Abstract
FXR agonists have demonstrated very promising clinical results in the treatment of liver disorders such as primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), and nonalcoholic steatohepatitis (NASH). NASH, in particular, is one of the last uncharted white territories in the pharma landscape, and there is a huge medical need and a large potential pharmaceutical market for a NASH pharmacotherapy. Clinical efficacy superior to most other treatment options was shown by FXR agonists such as obeticholic acid (OCA) as they improved various metabolic features including liver steatosis as well as liver inflammation and fibrosis. But OCA’s clinical success comes with some major liabilities such as pruritus, high-density lipoprotein cholesterol (HDLc) lowering, low-density lipoprotein cholesterol (LDLc) increase, and a potential for drug-induced liver toxicity. Some of these effects can be attributed to on-target effects exerted by FXR, but with others it is not clear whether it is FXR- or OCA-related. Therefore a quest for novel, proprietary FXR agonists is ongoing with the aim to increase FXR potency and selectivity over other proteins and to overcome at least some of the OCA-associated clinical side effects through an improved pharmacology. In this chapter we will discuss the historical and ongoing efforts in the identification and development of nonsteroidal, which largely means non-bile acid-type, FXR agonists for clinical use.
Keywords
1 Overview and Brief History of FXR Drug Discovery
Soon after the identification of bile acids to be likely the prevailing endogenous ligands of FXR (Makishima et al. 1999; Parks et al. 1999), the nuclear receptor drug discovery group around Timothy Willson at GlaxoSmithKline research site at Research Triangle Park, North Carolina, initiated a screen against a synthetic compound library derived from combinatorial chemistry (Maloney et al. 2000). This screen led to the identification of GW4064 (Fig. 3), the first synthetic FXR agonist with decent potency (30 nM in a cell-based assay) and selectivity against other nuclear receptors. In the first publication by this group, GW4064 was tested in Fisher rats to yield significant lowering of serum cholesterol and triglycerides in this animal model. Several publications that were released in the early 2000s and that mainly employed 6-ethyl-CDCA (now known as INT-747 or OCA) as a tool FXR agonist demonstrated strong hepatoprotective effects of FXR activation in various animal models of liver cholestasis and fibrosis (Fiorucci et al. 2004, 2005b; Pellicciari et al. 2002). This was the basis for the early Phase II trials of INT-747/OCA in PBC which is the most severe autoimmune-type liver disorder that presents with such sequelae. In 2005 and 2006, three independent publications were released where the respective authors showed that activation of FXR leads to beneficial insulin-sensitizing, glucose-, lipid-, and cholesterol-lowering effects in mice (Cariou et al. 2006; Stayrook et al. 2005; Zhang et al. 2006), thus implicitly suggesting a FXR agonist-based treatment for metabolic diseases such as type 2 diabetes (T2D). This spurred the efforts in the biopharmaceutical industry to find novel, improved synthetic FXR agonists with appropriate drug-like properties, and in the following 5 years, companies such as GlaxoSmithKline, Eli Lilly, F. Hoffmann-La Roche, Wyeth (now Pfizer), and Phenex Pharmaceuticals patented several novel derivatives of GW4064 (the so-called “hammerhead” class; see Fig. 2 and Gege et al. 2014) but also some novel chemotypes such as WAY-450. The reason why many major pharmaceutical companies have intensely worked on the medicinal chemistry of synthetic FXR agonists but failed to test them in human patients was likely that the pure antidiabetic effects of FXR agonists were limited in their efficacy and the lipid-lowering effects typically came with the liability that it was accompanied by HDLc lowering in most preclinical species tested including mice and monkeys (Evans et al. 2009; Hambruch et al. 2012; Hartman et al. 2009). Thus, a FXR agonist, just based on the preclinical findings, would not represent the ideal prototype of a novel antidiabetic or lipid-lowering drug.
On Jan 8, 2014, an absolute turning point in the drug discovery history of FXR agonists was reached: Intercept Pharmaceuticals released topline data from their FLINT trial, a Phase IIb study investigating OCA in a liver indication previously only known to hepatologists named nonalcoholic steatohepatitis or in brief NASH (Neuschwander-Tetri et al. 2015; Press Release 2014). OCA clearly improved the NASH liver histopathology in all relevant categories even in an interim analysis when roughly only a half of the 280 planned for patients had been analyzed. The stock price of Intercept skyrocketed on that day from 72 to 275 US$ which indicated the enormous market potential that was projected for NASH. We will discuss the application of FXR agonists in NASH later, but this single event boosted the commercial as well as academic drug discovery efforts enormously.
Companies with a declared focus on liver diseases such as Gilead Sciences, Novartis, and Allergan increased their efforts in finding appropriate FXR agonist drug candidates. These efforts gave rise to LJN452 aka tropifexor which is a very potent synthetic GW4064 derivative stemming from Novartis internal drug discovery (Tully et al. 2017) and to GS-9674 aka cilofexor (see Fig. 1 for structure of both compounds) which was originally made and patented by Phenex Pharmaceuticals but then sold to Gilead Sciences in a 470 M US$ transaction. These two compounds are clinically the two most advanced synthetic FXR agonists, followed by a second FXR agonist from Novartis, LMB763 aka nidufexor, which has a completely different structural motif compared to the isoxazole-type tropifexor and cilofexor. Allergan has licensed a compound from Akarna which is likely a derivative of the WAY-450 (Fig. 1) but has not officially initiated clinical development of this drug, probably because Allergan has partnered with Novartis, thereby gaining access to their two advanced FXR agonists.
