtures containing the active ingredient. Other topical formulations may take the form of a lotion, solution, cream, ointment or dusting powder, that may optionally be in the form of a skin patch.
According to a fourth aspect of the invention there is provided a process for the preparation of a pharmaceutical composition comprising bringing a cholestenoic acid or an inhibitor of an enzyme in the cholestenoic acid biosynthetic or metabolic pathway, and in particular a cholestenoic acid, in conjunction or association with a pharmaceutically or veterinarily acceptable carrier or vehicle.
According to a fifth aspect of the invention there is provided a method of treatment or prevention of neurodegenerative conditions which method comprises modifying the amount of specific cholestenoic acids in an individual. In particular, the amount of 3β,7α-dihydroxycholest-5-en-26-oic (3β,7α-diHCA) is increased, and/or the amount of 3β,7β-dihydroxycholest-5-en-26-oic (3β,7β-diHCA) or 3β-hydroxycholest-5-en-26-oic (3β-HCA) is reduced.
In a particular embodiment, of the fifth aspect, a cholestenoic acid, a cholestenoic acid precursor or an inhibitor of an enzyme in the cholestenoic acid biosynthetic or metabolic pathway, or a pharmaceutically acceptable salt thereof, is administered to an individual in need thereof.
In particular, the method is for treating a neurodegenerative condition. In particular a cholestenoic acid is administered to an individual in need thereof.
Suitable neurodegenerative conditions that may be treated in this way include Parkinson's disease, Alzheimer's disease, mild cognitive impairment (MCI), frontotemporal dementia, dementia, multiple sclerosis, motor neuron disease, Huntingdon's disease, epilepsy, anxiety disorders (including panic disorders and posttraumatic stress disorder (PTSD)), depression, alcohol disorder, drug abuse, growth retardation and cachexia.
In particular, the method of the invention may be used to treat a motor neuron disease. Without being bound by or limited to any classification system, suitable motor neuron diseases treated in this way include but are not limited to amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), progressive bulbar palsy (PBP), Pseudobulbar palsy (BP), spinal muscular atrophy (SMA), hereditary spastic paresis (HSP) and cerebrotendinous xanthomatosis (CTX). In particular, the method of the invention may be used to treat ALS.
Similarly, the cholestenoic acid or enzyme inhibitor is suitably administered in the form of a pharmaceutical composition as described above.
The amount of cholestenoic acid or enzyme inhibitor administered will vary depending upon factors such as the specific nature of the cholestenoic acid used, the size and health of the patient, the nature of the condition being treated etc. in accordance with normal clinical practice. Typically, a dosage in the range of from 0.01-1000 mg/Kg, for instance from 0.1-10 mg/Kg, would produce a suitable therapeutic effect.
Dosages may be given by constant infusion, in single dose regimens, split dose regimens and/or in multiple dose regimens lasting over several days. Effective daily doses will, however vary depending upon the inherent activity of the active ingredient, such variations being within the skill and judgment of the physician. The cholestenoic acid or enzyme inhibitor may be used in combination with or alternating with one or more other active agents, such as one or more pharmaceutically active agents or may be integrated into courses of physical treatment modality, such as surgery or physiotherapy
In each of the first to fifth aspects of the invention the reagent is preferably the cholestenoic acid, 3β,7α-dihydroxycholest-5-en-26-oic (3β,7α-diHCA).
It is further envisaged that the cholestenoic acid is 3β,7α-dihydroxycholest-5-en-26-oic (3β,7α-diHCA) alone or in combination with 25-hydroxycholesterol (25-HC).
The applicants' discovery of the importance of various forms of cholestenoic acid in neurodegenerative conditions also allows for diagnosis of such conditions to be made. Thus, in a sixth aspect of the invention there is provided method for diagnosing or predicting a predisposition towards a neurological disorder, which method comprises determining the absolute or relative amounts of specific forms of cholestenoic acid and/or a cholestenoic acid precursor present in a biological sample, and relating the result to the presence or absence of or a predisposition towards development of a neurological disorder. In particular, the presence or amount of cholestenoic acid selected from 3β,7α-diHCA, 3β,7β-diHCA, 7α,26-diHCO, 7α,26-diHC and 3β-HCA is determined as a basis of the diagnostic method. In accordance with the work reported here, it is assumed that a lack of or reduction in the levels of any of these or elevated levels of 3β-HCA or 3β,7β-diHCA, may be indicative of a neurodegenerative condition.
In a seventh aspect, the invention provides the use of a cholestenoic acid and/or a cholestenoic acid precursor as a diagnostic biomarker for the degree of or progression of CNS disease or a predisposition to CNS disease including but not limited to a neurodegenerative condition as described above. In accordance with normal practice, the level of the specific cholestenoic acid used as such a biomarker may be compared to levels found in samples from normal individuals either once or repeatedly and variance of this amount may be interpreted as being an indicator of the presence of or likelihood of or progression of disease or as an index of therapeutic response or for the stratification of patients in clinical trials. In particular, the cholestenoic acid is selected from those listed above.
According to an eighth aspect of the invention there is provided compound of general formula (I)
or a pharmaceutically acceptable salt thereof, wherein:
It is envisaged that R1 is deuterium and R2-R4 is hydrogen. As an alternative it is envisaged that R2 is deuterium and R1, R3, R4 is hydrogen.
In an alternative arrangement each of R1 and 2 are deuterium and R3 and R4 are hydrogen.
It is similarly envisaged that R3 and/or R4 is deuterium giving additional arrangement of isotopes.
Preferably the compound of formula (I) is a cholestenoic acid.
It is preferred that the compound of formula (I) is deuterated 3β,7α-dihydroxycholest-5-en-26-oic acid (3β,7α-diHCA) and pharmaceutically acceptable salts thereof.
It is envisaged that any atom not designated as deuterium is to be considered to be present at or close to its natural abundance.
Alternatively any hydrogen atom may optionally be substituted for a deuterium atom at an abundance that is greater than the natural abundance of deuterium.
The compounds are useful for treating both humans and other mammals for example farm animals or pets and so according to a ninth aspect of the invention there is provided a compound of formula (I) for use in medicine and in particular for the treatment of neurodegenerative diseases.
According to a tenth aspect there is provided the use of a compound of general formula (I) in the preparation of an agent for the treatment of neurodegenerative conditions.
Furthermore according to an eleventh aspect of the invention there is provided a method for the treatment of neurodegenerative diseases, the method comprising administering to a patient in need of such treatment, an effective amount of a compound of general formula (I).
According to a fifth aspect of the invention there is provided a process for the preparation of a pharmaceutical composition comprising bringing a compound of general formula (I) in conjunction or association with a pharmaceutically or veterinarily acceptable carrier or vehicle.
To conclude, this invention, in the eighth to eleventh aspects, relates to deuterated compounds, and in particular to forms of 3β,7α-dihydroxycholest-5-en-26-oic acid (3β,7α-diHCA) and pharmaceutically acceptable salts thereof. This invention also provides compositions comprising a compound of this invention and the use of such compositions in methods of treating diseases and conditions that are beneficially treated by administering a deuterated form of 3β,7α-dihydroxycholest-5-en-26-oic acid (3β,7α-diHCA) to treat illnesses such as motor neurone disease.