Although most companies that have FXR agonists in clinical development run trials in PBC and PSC, NASH is clearly the most appealing indication given its enormous market potential (see Sect. 5). Twenty years after its discovery as a bile acid receptor and an important regulator of liver protection and intermediary metabolism, FXR has been torn out of the academic atmosphere into the limelight of pharmaceutical companies and investors’ interests. Still, there are several open questions, in particular with regard to the known and well-documented side effects of FXR agonist-based therapies, such as:
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Are the signs of pruritus that are now documented not only for OCA but also for synthetic nonsteroidal FXR agonists really a direct outcome of FXR activation? Is it common to all FXR agonists, or are there differences depending on the chemical nature (steroidal versus nonsteroidal)?
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Given that for FXR activation beneficial effects on steatosis, inflammation, fibrotization, and liver endothelial recovery are published: Will a pure FXR agonist-based therapy suffice for NASH, in particular?
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Are there any means to dial in certain desired effects into and undesired effects out of FXR agonists? If so, what could be the rationale behind such efforts?
2 Structural Diversity of Nonsteroidal FXR Agonists
Figure 1 lists all published FXR agonists that have reached Phase I human clinical testing at least. Two of them, WAY-450 and Px-102/104, have been abandoned for undisclosed reasons, but the remaining eight ones are in active clinical trials. Among these substances two (OCA and EDP-305) have a steroid scaffold, whereas the other ones are fully synthetic nonsteroidal structures. Figure 2 represents a kind of “evolutionary tree” of nonsteroidal FXR structures. What can be nicely observed is that the release of positive Phase IIb “FLINT” trial data from OCA in NASH patients in January 2014 has triggered a burst of synthetic and – with a due delay of 2–3 years – also of patenting activities of novel synthetic FXR agonists.
The richest class of nonsteroidal FXR agonists is the so-called “hammerhead” class of trisubstituted isoxazole core compounds pioneered by the aforementioned GW4064. GW4064 could serve only as a tool compound since its bioavailability and its in vivo half-life were very limited. Beyond its pharmacokinetic (PK) limitations, the chemical structure of GW4064 harbors a stilbene moiety that could appear as a photolabile and potential metabolite soft spot. Such stilbene moieties are known to potentially demonstrate potent estrogenic effects, and indeed it was found that GW4064 changes muscle metabolism by acting on estrogen-related receptor alpha (ERRα) independent from its activity on FXR (Dwivedi et al. 2011). Moreover, GW4064 was also found to inhibit breast and Leydig tumor cell growth by inhibiting aromatase expression which is another indication of estrogen-like activity of GW4064 (Swales et al. 2006). Various attempts mainly by GlaxoSmithKline, Lilly, and Phenex have been undertaken to replace the photolabile and potentially pro-estrogenic stilbene moiety of GW4064 (reviewed in Gege et al. 2014) by at least equipotent linkers to yield more stable and drug-like compounds. The first to reach Phase I testing was Px-102 from Phenex where the double bond linker was replaced by a cyclopropyl moiety. In this constellation, potentially four stereoisomers, the cis- and trans-substituted cyclopropyls and their enantiomers, are possible, but the cis isomer can be ruled out for the energetically unfavored sterical conformation that this isomer would induce. Phenex tested the racemic version, Px-102 (also published as PX20606 in scientific literature), in two human Phase I studies in healthy volunteers first during 2011 and 2012 (NCT01998672, NCT01998659) and switched to the eutomer, Px-104, for a small exploratory Phase II study (NCT01999101) after which further development was abandoned for undisclosed reasons. In a subsequent publication, Kinzel et al. laid out their intention to replace the hydrophobic and stereocenter-containing cyclopropyl with four-membered ring systems and to introduce more polarity into the hydrophobic aromatic ring systems (Kinzel et al. 2016). This was achieved by replacing the terminal COOH-bearing aryl with a heteroaryl such as pyridine by using the four-membered N-containing azetidinyl as the middle linker element and by further adding a hydroxy group to this cyclic system which is said to engage into a polar interaction and also to increase hydrophilicity. These improvements are best exemplified in the clinical development candidate GS-9674 which was acquired by Gilead Sciences and is now officially termed “cilofexor” (Fig. 3).
From this “early” period of FXR synthetic activity from 1999 till 2014, only cilofexor and the Lilly compound LY2562175 (Genin et al. 2015) survived as clinical candidates. LY2562175 was tested in a European Phase I study and subsequently licensed to Terns Pharmaceuticals and renamed into TERN-101. The company plans to test it in a Phase II study in NASH patients in China. The novelty of this hammerhead isoxazole compound is that the aromatic ring attached to the oxymethylene linker of the isoxazole is replaced by a saturated piperidine ring and the carboxylic acid-bearing terminal part is embodied as a bicyclic ring (Fig. 4).
Building on this core motif of the Lilly compound, researchers at Novartis have modified the middle saturated piperidine by adding an ethylene bridge to form an 8-azabicyclo[3.2.1]octane system and by replacing the indole with a reverse benzothiazole. This yielded a single-digit nanomolar potent and fully efficacious FXR agonist with high in vivo efficacy called LJN452, now tropifexor, and is one of two Novartis compounds in Phase II testing in NASH patients and other indications. Several, mainly Asian biotech companies filed me-too patent applications, which exploit presumed free chemical space (Fig. 4). For HNC143 and HNC180, the X-ray structure bound to FXR was published (Wang et al. 2018). Novartis’ nidufexor is a completely novel chemotype, and only sparse data from a Phase I study with this drug candidate has been released which shows that it has medium potency but also displays HDLc-lowering effects (Huttner et al. 2017).
The replacement of the isoxazole moiety was already extensively investigated (reviewed in Gege et al. 2014) – 1,2,3-triazole or pyrazole first described by Lilly remained promising replacements for the isoxazole. Isothiazole was previously not covered, and two Asian companies recently claimed these structures (Fig. 5): KBP Bio Sciences focus on a me-too of an old Phenex application from 2009, while Jiangsu Hansoh Pharmaceuticals focus on a Phenex application covering cilofexor. Additional patent space is intended with isoxazoles by omitting the hydroxy element in the azetidine linker.
Enanta has taken a more universal approach to generate own intellectual property (Fig. 6): They use carboxylic acid bioisosteres to replace the terminal carboxylic acids of known hammerhead-type structures, thereby yielding compounds that are about equipotent to their carboxylic acid counterparts. The acid is involved in hydrogen bond interactions as well as in ionic interactions with arginines at this part of the ligand-binding domain (LBD), so it is possible to replace a normal carboxylic acid with a bioisostere. Enanta uses alkyl- or aryl-substituted sulfonyl-acyl amides with an acidic N-H. The aryl/alkyl substituent will presumably protrude out of the ligand-binding pocket into the water environment. This might explain why many small alkyl/aryl substituents are allowed for. It has to be kept in mind that this type of acid isostere unlike a real carboxylic acid cannot be taurine- or glycine-conjugated in vivo, an important feature that we need to consider when discussing their PK behavior (see below). The newer Enanta patent applications, however, take a different approach by replacing the O-methylene effectively with a N-methylene preferably on Novartis’ 8-azabicyclo[3.2.1]octane scaffold (WO2018/067704). The other new application introduces a urea system between the middle azabicyclo system and the terminal aryl ring. Here, even the terminal carboxylic acid or acid isostere is omitted (WO2018/081285) still furnishing FXR agonists within the best potency range (<10 nM). Ardelyx follows Enanta and Novartis, then, using their azabicyclo ring replacement in combination with the Novartis or with Enanta headgroups but with an ester linker instead of an O-methylene linker (WO2018/039384).
Another family of structures and patents with decent FXR agonist activity was built around the WAY-450, originally discovered at X-Ceptor Therapeutics (then called XL-335) and then acquired by Exelixis and finally by Wyeth. For WAY-450 several animal studies showed its significant potential in various applications (Evans et al. 2009; Flatt et al. 2009; Hartman et al. 2009; Zhang et al. 2009). WAY-450 suffered from insufficient aqueous solubility since it does not have a free ionizable group and attempts were undertaken to improve this shortcoming by introducing more polar elements and a terminal morpholine-based solubility handle (Lundquist et al. 2010). Akarna Therapeutics was the first “follower” to take WAY-450 as a template to come up with own improved but still related structures (Fig. 7), and Alios BioPharma is another company fishing in the same water as Akarna with a series of WAY-450 replacements. The Salk Institute has also filed a patent application on deuterated WAY-450 structures. However how these deuterated compounds differ from their normal hydrogen-decorated counterparts is not exemplified in the patent application nor published in a scientific journal.
Fexaramine (Fig. 8) is one of the earliest synthetic nonsteroidal FXR agonists that was discovered in the groups of Kyriacos C. Nicolaou and Ron Evans (Downes et al. 2003). Although it shows double-digit nM potency at FXR in in vitro assays, it has some features that prevent it from being drug-like such as an overall hydrophobicity and thus insolubility, two aniline substructures with a potential risk of mutagenicity (Ames et al. 1975), and the terminal aryl-acrylic acid ester which can act as a potential Michael acceptor (Amslinger 2010). However, fexaramine has become the prototype of an intestinally restricted FXR agonists (Fang et al. 2015) and has demonstrated to elicit potent beneficial metabolic effects (see discussion at the end of this section) while avoiding the side effects that come with liver FXR activation. Metacrine was set up as a commercial company to explore the potential of fexaramine further, and a key step in improving fexaramine was the replacement of the acrylate ester by various other moieties including aryl-aryl systems, aromatic acid amides, and oxy- or amino-methylene linker elements (with WO2017/049173 considered the most important one, since prosecuted in many countries). It is likely that Metacrine’s MET409, their clinical candidate which is currently in Phase I clinical testing, is based on a structure covered by this patent application although Metacrine has disclosed in 2018 five further international patent applications with further improvements on other areas of the molecule (e.g., saturate the inner aryl of the biphenyl moiety or substitute the cyclohexyl moiety).