An embodiment of the invention will now be described by way of example only with reference to the accompanying figures and Tables in which:
The firefly luciferase activity was normalized to Renilla luciferase activity, and the values are expressed as fold activation over the normalized basal Lxr or Fxr response element-luciferase activity set to 1. Data are means±SE (n=3), *, p<0.05; **, p<0.01 compared with vehicle treatment. Additional analysis was performed on the compounds 5α-cholestan-3β-ol, 5α-cholestan-3-one, cholest-4-en-3-one, cholesta-4,6-dien-3-one, 7α-hydroxycholesterol, 7α-hydroxycholest-4-en-3-one, 7β-hydroxycholesterol, and 7α, 12α-dihydroxycholest-4-en-3-one which are found at elevated levels in plasma of CTX patients. None of these compounds showed significant activity in the luciferase assay. Panel D shows a time-resolved (TR)-FRET Lxrβ coactivator assay used to determine the binding affinity of cholestenoic acids towards the Lxrβ-LBD. Data are means±SEM (n=3) and * represents significant difference (p<0.05) compared with vehicle treatment for cholestenoic acid concentrations of 10 μM and higher. Concentration is plotted on a log scale. Panel E illustrates that 3β,7α-diHCA, 3β,7β-diHCA and 3β-HCA induce significant increases in Abca1, Abcg1, and Srebf1 in SN4741 cells. Data are means±SE (n=3), *, p<0.05; **, p<0.01 compared to vehicle treatment.
In panel 3C one sees Islet-1 mRNA levels, in panel 3D, and panel 3E Islet-1 one sees protein levels (quantification and representative blot) and in panel 3F obe sees abca1 mRNA levels, after treatment of zebrafish with the compounds indicated. Data are means±SE (n=3), *, p<0.05; **, p<0.01 compared to vehicle-treated zebrafish. Other test compounds: 25-hydroxycholesterol (25-HC); 7α-hydroxy-3-oxocholest-4-en-26-oic acid (7αH,3O-CA); 3β-hydroxychol-5-en-24-oic acid (3βH-Δ5-BA). The cranial nerves (III, IV, V, VII, X) in the zebrafish are evolutionarily homologous to those in humans. Locus III contains the oculomotor neurons, IV contains trochlear neurons, V the trigeminal motor neurons, VII the facial motor neurons and X the cell bodies of the vagus nerve.
Table 1 shows: oxysterols and Cholestenoic Acids in Human CSF
Oxysterols and cholestenoic acids identified by LC-ESI-MSn in CSF following SPE and charge-tagging with GP-hydrazine. In the absence of authentic standards presumptive identifications based on exact mass, MSn spectra and retention time are given. Samples from 12 individual subjects (bluet) and a pool of fifteen subjects (black %) were analysed.
Table 2 shows oxysterols and Cholestenoic Acids in Human Plasma (Serum)
Oxysterols and cholestenoic acids identified by LC-ESI-MSn in plasma (serum) following SPE and charge-tagging with GP-hydrazine. In the absence of authentic standards presumptive identifications based on exact mass, MSn spectra and retention time are given. Control samples from 56 adults (bluet) and 3 children (blackj) were analysed. Data for six adults showing clinically pure HSP SPG5 plus one adult with complicated HSP SPG5 (purple*), three infants suffering from 07αHD (gold§), and four patients suffering from CTX (brown]}) are given. Clinical data is given in Table 3.
Table 3 shows: mutations in SPG5, 07AHD and CTX patients studied.
Table 4 shows cholestenoic Acids in Cyp7bl−/− and Cyp27αl-I—Mouse Brain and Plasma Cholestenoic acids identified by LC-ESI-MSn following SPE and charge-tagging with GP-hydrazine.
Reagents. HPLC grade water and solvents were from Fisher Scientific (UK) or Sigma Aldrich (UK). Authentic sterols, steroids, cholestenoic acids, bile acids and their precursors were from Avanti Polar Lipids (Alabama, USA), Steraloids Inc (Rhode Island, USA), Sigma Aldrich (UK), or from previous studies in our laboratories. Girard P (GP) reagent [1-(carboxymethyl)pyridinium chloride hydrazide] was from TCI Europe (UK) or synthesized in earlier studies, and cholesterol oxidase from Streptomyces sp was from Sigma-Aldrich. Certified Sep-Pak Ci8 200 mg solid phase extraction (SPE) cartridges were from Waters (UK). Leur-lock syringes were from BD Biosiences (UK). Patient Samples. Adult CSF and plasma samples were part of a GlaxoSmithKline study. Plasma/serum from CTX, SPG5 and 07AHD patients were from Barts and the London NHS Trust; St Mary's Hospital, Manchester; Institute of Child Health, London; Conegliano Research Center, Conegliano; Universita degli Studi di Napoli Federico II, Naples; and Kurume University School of Medicine, Kurume, Japan.
Animals. Lxrα−/−fi−/− mouse cell cultures were from the colony at the Department of Biosciences and Nutrition at Novum, Karolinska Institutet. Male and female wild-type and Lxrα−/−fi−/− mice were generated as previously described (Alberti et al. J. Clin. Invest 2001: 107(5), 565-73). Mice were back crossed onto a C57BL/6 background for 10 generations. Male Cyp7bl−/− mouse brain and plasma were from animals generated at the University of Edinburgh. Male mice homozygous for targeted disruption of the Cyp7bl gene congenic on the C57BL/6 genetic background (>15 generations backcrossed to C57BL/6) and wild-type littermate controls were generated from Cyp7bl-I+ crosses. Male Cyp27αl−/− mouse tissue and plasma was purchased from The Jackson Laboratory (ME, USA) strain B6.129-Cyp27αltmlElt/J. The Cyp27αl−/− colony was backcrossed to C57BL/6J inbred mice for approximately 12 generations by the donating investigator prior to sending to The Jackson Laboratory Repository. Upon arrival, mice were bred to C57BL/6J inbred mice for at least one generation to establish the colony. Wild type animals from the colony were used as controls.
Extraction of Sterols. Sterols were extracted from CSF, plasma or mouse brain into ethanol and fractionated by reversed phase SPE to give a cholestenoic acid and oxysterol rich fraction devoid of cholesterol.
Charge Tagging of Sterols. The sterols were charge-tagged with the GP-hydrazine to enhance their response when analysed by LC-ESI-MS and tandem mass spectrometry (MSn).
LC-ESI-MS on the LTQ-Orbitrap LC-ESI-MS and LC-ESI-MSn was performed using an Ultimate 3000 HPLC system (Dionex, Surrey, UK) linked to the ESI source of a LTQ-Orbitrap XL or LTQ-Orbitrap Velos (Thermo Fisher, San Jose, CA) mass spectrometer. Luciferase Reporter Assay. The ability of oxysterols and their acidic metabolites to activate several nuclear receptors i.e. Lxrα and β, Fxr, Vdr, Nurrl was tested in luciferase assays. Transient transfection studies were performed in the mouse neuronal cell line SN4741. This cell line was selected as the oxysterols and acidic metabolites tested were initially identified in CSF. Cells were plated in 24-well plates (5×105 cells/well) 24 hr before transfection and transfected with 1 μg of plasmid DNA/well complexed with 2 μL of Lipofectamine 2000 (Invitrogen). Cells were transfected with 400 ng of a Lxr-, Fxr-, Vdr- or Nurrl−responsive luciferase reporter construct, and 200 ng of Lxrα, Lxrβ, Fxr, Vdr or Nurrl. A reporter gene expressing the Renilla luciferase (pRL-TK, Promega) was co-transfected in all experiments as an internal control for normalization of transfection efficiency. After a 12 h incubation, the lipid/DNA mix was replaced with fresh 2.5% serum medium containing vehicle or appropriate ligand (10 μM), as specified in each experiment. The ability of cholestenoic acids to activate Lxr was confirmed in experiments with or without the Lxr inhibitor GGPP (10 μM) also added to the medium. Luciferase activities were assayed 24 h later using the Dual-Luciferase Reporter Assay System (Promega), following the manufacturer's protocol.
Lxrβ Ligand Binding Assay. For Lxrβ ligand binding activity measurement, the applicants used the LANTHASCREEN™ TR-FRET Lxrβ Coactivator Assay (Invitrogen). The assay uses a terbium (Tb)-labeled anti-GST antibody, a fluorescein-labeled coactivator peptide and the Lxrβ-LBD tagged with glutathione-S-transferase (GST). Binding of the agonist/ligand to Lxrβ-LBD causes a conformational change that result in an increase in the affinity of the Lxrβ for the coactivator peptide. The close proximity of the fluorescently labeled coactivator peptide to the Tb-labeled antibody causes an increase in the TR-FRET signal intensity. The TR-FRET ratio of 520/495 was calculated using a Victor multi label reader with an excitation wavelength of 340 nm and emission wavelengths of 520 nm and 495 nm. The activational capacity of potential ligands was tested in a 382-well polypropylene plate, following the manufacturer's protocol.
Quantitative PCR. Total RNA was extracted from SN4741 cells and zebrafish treated with the compounds of interest using the RNeasy Mini Kit (Qiagen), 1 μg was treated with RQ1 RNase-free DNase (Promega) and reverse transcribed using Superscript II Reverse Transcriptase (Invitrogen) and random primers (Invitrogen) (RT+ reaction). Parallel reactions without reverse transcriptase enzyme were done as a control (RT− reaction), and Sybergreen real-time quantitative PCR assays were performed. Expression levels were obtained by normalization with the value of the housekeeping gene encoding actin obtained for every sample in parallel assays.
Primary Brain Cultures. Brains from E11.5 mice were manually dissected, plated on poly-D-lysine (150,000 cells/cm2) and grown in serum-free N2 media consisting of 1:1 mixture of F12 and DMEM with 10 ng/mL insulin, 100 μg/mL apo-transferrin, 100 μM putrescine, 20 nM progesterone, 30 nM selenium, 6 mg/mL glucose, and 1 mg/mL BSA. Cells were treated for 3 days in vitro (DIV) with the compounds of interest, fixed with 4% PFA and processed for staining using appropriate antibodies.
For BrdU analysis, cells were treated with BrdU one hour after plating and media was replaced with fresh medium after 16 h. After a further 2 days in culture, cells were treated for 30 min with 2N HCl and then immunocytochemistry was performed to evaluate the number of double BrdU+;Islet1+ cells (a measure of motor neuron neurogenesis). Hoechst staining was performed by permeabilizing cells with a 0.3% Triton-X 100/PBS solution for 5 min followed by incubation with Hoechst 33258 (Sigma) for 10 min.
Antibodies and Detection Procedures. Cells were fixed in 4% PFA, washed in PBS and blocked in 5% normal goat serum/PBS for 1 h at room temperature. Primary antibodies were diluted in PBS (pH 7.4), 0.3% Triton X-100, 1% BSA and incubations were carried out overnight at +4° C. or at room temperature for 2 h. The antibodies used were anti-: BrdU (1:400; Abeam), Islet-1 (1:100; Developmental Studies Hybridoma Bank), cleaved caspase-3 (Asp175) (1:100; Cell Signaling Technology), tyrosine hydroxylase (TH;1:1000; Pel-Freeze) GABA (1:1,000; Sigma), Brn3a (1:250; Millipore), Nkx6.1 (1:200; Novus Biologicals), choline acetyltransferase (ChAT; 1:500; Millipore) and appropriate secondary antibodies (Jackson ImmunoResearch or Alexa). Cells positive for the corresponding marker were counted directly at the microscope at a magnification of 20 x. Cells were counted in every well, in eight consecutive fields (going from one side of the well to the other, passing through the center), in three different wells per experiment and in three different experiments per condition. Random pictures of the wells were taken for every condition to document the result, and representative pictures were subsequently selected to represent the quantitative data. Photos were acquired with a Zeiss Axioplan microscope and a Hamamatsu camera C4742-95 using the Openlab software.
Animals for in-utero injections and tissue preparation. Female wild-type CD-1 mice (25-35 g; Charles River Breeding Laboratories) were housed, bred, and treated according to the guidelines of the European Communities Council (directive 86/609/EEC) and the Society for Neuroscience (www.sfr.org), and all experiments were approved by the local ethical committee. Ethical approval for CD-I mice experimentation was granted by Stockholm Norra Djurforsoksetisks Namnd number N1 54/06, N273/11 and N370/09. For embryo analyses, wild type CD-I mice were mated overnight, and noon of the day the plug was considered E0.5. Embryos were dissected out of the uterine horns in ice-cold PBS, fixed in 4% paraformaldehyde (PFA) for 4 h to overnight, cryoprotected in 15-30% sucrose, frozen in Tissue-Tek Optimum Cutting Temperature (OCT) compound (Sakura Fine-Tek) on dry ice, and stored at −80° C. until use. Serial coronal 14-μπι sections of the brain were obtained on a cryostat.
Immunohistochemical analysis of sections. Ten sets of 14 μπι serial coronal sections were cut on a cryostat. No. 1 and 6 sets were subjected to immunohistochemistry. Sections were pre-incubated for 1 h in blocking solution followed by incubation at 4° C. overnight with following primary antibodies: anti-TH (1:750, Pel-Freeze), anti-Islet-1 (1:100; Developmental Studies Hybridoma Bank), anti-cleaved caspase-3 (Aspl175) (1:100; Cell Signaling Technology). After washing, slides were incubated for 1-2 h at room temperature with the appropriate fluorophore-conjugated (Cy2-, Cy3- and Cy5-, 1:300, Jackson Laboratories; Alexa488-, 555-, and 647-, 1:1000, Invitrogen) secondary antibodies. Confocal pictures were taken with a Zeiss LSM5 Exciter or LSM700 microscope.
In utero intraventricular injections. E11.5 pregnant females were deeply anesthetized using Isofluorane (ISOFLO®, Abbott Labs) and the uterine horns were accessed through an abdominal incision. 1 μL of the different cholestenoic acids studied (5 mM) or vehicle solution (isopropanol, 50% v/v) was injected into the cerebral aqueduct. The uterine horns were replaced into the abdominal cavity, which was then closed with sutures. Embryos were analyzed 48 h later.
Zebrafish Strain and Mainrenance Zebrafish were raised on a 14/10 h day/night cycle and were kept at 28.5° C. Islet-1:GFP transgenic embryos were obtained via natural spawning and staged in hours or days post fertilization (hpf and dpf) according to Kimmel et al). Embryos older than 24 hpf were treated with 0.03% phenylthiourea (PTU) to inhibit pigmentation. MO injections were performed using splice site specific zebrafish 1xr MO. MO injected embryos were immediately dechorionated and transferred to a 96 well plate and exposed to dimethyl sulfoxide (DMSO) treated or ligand treated medium.
Compound Exposure. Islet-1:GFP embryos were collected by natural mating and immediately (at 1 cell stage) dechorionated and transferred to a 96 well plate. Each of the compounds tested were obtained as 10 mM stock and diluted in embryo medium to a final concentration of 10 μM, and 200 μL was added to each well. DMSO or propan-2-ol treated embryo medium was taken as control. Ligand solutions were replaced every 12 h with fresh ligand solution prepared in PTU treated embryo medium. Embryos were collected at 48 hpf, fixed for 4 h at room temperature with 4% paraformaldehyde and then washed and kept in PBST. Immunocytochemistry was performed using an anti-GFP antibody and fluorescence was viewed and photographed using a Zeiss Axioplan compound microscope and a Zeiss Axiocam digital camera.