Further independent FXR structures are summarized in Fig. 8. Noteworthy is the identification of MFA-1 as a potent FXR agonist (Soisson et al. 2008). Although MFA-1 has a steroidal structure, it binds to FXR very differently from bile acids in that it is appr. 180° rotated as was demonstrated by X-ray analysis of a FXR-MFA-1 cocrystal structure. As early as 2003, another set of FXR agonists was disclosed in a patent application by a team from Lion Bioscience which later appeared as founders of Phenex Pharmaceuticals (WO2003/016280; WO2003/015777; WO2003/016288). These patent applications cover structures centered around screening hit TR0800012996 that was described with a potency of 0.5 μM in a cellular reporter assay. This compound contains a central, trisubstituted 2-aminopyridine moiety, and compounds with the same core structure are claimed in WO2003/016280. Replacement of the central 2-aminopyridine with 2-aminopyrimidines or with 2-aminothiazoles yielded compounds of similar potency, but none of them was clinically developed. In 2011, a team from Roche published series of substituted benzimidazole FXR agonists (Richter et al. 2011a, b). They were later shown to elicit certain lipid-lowering effects but appeared to be partial FXR agonists with limited in vivo potency compared to isoxazole-hammerhead-type FXR agonists (Gardès et al. 2013).
Worth mentioning is also the Poxel/Enyo compound EYP001 since this molecule is in Phase II testing for hepatitis B infection (HBV) as well as for NASH treatment. Unfortunately, there is no peer-reviewed publication on this compound, and the patent applications are not very informative with regard to its pharmacological properties.
In summary, the drug discovery industry has undertaken serious efforts to identify, optimize, and finally develop nonsteroidal FXR agonists all the way from initial hit identification up to late-stage clinical trials. The one series that is really outstanding are the hammerhead-type isoxazole core structure compounds, pioneered and first exemplified by GW4064. This chemical motif has the advantage of providing decent potency along with FXR selectivity due to its unique binding mode (see detailed discussion in Gege et al. 2014). The fact that this series has given rise to at least four different clinical candidates (Px-102, cilofexor, tropifexor, TERN-101) and that several mainly Asian followers try to copy this motif in own patent applications shows that this type of FXR agonist seems to have unique and superior properties. However, it needs further detailed investigation to uncover what exactly makes this particular structural motif so favorable.
Nevertheless, there are several other compounds of independent structures in clinical development, and it remains to be shown which ones will ultimately arrive in mankind’s pharmacopeia.
3 Pharmacology of Nonsteroidal FXR Agonists
In this section we provide an overview on assay formats including considerations on easiness of setup, information content and limitation, as well as meaningfulness and positioning in the drug discovery cascade.
A screen or a medicinal chemistry effort starts with the desired properties of the target molecule and with the assay setup. There are various standard formats for nuclear receptor assays, and since there are many reference agonists for FXR commercially available, there are no special efforts or requirements necessary to launch a screening campaign for FXR agonists. The FXR agonists currently in clinical development all have potencies of ≤100 nM in typical biochemical assays – a potency in the low double-digit or even single-digit nanomolar range is probably desirable. There are various modalities for FXR agonists that need to be considered (partial or selective agonism, transporter dependency, tissue selectivity, etc.) but this will be discussed in subsequent sections.
In general, nuclear receptors such as FXR bind to their cognate response genes promoters as heterodimers with retinoid X receptor (RXR), the universal permissive heterodimer partner for type 2 nuclear receptors such as FXR, liver X receptors (LXRs), peroxisome proliferator-activated receptors (PPARs), vitamin D receptor, or pregnane X receptor (PXR). All nuclear receptors undergo a conformational rearrangement upon ligand binding in that the C-terminal Helix 12 which is located at the outside surface of the ligand-binding domain is “locked” into the active conformation through appropriate interactions with the agonist (Brzozowski et al. 1997; Gronemeyer et al. 2004). The active conformation is the conformation that allows for so-called coactivator proteins to bind to the RXR-FXR heterodimer and adapt it to other chromatin remodeling factors that are necessary to open the chromatin around the transcriptional start site for the initiation of transcription. This is a very complex event that involves up to 50 different proteins in a certain temporal and spatial order which is beyond the scope of this book chapter to be described in all details but reviews summarize these events well (Bulynko and O’Malley 2011; Burris et al. 2013; Kremoser et al. 2007).