The metabolic stability of the deuterated compounds can be compared to that of unlabelled compounds by in vitro incubation (i) with the enzyme HSD3B7 to assess the propensity for oxidation at C-3, (ii) with the enzyme HSD11B1, a putative 7-hydroxy epimerase, and (iii) with peroxisome preparation to asses the propensity for side-chain shortening; followed by mass spectrometry analysis to determine the rate of loss of substrate and the rate of formation products. Similar experiments can be performed with primary cultures of mouse and hμ an hepatocytes, and of mouse neurons, glial and Schwann cells.
To asses in vivo metabolism in brain, mice can be injected with both deuterated compounds and also with analogues, 13C labelled compounds. The presence of both substrates and products can be measured in cerebrospinal fluid and brain tissue, and in both the carotid artery and jugular vein, by mass spectrometry providing a measure of the relative rate of metabolism.
As shown in
Statistical analysis was performed by Student's t-test and Mann-Whitney test using Prism4 (Graphpad Software, La Jolla, CA; www.graphpad.com). p<0.05 was considered a statistically significant difference (*), p<0.01 (**). Data represent mean±SE.
As discussed previously, cholesterol metabolites have the capacity to activate Lxrs but the applicants set out to identify what these metabolites are and how they act in plasma and cerebrospinal fluid (CSF) of patients with human diseases associated with motor dysfunction, cerebrotendinous xanthomatosis (CTX) and hereditary spastic paresis (HSP) type 5 (SPG5) and patients with oxysterol 7α-hydoxylase deficiency (07αHD). These diseases result from mutations in the cytochrome P450 (CYP27A1, CYP7B1 and CYP7B1) genes, respectively. The enzymes coded by these genes are responsible for (25R),26-hydroxylation of cholesterol and 7α-hydroxylation of oxysterols, respectively, reactions that generate further oxysterols and ultimately cholestenoic acids (
Cholestenoic acids are abundant in human plasma and human CSF (cerebral spinal fluid) also contains cholestenoic acids. Surprisingly, in CSF the levels of cholestenoic acids were higher than those of oxysterols. The exact identity of 16 oxysterols and downstream metabolites, including cholestenoic acids in human CSF are shown in Table 1. The most abundant of these metabolites were 7α-hydroxy-3-oxocholest-4-en-26-oic acid (7αH,3O-CA), 3β-hydroxycholest-5-en-26-oic acid (3β-HCA) and newly identified in CSF 3β,7α-diHCA and 3β,7β-dihydroxycholest-5-en-26-oic acid (3β,7β-diHCA) (19.48-0.25 ng/mL,
Low levels of 26-HC have been found in human and mouse brain and in human CSF conversely, 7αH,3O-CA, a metabolic product of 26-HC, is exported from brain to blood in human. The CSF to plasma ratios for 7αH,3O-CA and 26-HC has been found to be >1:6 and <1:130, respectively. These ratios are consistent with the hypothesis that while 26-HC is imported into the CNS, 7αH,3O-CA, the most abundant sterol metabolite found in CSF, is synthesized in the CNS.
Reduced Levels of 7α-Hydroxylated Cholestenoic Acids in Plasma/Serum of Human Patients with CTX Cerebrotendinous-Xanthomatosis and SPG5 Hereditary Spastic Paraplegia
Two human diseases that can present with upper motor neuron signs, CTX and SPG5, result from mutations in CYP27A1 and CYP7B1, respectively, two of the genes encoding enzymes required for extrahepatic synthesis of 7αH,3O-CA and its precursor 3β,7α-diHCA (
The applicants also examined patients with SPG5, a disease resulting from mutations in the CYP7B1 gene encoding the oxy sterol 7α-hydroxylase responsible for the extrahepatic 7α-hydroxylation of side-chain oxidised sterols. In agreement with this, a 6-9 fold increase in 26-HC was been described in plasma of SPG5 patients. Here the applicants studied six patients showing pure SPG5 and one adult showing complex SPG5. In all cases elevated levels of the CYP7B1 substrates 25-HC, 26-HC and 3β-HCA (
The applicants were also able to analyse CSF from three patients with SPG5 and two health carriers, heterozygotes, with a single mutation in CYP7B1 (data in Table SI and patient description in Table 3). As observed in plasma, levels of 25-HC, 26-HC and 3β-HCA were elevated in the patient CSF while 3β,7α-diHCA was reduced. This indicates that, for these metabolites, plasma represents a good surrogate for CSF.
The applicants also investigated the plasma oxysterol and cholestenoic acid profile of three infants with mutations in CYP7B1 (Table 2) resulting in oxysterol 7α-hydroxylase deficiency (07AHD) and neonatal liver disease), as well as SPG5 in adults. The first identification of CYP7B1 mutations were found in a child with severe cholestasis, defining an inborn error of bile acid biosynthesis. As expected by the absence of functional CYP7B 1 in these patients, the applicants found very low plasma levels of 3β,7α-diHCA and elevated levels of 3β-HCA, as described above for SPG5 (
In order to gain insights into the mechanism by which alterations in cholesterol metabolism causes neurological disease, the applicants studied whether any of the cholestenoic acids present at high levels in control human CSF and deregulated in CSF or plasma of SPG5 or CTX patients work as Lxr ligands. The applicants thus focused on 3β-HCA, 3β,7α-diHCA its isomer 3β,7β-diHCA and 7αH,3O-CA (
In order to confirm that the cholestenoic acids are indeed agonists to Lxr the applicants tested the Lxrβ activational capacity of either 3β,7α-diHCA, 3β,7β-diHCA or 3β-HCA in combination with 22R-HC. No additive effect was observed, indicating that the acid molecules work via the same mechanism as 22R-HC (
In order to show that specific cholestenoic acids directly interact with Lxr the applicants performed a binding and activation fluorescence resonance energy transfer (FRET) assay in which ligand binding to the ligand binding domain (LBD) of Lxrβ recruits a fluorescent coactivator. The applicants found that 3β,7α-diHCA induced FRET in a dose dependent manner to a higher degree than the other acids (
Having established that cholestenoic acids that are altered in CTX or SPG5 can activate Lxrs in vitro, the applicants next sought to identify their effect in vivo. In particular, the applicants focused on the expression of Islet-1, a transcription factor expressed in all postmitotic motor neurons and required for multiple aspects of motor neuron development, including motor neuron specification, motor column formation, axonal growth and maintenance of spinal motor neuron identity. The applicants used transgenic zebrafish embryos expressing green fluorescent protein (GFP) driven by the Isl1 gene promoter/enhancer sequences (Tg[Isl1/:GFP]) to screen for biologically active compounds in vivo. Previous studies have indicated that Islet-1 protein is required for the formation of zebrafish primary motor neurons and is conserved throughout vertebrate evolution. Treatment of transgenic zebrafish embryos with 3β-HCA and 3β,7β-diHCA, two weak Lxr ligands, as well as CDCA, the most potent Fxr ligand, had a deleterious toxic effect which impaired the survival of the fish and precluded further in vivo analysis. The applicants also found that the Lxr agonists 3β,7α-diHCA and 3 βH, 7O-CA increased Islet-GFP expression (