A typical assay cascade starts with a biochemical, i.e., cell-free assay, where a recombinant full-length version or more typical the LBD in this case of FXR provides the backbone. From our own experience, the FXR-LBD can be expressed in normal E. coli expression systems without any special needs for posttranslational modifications or proper folding. The conformational switch, i.e., the changes adopted by Helix 11 and 12 upon agonist ligand binding, is exploited for typical biochemical assay formats. The most common one for nuclear receptors such as FXR is the homogeneous time-resolved fluorescence energy transfer (HTR-FRET) assay (Glickman et al. 2002). In the FRET format, a recombinantly expressed FXR-LBD is combined with a peptide of approx. 20 amino acids that contains an LXXLL sequence (where X can be any amino acid) taken from one of the known canonical nuclear receptor coactivators (e.g., SRC-1, TIF-2, NCoA3). The LXXLL sequence is able to interact with the agonist conformation where Helix 11 and 12 are coordinated by the agonist ligand. In order to elicit a FRET signal, an appropriate fluorescence donor and acceptor pair is needed. Typically, a europium cryptate, a rare earth metal aromatic system chelate, is used as a donor and a red light-emitting acceptor such as XL665, a phycobilliprotein, or allophycocyanin (APC) as an acceptor. N-terminally fused to the FXR-LBD is a generic tag, typically a Glutathione S-transferase (GST) domain, which is recognized by an anti-GST antibody conjugated to the Eu-cryptate. The coactivator peptide with the LXXLL-motif is typically biotinylated which allows for the use of a generic streptavidin-APC complex to bind to it and thereby provides a fluorescent tag. Once an agonist ligand induces the appropriate conformation, the LXXLL-coactivator peptide binds to the activated FXR-LBD bringing the peptide-biotin-streptavidin-APC complex into close proximity with the GST-anti-GST Eu-cryptate, thus generating a rather long-lived (i.e., a few milliseconds) FRET signal. This can be easily read out by appropriate fluorescent readers. The respective reagents are all commercially available. This FRET assay format is probably best-suited for the cost-effective and robust screening of compound libraries for novel FXR agonists.
Alternative biochemical formats are AlphaScreen® or scintillation-proximity assays (SPA). Whereas the AlphaScreen® setup is basically similar to the FRET setup with just using the generation of a short-lived singlet oxygen to induce light emission in a receptive bead in close proximity, the SPA is a classical ligand-binding assay. SPA just relies on the displacement of a radioactively (i.e., 3H) labelled reference ligand. The radioligand induces scintillation signals when the FXR-LBD is bound to an appropriate scintillator bead or plate. Upon its displacement by a new incoming and unlabeled FXR agonist, the scintillation signal is weakened. Using appropriate dose-response curves and Scatchard analysis, one can derive a binding constant Ks for the new ligand. However, the key disadvantage of the SPA format is that it does not discriminate between agonists, partial agonists, and antagonists which can also turn into an advantage, of course, if such type of ligands is sought.
The biochemical assays are normally used as a primary screening tool, while cellular reporter assays are used as secondary assays for hit verification and also to determine generic properties of identified hits such as cell membrane permeation or cytotoxicity. These cellular reporter assays employ the transcription factor properties of nuclear receptors. FXR recognizes a certain DNA hexamer motif, a so-called IR-1 element, which is an inverted repetition of the ACCTCA with one nucleotide as a spacer in between. Therefore, it is possible to take a part of an active promoter region from a known FXR direct target gene, e.g., IBABP (ileal bile acid-binding protein) or SHP (small heterodimer partner), and put it in front of a reporter gene that will then be transcribed in an FXR-dependent manner. In an ideal setup for such a direct reporter gene (DR) assay, one has a plasmid encoding full-length FXR and RXR and another one with the appropriate FXRE (IR-1 containing) promoter-reporter construct (typically Firefly luciferase). Bringing both into a cell line (e.g., HEK293 cells) by transient transfection yields a fully functional cellular FXR assay. An even more simplified yet fully functional reporter assay, the so-called Gal4-hybrid or mammalian-one-hybrid format, uses only the FXR-LBD fused to the DNA-binding part of the Gal4 transcription factor from yeast. The resulting hybrid protein is inactive, but upon FXR ligand binding will recruit the necessary coactivators to initiate transcription from Gal4-promoter containing genes. The same luciferase reporter constructs can be used in this format just with a Gal4 promoter instead of an FXRE-containing one.
As a tertiary assay which would be even closer to the native state of FXR, one can use cell lines that endogenously express FXR in an appropriate context and where activation of FXR by an agonist would drive transcription of native direct target genes. Unfortunately, most liver-derived cell lines such as HepG2 or HuH7 human hepatoma cells have only little functional FXR under normal conditions, but they can be “boosted” by just adding full-length recombinant FXR by transfection and selection for stably overexpressing clones.
With regard to cellular assays, it should be noted that cell membrane permeability is a key requirement for appropriate readouts for potency and efficacy of FXR agonists. That can be simply monitored by testing the natural ligands such as chenodeoxycholic acid (CDCA) and its tauro conjugate tauro-CDCA. Even in the first papers that describe FXR as a bile acid sensor (Makishima et al. 1999; Parks et al. 1999), it is noted that for tauro-CDCA to elicit a signal in a cellular reporter assay, a functional bile acid transporter has to be added. The Na+-taurocholate cotransporting polypeptide (NTCP, SLC10A1) is the natural bile acid transporter for the import of conjugated bile acids from the sinusoids into hepatocytes and is therefore well suited to provide this functionality when co-transfected into the reporter assay cell line.