3β,7α-diHCA Promotes Rodent Motor Neuron Survival In Vitro, while 30,70-diHCA and 3β-HCA are Toxic
In order to determine the functional relevance of our findings in mammalian cells, it was examined first whether any of the cholestenoic acids present in human CSF and implicated in motor neuron disease were also capable of regulating Islet-1 expression in mouse primary brain progenitor cultures. The applicants first performed dose response analysis of the cholestenoic acids of interest, using a wide range of concentrations; while, 3β,7β-diHCA and 3β-HCA caused the loss of Islet1+ cells in the cultures, 3β,7α-diHCA and 3βH, 7O-CA increased the number of Islet1+ oculomotor cells in the cultures (
Furthermore, neither 3β,7α-diHCA nor 3β-HCA altered the total number of Nkx6.1+ cells in the cultures (
In order to unequivocally examine whether the effects of 3β,7α-diHCA and 3βH, 7O-CA are indeed specifically mediated by Lxr receptors in the rodent brain, primary ventral midbrain cultures from Lxrα−/−β−/−mice were performed. Remarkably, the effects of the two cholestenoic acids on Islet1+ neurons were eliminated, confirming that 3β,7α-diHCA and 3βH, 7O-CA regulate the number of Islet-1+ cells in the rodent brain through Lxrs (
Neurogenesis was examined in bromodeoxyuridine (BrdU) pulse-chase experiments, where neuronal progenitors in primary cultures are labeled with a pulse of BrdU at the beginning of the experiment and then are examined for their differentiation into motor neurons, as assessed by the acquisition of Islet-1 expression, a marker for motor neuron fate. Surprisingly, none of the cholestenoic acids studied affected the number of double BrdU+;Islet+ cells, indicating that they do not promote motor neuron neurogenesis. Similarly, none of the cholestenoic acids affected the total number of BrdU+ cells in the cultures, indicating that they do not modulate proliferation. Finally, it was tested whether cholestenoic acids regulate neuronal survival as assessed by active caspase-3 staining to detect the number of cells undergoing apoptosis in the cultures. Interestingly, treatment with 10 μM 3β,7α-diHCA decreased the number of active caspase-3+ cells (
The survival promoting effect of 3β,7α-diHCA was completely blocked by co-incubation with the Lxr inhibitor GGPP, indicating that the survival effect of this acid is mediated by Lxr (
In contrast, 10 μM 3βH, 7O-CA did not affect the number of active caspase-3+ cells, indicating that the increase in Islet1+ cells is not due to increased survival, but rather neuronal differentiation. Cholestenoic acids that reduced the number of Islet1+ cells were also examined for their capacity to induce cell death in motor neuron cultures. Interestingly, the applicants found that lower concentrations, 2 μM, of either 3β,7β-diHCA or 3β-HCA increased the number of active caspase-3+ cells. Moreover, no surviving cells were detected when cultures were treated with 10 μM 3β,7β-diHCA or 3β-HCA. The cell death effects of these two acids (at 2 μM), unlike the survival-promoting effects of 3β,7α-diHCA, were not blocked by GGPP, indicating that the survival-promoting effects of 3β,7α-diHCA, but not the toxic effects of 3β,7β-diHCA or 3β-HCA, are mediated by Lxr. As a final experiment the applicants tested whether 3β,7α-diHCA could reduce the toxic effect of 3β,7β-diHCA or 33-HCA. The applicants found that addition of 10 μM 3β,7α-diHCA reversed the loss of Islet1+ cells in the cultures treated with either 2 μM 3β,7β-diHCA or 3β-HCA (
In order to examine whether there is competition between the effects of the cholestenoic acids studied the applicants performed several studies. In a binding and activation FRET assay, when 3β,7α-diHCA at its most potent concentration, was used together with increasing concentrations of 3β,7β-diHCA or 3β-HCA, the effect of 3β,7α-diHCA was reduced to that of the latter acids, indicating that there was competition between the various cholestenoic acids for binding to the LBD of Lxrβ (
Thus, combined, our results indicate that cholestenoic acids regulate the number of Islet+ motor neurons by controlling the expression of Islet-1 (3βH, 7O-CA), and regulating neuronal survival in a positive (3β,7α-diHCA) or a negative (3β,7β-diHCA and 3β-HCA) manner.
Cyp7bl Knockout Mice Exhibit Elevated Levels of 25-HC and do not Suffer from Motor Neuron or Liver Disease
In light of the human diseases associated with mutations in the CYP7B1 and 27A1 genes the applicants decided to examine the sterol profiles of Cyp7bl and Cyp27αl knockout mice (Cyp7bl−/− and Cyp27αl−/−, respectively). As neither Cyp7bl−/− nor Cyp27αl−/− mice suffer from motor neuron dysfunction. The plasma and brain tissue samples from adult male knockout animals were analysed to search for the presence of the survival-promoting acid. LC-ESI-MS spectra of the Cyp7bl−/− mouse revealed an absence of 3β,7α-diHCA, its 7β-isomer and also 3 βH, 7O-CA both in plasma (n=9) and in whole brain tissue (n=7). An active bile acid biosynthesis pathway was maintained in these animals as indicated by the presence of 7αH,3O-CA in plasma (
The applicants next analysed the oxysterol and cholestenoic acid profile of whole brain tissue 50 of Cyp27αl−/− adult male mice and found an absence of 3β,7α-diHCA, 3βH, 7O-CA and also 25-HC. The levels of 7α-HC and 7α-HCO; 7α, 12a-diHC and 7α, 12a-diHCO; 7α, 25-diHC and 7α, 25-diHCO; and also of a trihydroxycholesterol and a trihydroxycholest-4-en-3-one, probably 7α, 12α, 25-trihydroxycholesterol (cholest-5-ene-3β,7α, 12α, 25-tetrol, 7α, 12α, 25-triHC) and 7α, 12α, 25-trihydroxycholest-4-en-3-one (7α, 12α, 25-triHCO), were elevated in the brain of the knockout mouse (
3β,7α-diHCA Promotes Rodent Motor Neuron Survival In Vivo
In order to demonstrate motor neuron survival in vivo in a mammalian system, 3β,7α-diHCA was injected into the cerebral aqueduct of E11.5 mice in utero and brain sections were analyzed at E13.5 for Islet1-, TH- and ac3-positive cells. The applicants observed an increase in the number of Islet+ oculomotor neurons but not of TH+ neurons (
In addition the applicants occasionally observed double-positive Islet1+;ac3+ cells (
Thus, our results show that specific cholestenoic acids have similar positive or negative effects on Islet1+ motor neurons both in vivo and in vitro.
The studies reported here show that cholestenoic acids are not mere intermediate metabolites of bile acid biosynthesis, but are rather a diverse family of bioactive compounds, capable of regulating nuclear receptor function. As such, cholestenoic acids were found to specifically activate Lxr and elicit an exquisite array of functions ranging from the regulation of Islet-1 expression, to the positive and negative regulation of motor neuron survival both in vitro and in vivo. Moreover, our study identifies cholestenoic acids in human CSF to be deregulated in plasma of patients with monogenetic motor neuron dysfunction, specifically CTX and SPG5 patients. Importantly, an absence of neuroprotective cholestenoic acids was found in CTX, while a combination of decreased neuroprotective and increased toxic cholestenoic acids was detected in SPG5. These results thus identify cholestenoic acids as key regulators of motor neuron function in development and disease.
Cholesterol is present at high levels in the CNS of vertebrates and is metabolized in brain predominantly to 24S-HC which accounts for about two thirds of brain cholesterol metabolism. Low levels of 26-HC have been found in human and mouse brain and in human CSF where it may be imported from the blood. Conversely, 7αH,3O-CA, a metabolic product of 26-HC, is exported from brain to blood in human. Four intermediates in the biosynthesis of 7αH,3O-CA from 26-HC via CYP27A1 and CYP7B1 to be present in CSF i.e. 3β,7α-diHCA, 3β-HCA, 7α,26-diHCO and 7α,26-diHC (
In human CSF and plasma the applicants also identified 3β,7β-diHCA but not its 3-oxo-4-ene metabolite. This result is in agreement with the earlier finding that hydroxysteroid dehydrogenase (HSD) 3B7, which acts on 7α-hydroxylated C27 sterols converting them to their 3-oxo-4-ene analogs, is expressed in brain but does not metabolize 7β-hydroxylated sterols. The absence of such a metabolic sink may thus account for the comparatively high level of 3β,7β-diHCA in human CSF (Table 1). Importantly, here the applicants show that 3β,7β-diHCA and 3βH, 7O-CA, the necessary intermediate in the epimerization reaction from the 3β,7α-isomer, also work as Lxr ligands (
Classical studies by Lehmann et al and Janowski et al defined the general structural requirements of steroidal Lxr ligands to be a 3β-hydroxy-5-ene function in the ring system and a hydroxy, oxo or epoxide function on the C-17 side-chain. The side-chain functions have more recently been extended to include a carboxylic acid group, a functional group which is also present in the synthetic non-steroidal Lxr ligand GW3965. In the current study, the applicants confirm the Lxr-activational capacity of cholestenoic acids with a 3β-hydroxy-5-ene structure in neural cells, and show that despite the introduction of either a 7α- or 7β-hydroxy or a 7-oxo group, Lxr activity is maintained. On the contrary, the 3-oxo-4-ene equivalents of these acids are not Lxr ligands. Thus, 3β-HCA, 3β,7α-diHCA, its 7β-isomer, and the necessary 7-oxo intermediate in the epimerization reaction, are all Lxr ligands. Moreover, none of these acids were found to activate Fxr, Vdr or Nurrl in neural cells, thereby confirming the specificity of their effect on Lxr.
A number of studies in recent years have linked Lxr to neuronal degeneration. These studies have utilized Lxrβ−/− and Lxrα−/−β−/−mice. Indeed, both Lxr isoforms are expressed in brain and the knock-out mice show progressive accumulation of lipids in brain, abnormal blood brain barrier, increased reactive microglia, astrogliosis and degeneration of adult spinal cord motor neurons. Interestingly, a decrease in the number of oculomotor neurons was also detected during development in the Lxrα−/−β−/−mice at E11.5. However, it was not known whether Lxr ligands regulate the development of motor neurons in vivo. Moreover, the identity of endogenous brain Lxr ligands that regulate motor neuron function was unknown. Here the applicants used zebrafish to study the in vivo function of acid Lxr ligands newly identified in human CSF, 3β,7α-diHCA and 3β,7β-diHCA, and the 7-oxo intermediate in their epimerization, on Islet1+ cranial motor neurons. While metabolites that did not activate Lxr, such as 7αH,3O-CA, did not regulate the expression of Islet-1, the applicants found that two Lxr ligands 3β,7α-diHCA and 3βH, 7O-CA enhance the expression of Islet-1 transcript and protein in zebrafish embryos (
The fact that the upper motor neuron phenotype of SPG5 and CTX often presents in the adolescent or adult argues against the Lxr ligands presented here being exclusively essential for motor neuron development. However, the applicants know that deletion of Lxrs results in motor neuron loss (1). Thus, the lack of an early phenotype in human patients with SPG5 or CTX indicates that other endogenous Lxr ligands are present in the brain and contribute to motor neuron development. Indeed the Cyp7bl−/− and Cyp27αl−/− mice, do not suffer from motor neuron disease despite an absence of 3β,7α-diHCA in brain and plasma (Table 4). This finding suggest that while the developmental function of the cholestenoic acids identified here is redundant with that of other Lxr ligands, the accumulative effect of altered levels of cholestenoic acids over extended periods of time, as in SPG5 and CTX, may play a decisive role in motor neuron disease.
To summarize, in this study the applicants identified 3β,7α-diHCA and 3β,7β-diHCA as Lxr ligands present in human CSF. Of these, 3β,7β-diHCA, and the previously identified Lxr ligand, 3β-HCA, were found to cause cell death, and the latter was present at high levels in patients with SPG5. Instead, 3β,7α-diHCA, which was found at low levels in SPG5 and was absent from CTX patients, promoted motor neuron survival, while 3βH, 7O-CA regulated Islet-1 expression levels. Thus, our results uncover several novel functions of cholestenoic acids and identify them as Lxr ligands and as key regulators of motor neuron function in development and disease. Moreover, our study reveals that an orchestra of Lxr ligands regulates the development and survival of motor neurons. The results show that some specific cholestenoic acids selectively work on motor neurons, via Lxr, to regulate the balance between survival and death. These findings provide a mechanism for motor neuron dysfunction, for example in CTX and SPG5 and suggest that efforts aimed at restoring the balance between toxic and pro-survival Lxr ligands, such as administration of 3β,7α-diHCA or, in some cases, 25-HC may thus find a therapeutic application to prevent motor neuron loss and the treatment of motor neuron disease.
The concentration of deuterium at a designated position may be defined by the deuterium enrichment factor. “Deuterium enrichment factor” in the context used herein refers to the ratio between the deuterium isotopic abundance and the natural abundance of deuterium, each relative to hydrogen abundance.
If a substituent in a compound of this invention is denoted as deuterium, such compound has a deuterium enrichment factor for each designated deuterium atom of:
As stated according to a first aspect of the invention the compounds are of general formula (I) but there may be following variations of the compound or a pharmaceutically acceptable salt thereof, where R1-R4 are each independently selected from H and deuterium; and
These compounds are shown as compound (II) to (IX):
For the compound of the invention there may be isotopic variation among the constituent atoms of the molecules. Thus the extent of deuteration at each designated position is not necessarily the same. By way of example, when a compound of the present invention contains multiple deuterium atoms, the deuterium enrichment factor for one designated deuterium atom may be 3000 and the deuterium enrichment factor for another designated deuterium atom may be 5000. Such a compound is considered therefore to have a deuterium enrichment factor of 3000.
The structural formulae shown herein may or may not indicate whether atoms at certain positions are isotopically enriched with respect to deuterium. Thus in a general embodiment, when a structural formula is silent with respect to whether a particular position is isotopically enriched, it is to be understood that the stable isotopes at that particular position are present at, or close to, natural abundance or alternatively “D” indicates that the stable isotopes at that particular position are deuterium enriched.
The structural formulae shown herein may or may not indicate specific enantiomers for chiral centres where such chiral centres exist. Where a chiral centre exists but enantiomeric specificity is not designated it is understood that both or all chiral enantiomers are encompassed by the formula. As such, compounds of the present invention can exist as either individual enantiomers, or mixtures of the two enantiomers. Accordingly, a compound of the present invention may exist as either a racemic mixture or a scalemic mixture, or as individual respective stereoisomers that are substantially free from another possible stereoisomer. The term “substantially free of other stereoisomers” as used herein means less than 25% of other stereoisomers, preferably less than 10% of other stereoisomers, more preferably less than 5% of other stereoisomers and most preferably less than 2% of other stereoisomers are present. Methods of obtaining or synthesizing an individual enantiomer for a given compound are known in the art and may be applied as practicable to final compounds or to starting material or intermediates.