Is a bile acid transporter needed also for synthetic FXR agonists, as they are all structurally very unrelated to the bile acid steroid nucleus?
This question can only be answered when the pharmacokinetics and the in vivo metabolic fate of synthetic FXR agonists are carefully analyzed. We have performed systematic analysis of plasma pharmacokinetics and liver-to-plasma compound level ratios comparing homologous series of isoxazole “hammerhead” type of compounds in mice (Kinzel et al. 2016) and collected bile from the gallbladder of FXR agonist-treated mice to compare the metabolite spectrum of FXR agonists in plasma, liver, and bile. It should be noted that all “hammerhead”-type FXR agonists contain an acidic function and the terminal end of the stretched molecule, typically a normal, mostly aromatic carboxylic acid, but sometimes (i.e., Enanta compound from Fig. 1) this is replaced by an acid isostere such as a tetrazole or an acyl-sulfonamide. We have found that isoxazole-type FXR agonists with carboxylic acids are often conjugated with taurine in vivo which is one special form of Phase II metabolization. The degree of taurine conjugation varies with the degree of lipophilicity of such compounds; the more lipophilic, the more taurine-conjugated. In this aspect, this type of synthetic FXR agonist seems to mimic the conformation of hydrophobic, unconjugated bile acids since they are recognized by the two bile acid-conjugating enzymes Bile acyl-CoA synthetase (BACS) and Bile acyl-CoA-aminotransferase (BAAT) to about the same extent as natural unconjugated bile acids.
As mentioned before, taurine conjugates of bile acids but also of synthetic FXR agonists cannot penetrate the cell membrane due to the permanent negative charge of the taurine sulfonic acid function. However, when adding NTCP to a cellular reporter assay, taurine-conjugated compounds show about the same potency and efficacy compared to their unconjugated counterparts. The reason for this is that the taurine is added to the carboxylic acid which is located just to the opposite of the ligand molecule where the key agonist interaction between Helix 11 and 12 takes place. It is the threefold-substituted isoxazole “hammerhead” that locks the active conformation, whereas the acid part is engaged in some hydrogen bond interaction and potentially ionic interactions with arginine residues at the other side of the ligand-binding pocket [see Gege et al. (2014) and references therein for an in-depth discussion]. An extension of the acid by an amide-type prolongation is well permitted and the taurine therefore well accepted. The sulfonic acid part reaches out into the water environment and does not need to be desolvated which means there is no free enthalpy cost for the ligand binding of a taurine conjugate as opposed to an unconjugated carboxylic acid.
However, unlike taurine- or glycine-conjugated bile acids which have a very high degree (>95%) of reuptake in the intestine and thus enterohepatic circulation, we have found that taurine-conjugated synthetic “hammerhead”-type of isoxazoles are not bioavailable. Thus it seems as if they are not accepted as substrates of the intestinal bile acid uptake transporter apical sodium-dependent bile salt transporter (ASBT/IBAT).
In summary, synthetic FXR agonists, despite their chemical unrelatedness to the bile acid steroid nucleus, may adopt features of bile acid-type metabolization, i.e., Phase II metabolization such as taurine conjugation, acyl glucuronidation, and less Phase I metabolization, e.g., hydroxylation. However, the degree of “chemical mimicry” varies in that they seem to be accepted as substrates for hepatocyte-borne bile acid transporters, e.g., the sinusoidal NTCP or the canalicular BSEP but not necessarily for intestinal transporters. There are also synthetic FXR agonists that do not show a bile acid-like PK behavior. It seems as if the individual differences depend on the exact conformation of the ligand in the pocket but also on the degree of lipophilicity of the ligand (the more hydrophobic, the more similar to the bile acid pattern).
Another important aspect from the PK and tissue distribution point of view is that the main sites of action of FXR agonists by definition are the small intestine and the liver. Carboxylic acid-bearing synthetic FXR agonists are the ones that are most active in vivo, but they are also prone to be actively taken up by liver transporter such as NTCP as discussed before or more unspecific organic anion transporting polypeptide (OATP) transporters. We have observed huge variations in the plasma-to-liver ratios of different synthetic FXR agonists (Kinzel et al. 2016), but a prevalent motif is that FXR agonists that tend to be enriched in the liver turn out to show very potent in vivo effects but this includes also the side effects (see discussion at the very end: intestinal versus liver activity of FXR agonists). This is mentioned here because it means in consequence that plasma pharmacokinetics are misleading as guidance of active FXR agonist exposure and the liver residence time may be far longer than the actual plasma lifetime.
4 Applications for Nonsteroidal FXR Agonists: Preclinical Studies
Based on the biological roles of FXR in bile acid (BA) metabolism and whole-body energy homeostasis, FXR agonists are gaining attention as potential therapeutic agents in hepatobiliary disease. The following paragraph will highlight several preclinical studies and experimental models, focusing in particular on those relevant for currently ongoing clinical studies and indications.
4.1 Cholestatic Liver Diseases and BA Dysregulation
FXR is highly expressed in tissues that participate in BA metabolism such as the liver and intestine. Upon activation by conjugated and unconjugated bile salts, FXR regulates BA homeostasis by controlling genes involved in BA synthesis, secretion, conjugation, transportation, absorption, and detoxification (Mazuy et al. 2015; Wang et al. 2008a). Thereby FXR critically regulates bile formation and BA enterohepatic circulation. BAs and FXR play a pivotal role in regulating hepatic inflammation and regeneration as well as in regulating extent of inflammatory responses, barrier function, and prevention of bacterial translocation in the intestine. Thus, FXR has been considered as a promising target for the treatment of disorders of the biliary and gastrointestinal tract such as cholestatic liver diseases and inflammatory bowel disease (IBD) (Gadaleta et al. 2010).
The chronic cholestatic diseases PBC and PSC are characterized by defective BA flow from the liver to the intestine. The impaired bile secretion and transport result in intrahepatic accumulation of bile acids and cause fibrosis, inflammation, and cirrhosis. Additionally, the amount of BAs within the intestinal lumen is often decreased, which may lead to insufficient FXR-dependent FGF19 secretion by the enterocyte, thereby inducing a vicious circle of increased hepatic BA synthesis and progressive liver damage. FXR activation reduces the BA pool size, which represents one of the most important factors in cholestasis, by downregulating cytochrome P450 family 7 subfamily A member 1 (CYP7A1) expression via a synergistic mechanism that involves hepatic and intestinal FXR (Inagaki et al. 2005; Kim et al. 2007; Sinal et al. 2000). Several animal models support the role of FXR in the pathogenesis of cholestasis. In two models of acute cholestatic liver injury in rats (bile duct ligation (BDL) and α-naphthylisothiocyanate (ANIT) toxicity), the synthetic FXR agonist GW4064 markedly reduces bile duct proliferation, inflammation, and liver injury (Liu et al. 2003). Moreover, both GW4064 and OCA were able to protect rats from ethinyl-estradiol-induced cholestasis (Fiorucci et al. 2005a). Back then, the proposed protective effect of FXR was ascribed only to the reduced expression of CYP7A1 and NTCP and the induction of multidrug resistance-associated protein 2 (MRP2) and bile salt export pump (BSEP) in the liver. Today, evidence has shown a critical involvement of intestinal FXR activation leading to an additional repression of liver CYP7A1 expression, thereby further reducing BA pool size. Furthermore, FXR has been implicated to protect against pathogen-associated molecular patterns (PAMP) recognition and inflammatory signaling via downregulation of NF-kB pathways (Chignard and Poupon 2009; Wang et al. 2008b) adding an anti-inflammatory component to the pro-choleretic effects of FXR activation.
4.2 Inflammatory Bowel Disease (IBD)
IBD represents a group of disorders characterized by chronic intestinal inflammation. Currently, it is believed to result from dysregulation of the mucosal immune system, compromised intestinal epithelial barrier function, and an atypical, unhealthy gut microbiome (Podolsky 2002). Implicating FXR function as an important determinant in IBD, chemical-induced intestinal inflammation was shown to be reinforced in Fxr −/− mice, and genetic variation of FXR is reported to be associated with human IBD (Attinkara et al. 2012). Moreover, the steroidal FXR agonist OCA was able to protect mice from chemical-induced intestinal inflammation (Gadaleta et al. 2011). FXR alleviates inflammation and preserves the integrity of the intestinal epithelial barrier. Activated FXR also limits bacterial overgrowth and prevents bacterial translocation in the intestinal tract (reviewed in Ding et al. 2015; Gadaleta et al. 2010).
4.3 Metabolic Liver Diseases Nonalcoholic Fatty Liver Disease (NAFLD)/NASH
NAFLD presents a spectrum of liver diseases initiated with excess accumulation of lipids in the hepatocytes. NAFLD starts with simple benign hepatic steatosis, progresses further to NASH, characterized by liver steatosis in addition to signs of inflammation and ballooning of the liver cells, and ultimately leads to NASH-induced liver fibrosis and cirrhosis, the common end stage of most chronic liver diseases. At the stage of NASH, the risk of hepatocellular carcinoma (HCC) is substantially increased (Torres et al. 2012). From the etiology, NASH is closely linked to features of the metabolic syndrome such as obesity, hypertriglyceridemia, low high-density lipoprotein levels, hypertension, and elevated fasting plasma glucose (Yki-Järvinen 2014), and it is suggested that a bi-directional mutual relationship between NASH and various components of metabolic syndrome or even overt T2D exists (Lonardo et al. 2018). Along with the tight control of BA metabolism exerted by FXR, accumulating data demonstrate that FXR also plays an essential role in maintaining lipid and glucose homeostasis (Cariou et al. 2006; Lee et al. 2006; Ma et al. 2006; Stayrook et al. 2005; Zhang et al. 2006) (reviewed in Lefebvre et al. 2009; Zhang and Edwards 2008) and that modulation of FXR can have significant influence on metabolic homeostasis. FXR is regarded as a promising therapeutic target for obesity and NASH, and the activation of FXR showed beneficial effects on various metabolic diseases, including fatty liver diseases, T2D, dyslipidemia, and obesity (Chávez-Talavera et al. 2017; Teodoro et al. 2011).