Compounds of the present invention may be formulated to treat or provide a therapy for a range of motor neurone diseases. “Treat” or “therapy” means decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease (e.g., a disease or disorder delineated herein), lessen the severity of the disease or improve the symptoms associated with the disease. Additionally, it includes prophylactic use. The treatment or therapy may be on humans or animals.
The compound will be formulated to contain deuterated compound in an amount such that, depending on the route of administration and prescribed frequency, the required daily or weekly dosage of deuterated compound is provided. The composition will be formulated to allow for daily administration or so it can be incorporated in a treatment regime. The compounds may be used on their own or formulated in combination with other therapeutic compounds for the treatment of motor neurone disease.
“Motor Neurone Disease” in the context used herein is understood to be a disorder selected from Amyotrophic Lateral Sclerosis (ALS), Primary Lateral Sclerosis (PLS), Progressive Lateral Sclerosis (PLS), Progressive Bulbar Palsy (PBP), pseudobulbar palsy, Spinal Muscular Atrophy (SMA), spinobulbar muscular atrophy, Charcot-Marie-Tooth disease and other degenerative disorders of upper and/or lower spinal motor neurones irrespective of whether they are classified as “motor neurone disease” in commonly used disease classification systems such as Medical Subject Headings (MeSH) or the International Statistical Classification of Diseases and Related Health Problems (ICD-10). It is also understood that motor neurone diseases may be known by alternative names and/or is subject to different classification in territories other than the United Kingdom (for example Lou Gehrig's disease in the United States).
When they are used, the compounds are included in a “pharmaceutically acceptable” carrier that means a component that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other mammals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
Such carriers include “pharmaceutically acceptable salts” means any non-toxic salt that, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound of this invention. A “pharmaceutically acceptable counter-ion” is an ionic portion of a salt that is not toxic when released from the salt upon administration to a recipient. Acids commonly employed to form pharmaceutically acceptable salts include inorganic acids such as hydrogen bisulfide, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid, as well as organic acids such as para-toluenesulfonic acid, salicylic acid, tartaric acid, bitartaric acid, ascorbic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, lactic acid, oxalic acid, para-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid and acetic acid, as well as related inorganic and organic acids.
A salt of a compound of this invention is formed between an acid and a basic group of the compound, such as an amino functional group, or a base and an acidic group of the compound, such as a carboxyl functional group. Pharmaceutically acceptable salts thus include but are not limited to sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylene sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, [beta]-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and other salts. Pharmaceutically acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and especially those formed with organic acids such as maleic acid. The pharmaceutically acceptable salt may also be a salt of a compound of the present invention having an acidic functional group, such as a carboxylic acid functional group, and a base. Exemplary bases include, but are not limited to, hydroxide of alkali metals including sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals, such as aluminium and zinc; ammonia, organic amines such as unsubstituted or hydroxyl-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH—(Ci-C6)-alkylamine), such as NJM-dimethyl-N-(2-hydroxyethyl) amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; morpholine; thiomorpholine; piperidine; pyrrolidine; and amino acids such as arginine, lysine, and the like. The invention also provides pharmaceutical compositions comprising an effective amount of a compound of Formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII) or (IX) (e.g., including any of the formulae herein), or a pharmaceutically acceptable salt of said compound; and a pharmaceutically acceptable carrier. The carrier(s) are “acceptable” in the sense of being compatible with the other ingredients of the formulation and, in the case of a pharmaceutically acceptable carrier, not deleterious to the recipient thereof in an amount used in the medicament.
Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminium stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene−block polymers, polyethylene glycol and wool fat. It is also possible to, enhance the bioavailability if the compound using an amorphous form of the compound optionally formulated with a poloxamer, such as LUTPvOL™ and PLURONIC™ (BASF Corporation), or block copolymers of ethylene oxide and propylene oxide.
The compound of the present invention may exist in a number of different crystalline polymorphs and the polymorph(s) with optimal pharmaceutical properties can be selected so all crystalline polymorphs of the compound of the present invention are incorporated herein.
The pharmaceutical compositions of the invention include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) intrathecal or other intraspinal administration. In certain embodiments, the compound of the formulae herein is administered transdermally (e.g., using a transdermal patch or iontophoretic techniques). Other formulations may conveniently be presented in unit dosage form, e.g., tablets, sustained release capsules, and in liposomes. The molecule to be administered is brought into association with ingredients such as the carrier which may be a liquid carrier, liposomes or finely divided solid carriers, or both, and then, if necessary, shaping the product. Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, sachets, or tablets each containing a predetermined amount of the active ingredient; a powder or granules; a solution or a suspension in an aqueous liquid or a nonaqueous liquid; an oil-in-water liquid emulsion; a water-in-oil liquid emulsion; packed in liposomes; or as a bolus, etc. Soft gelatin capsules can be useful for containing such suspensions, which may beneficially increase the rate of compound absorption. In the case of tablets for oral use, carriers that are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or colouring agents may be added, for example if the composition is included in a lozenges a flavouring which is usually sucrose and acacia or tragacanth is used and for pastilles the active ingredient is included with an inert basis such as gelatin and glycerin, or sucrose and acacia.
Compositions suitable for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions which may include suspending agents and thickening agents.
The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets. Such injection solutions may be in the form, for example, of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenter ally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant.
The pharmaceutical compositions of this invention may be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, 50 beeswax and polyethylene glycols.
The pharmaceutical compositions of this invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
Topical administration of the pharmaceutical compositions of this invention is especially useful when the desired treatment involves areas or organs readily accessible by topical application. For topical application topically to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax, and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. The pharmaceutical compositions of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically-transdermal patches and iontophoretic administration are also included in this invention.
Application of the subject therapeutics may be local, so as to be administered at the site of interest. Various techniques can be used for providing the subject compositions at the site of interest, such as injection, use of catheters, trocars, projectiles, pluronic gel, stents, sustained drug release polymers or other device which provides for internal access. Accordingly, the compounds of this invention may be incorporated into compositions for coating an implantable medical device, such as prostheses, artificial valves, vascular grafts, stents, or catheters. Suitable coatings are typically biocompatible polymeric materials such as a hydrogel polymer, polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinyl acetate, and mixtures thereof. The coatings may optionally be further covered by a suitable topcoat of fluorosilicone, polysaccharides, polyethylene glycol, phospholipids or combinations thereof to impart controlled release characteristics in the composition.
The compound of the present invention is administered in an effective amount which means that when administered in a proper dosing regimen, the amount is sufficient to treat the target disorder and this amount can vary according to the height and weight of the subject. Effective doses of the compounds of the present invention will also vary, as recognized by those skilled in the art, depending on the disease(s) treated, the severity of the disease, the route of administration, the sex, age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician.
It is also possible to co-administer the compound of the invention with one or more second therapeutic agents or treatments. The term “co-administered” as used herein means that the second therapeutic agent may be administered together with a compound of this invention as part of a single dosage form (such as a composition of this invention comprising a compound of the invention and an second therapeutic agent as described above) or as separate, multiple dosage forms. Alternatively, the additional agent may be administered prior to, consecutively with, or following the administration of a compound of this invention. It is possible that where a second therapeutic agent is administered to a subject, the effective amount of the compound of the invention or the second therapeutic agent that is required is less than its effective amount than when the agent or compound are administered on their own so by being able to give less drugs to the individual undesired side effects associated with high doses of either agent may be minimized. Other potential advantages include improved dosing regimens and/or reduced drug cost. The present invention overcomes problems of the prior art as there is a reduction in metabolism of the compound compared with that of its non-deuterated parent compound. This has the surprising effect that the specific deuterated compounds described are more neuroprotective compared with their non-deuterated parent compounds and also give rise to lower levels of neurotoxic metabolites so making the compound particularly attractive for administering to patients that have a long term illness.
It is to be understood that the above embodiments have been provided only by way of exemplification of this invention, such as those detailed below, and that further modifications and improvements thereto, as would be apparent to persons skilled in the relevant art, are deemed to fall within the broad scope and ambit of the present invention described. Furthermore where individual embodiments are discussed, the invention is intended to cover combinations of those embodiments as well. The systems shown and described are not limited to the precise details and conditions disclosed. Method steps provided may not be limited to the order in which they are listed but may be ordered any way as to carry out the inventive process without departing from the scope of the invention. Furthermore, other substitutions, modifications, changes and omissions may be made in the design, operating conditions and arrangements of the exemplary embodiments without departing from the scope of the invention as expressed in the appended claims.
7α-Hydroxy-3-oxochol-
7α-Hydroxy-3-oxochol-
(7αH,3O-Δ
4
-BA)
7α-Hydroxy-26-nor-
7α-Hydroxy-26-nor
Cholest-5-ene-3β,24S-
Cholest-5-ene-3β,25-
Cholest-5-ene-3β,26-
Cholest-5-ene-3β,7β-
(7β-
7β-HC)
3β-Hydroxycholest-5-en-
3β-Hydroxycholest-5-
7α-Hydroxycholest-4-en-
Cholest-5-ene-3β,7α-
(7α-
7α-HC)
Cholest-4-ene-3β,6-diol
or Cholest-5-ene-3β,6-
3β-Hydroxycholesta-
3β-Hydroxycholest-5-
en-26-oic acid (3β-
7α,25-Dihydroxycholest-
7α,25-
3-one (7α,25-diHCO)
7α,25-Dihydroxycholest-
3β,7α,25-triol
(7α,25-
7α,25-diHC)
7α,26-Dihydroxycholest-
7α,26-
3-one (7α,26-diHCO)
7α,26-Dihydroxycholest-
3β,7α,26-triol
(7α,26-
7α,26-diHC)
24-Hydroxy-3-
24-Hydroxy-3-
25-Hydroxy-3-
25-Hydroxy-3-
7β-Hydroxy-3-
3β,7β-
26-oic acid (3β,7β-
22,25-Dihydroxycholest-
3β,22,25-
7α-Hydroxy-3-
7α-Hydroxy-3-
acid (7αH,3O-CA)
7α-Hydroxy-3-
3β,7α-
26-oic acid (3β,7α-
7α,24(or26),25-
7α,24(or26),25-
7α-Hydroxy-3,24-
7α-Hydroxy-3,24-
7α,24-Dihydroxy-3-
7α,24-Dihydroxy-3-
7α,25-Dihydroxy-3-
7α,25-Dihydroxy-3-
indicates data missing or illegible when filed
7α-Hydroxy-3-oxochol-
7α-Hydroxy-3-oxochol-4-en-
24-oic acid (7αH,3O-Δ
4
-BA)
7α-Hydroxy-3-oxochol-
3β,7α-Dihydroxychol-5-en-24-
oic acid (3β,7α-diH-Δ
5
-BA)
3β-Hydroxycholest-5-en-24-
7α-Hydroxy-26-nor-
7α-Hydroxy-26-nor-cholest-4-
Cholest-3-ene-3β,24S-diol
Cholest-5-ene-3β,25-diol
Cholest-5-ene-3β,26-diol
7β-Hydroxycholest-4-
7β-Hydroxycholest-4-en-3-one
(7β-HCO)
7β-Hydroxycholest-4-
Cholest-5-ene-3β,7β-diol
(7β-Hydroxycholesterol, 7β-
HC)
3β-Hydroxycholest-5-
3β-Hydroxycholest-5-en-7-one
7α-Hydroxycholest-4-
7α-Hydroxycholest-4-en-3-one
(7α-HCO)
7α-Hydroxycholest-4-
Cholest-5-ene-3β,7α-diol
(7α-Hydroxycholesterol, 7α-
HC)
Cholest-4-ene-3β,6-diol or
Cholest-5-ene-3β,6-diol
3β-Hydroxycholesta-5,7-dien-
3β-Hydroxycholest-5-en-26-
oic acid (3β-HCA)
7α,25-Dihydroxycholest-
7α,25-Dihydroxycholest-4-en-
3-one (7α,25-diHCO)
7α,26-Dihydroxycholest-
7α,26-Dihydroxycholest-4-en-
3-one (7α,26-diHCO)
7α,26-Dihydroxycholest-
Cholest-5-ene-3β,7α,26-triol
(7α,26-Dihydroxycholesterol,
7α,26-diHC)
7α,12α-
7α,12α-Dihydroxycholest-4-
en-3-one (7α,12α-diHCO)
7α,12α-
Cholest-5-ene-3β,7α,12α-triol
(7α,12α-Dihydroxycholesterol,
7α,12α-diHC)
24-Hydroxy-3
24-Hydroxy-3-oxocholesta-
7β-Hydroxy-3-
3β,7β-Dihydroxycholest-5-en-
26-oic acid (3β,7β-diHCA)
22,25-Dihydroxycholest-
3β,22,25-Trihydroxycholest-5-
7α-Hydroxy-3-
7α-Hydroxy-3-oxocholest-4-
en-26-oic acid (7αH,3O-CA)
7α-Hydroxy-3-
3β,7α-Dihydroxycholest-5-en-
26-oic acid (3β,7α-diHCA)
7α,24(or26),25-
7α,24(or26),25-
7α,24-Dihydroxy-3-
7α,24-Dihydroxy-3-
indicates data missing or illegible when filed
Genet 2012;
Pediatr
Gastroenterol
Nutr 2008;
Inherit Metab
Dis 2010;
| Number | Date | Country | Kind |
|---|---|---|---|
| 1303589.4 | Feb 2013 | GB | national |
| 1316050.2 | Sep 2013 | GB | national |
This application is a continuation of U.S. application Ser. No. 16/951,963 filed Nov. 18, 2020, which is a continuation-in-part of U.S. application Ser. No. 15/848,664, filed Dec. 20, 2017, which is a continuation of U.S. application Ser. No. 14/917,877, filed Mar. 9, 2016, which is a U.S. National Stage filing of International Application No. PCT/GB2014/000356, filed Sep. 10, 2014 which claims priority to Great Britain Patent Application No. 1316050.2, filed Sep. 10, 2013; U.S. application Ser. No. 16/951,963, filed Nov. 18, 2020, is also a continuation-in-part of U.S. application Ser. No. 16/297,271, filed Mar. 8, 2019, which is a divisional of U.S. application Ser. No. 14/770,313_filed Aug. 25, 2015, which is a U.S. National Stage filing of international Application No. PCT/GB2014/050572, filed Feb. 26, 2014, which claims priority to Great Britain Patent Application No, 1303589.4, filed Feb. 27, 2013, the contents of each of which are hereby incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14770313 | Aug 2015 | US |
| Child | 16297271 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16951963 | Nov 2020 | US |
| Child | 18157777 | US | |
| Parent | 14917877 | Mar 2016 | US |
| Child | 15848664 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15848664 | Dec 2017 | US |
| Child | 16951963 | US | |
| Parent | 16297271 | Mar 2019 | US |
| Child | 16951963 | US |