Fxr −/− mice display elevated serum cholesterol and triglyceride levels and an excessive accumulation of fat in the liver (Lambert et al. 2003; Sinal et al. 2000). Furthermore, they develop signs of insulin resistance as shown by hyperglycemia, impaired glucose tolerance, and severely blunted insulin signaling in both liver and muscle (Stayrook et al. 2005; Zhang et al. 2006). Activation of FXR by BAs or synthetic agonists lowers plasma triglyceride levels by a mechanism that involves the repression of hepatic transcription factor sterol regulatory element-binding protein 1c (SREBP-1c) expression and its lipogenic target genes in mouse primary hepatocytes and liver (Watanabe et al. 2004; Zhang et al. 2009). In the Zucker (fa/fa) rat, a model for NAFLD, where liver steatosis, diabetes, insulin resistance, and obesity occur due to a loss-of-function mutation of the leptin receptor, FXR activation by OCA protected against liver steatosis, body weight gain, and reversed insulin resistance (Cipriani et al. 2010). In addition, WAY-450 attenuated fructose-induced hepatic steatosis through the suppression of inflammation and the hepatic lipid droplet protein in mice (Liu et al. 2014). GW4064 improved insulin resistance in ob/ob mice, a genetic obesity model, and differentiated 3T3-L1 adipocytes displayed an enhanced insulin signaling and insulin-stimulated glucose uptake upon FXR agonist treatment (Cariou et al. 2006). Fexaramine also reduced obesity and promoted adipose tissue browning in mice (Fang et al. 2015). Beyond the individual metabolic effects, FXR agonists also improved liver histology in all parameters with reductions in fibrosis and steatosis and anti-inflammatory effects, in addition. In mice on methionine and choline deficient diet, WAY-450 reduced liver inflammation and fibrosis without triglyceride accumulation (Zhang et al. 2009). Furthermore, in STAM™ mice, a chemical-induced NASH model that develops manifest NASH at 8 weeks, Px-102 treatment was shown to improve liver histology, along with reduced inflammation and lipid disposition (Kremoser et al. 2011). The successor molecule to Px-102, cilofexor, also led to a significant decrease in liver steatosis and fibrosis in C57BL/6 mice kept on a fast-food diet (Liles et al. 2016).
4.4 Liver Fibrosis, Cirrhosis, and Portal Hypertension
In NAFLD, the progression from simple steatosis to NASH is determined by the initiation of the fibrotic response. As in NAFLD patients, it was shown that fibrosis stage but not NASH predicts clinical outcomes in terms of mortality (Dulai et al. 2017; Hagström et al. 2017). This is why fibrosis reduction or NASH resolution without worsening of fibrosis is the clinical endpoint which is accepted by the regulatory agencies FDA and EMA (Filozof et al. 2017; Hannah et al. 2016). Hepatic stellate cells (HSCs) are the main regulators of extracellular matrix (ECM) production and play an essential role in the development of fibrosis, and activation of HSCs is critical for initiation and progression of liver fibrosis (Tsuchida and Friedman 2017).
FXR expression was reported to be low in human HSCs (Fickert et al. 2009), but the roles of FXR in HSC biology were demonstrated as treatment with OCA increased the PPARγ mRNA levels in HSCs and in rodent models of liver fibrosis, leading to the inhibition of the HSCs activation (Fiorucci et al. 2004, 2005c). In addition, activation of the FXR-SHP regulatory cascade by OCA mediated inhibition of HSCs and promoted the resolution of liver fibrosis (Fiorucci et al. 2005b). Furthermore, in fibrotic liver tissues of humans and mice, FXR expression was reduced, and activation of FXR reduced mitochondrial dysfunction and oxidative stress and increased hepatocyte survival by repression of miR-199a-3p targeting liver kinase B1 (LKB1) (Lee et al. 2012). A recent study reported that inducing FXR signaling by GW4064 activated the hepatic inositol-requiring enzyme 1α/X-box binding protein 1 pathway of the unfolded protein response (Liu et al. 2018), suggesting the possible role of FXR as regulator for endoplasmic reticulum (ER) stress, a stimulator of fibrosis development. FXR activation by GW4064 in isolated rat HSCs led to an inhibition of the endothelin-1-mediated contraction and trans-differentiation (Li et al. 2010) and increased the miR-29a promoter activity responsible for the inhibition of ECM production in HSCs (Li et al. 2011).
There is a gradual transition from a fibrotic NASH liver toward a cirrhotic liver. A key hallmark of cirrhosis is portal hypertension, i.e., the increase in the hepatic vein pressure gradient (HVPG) as a reflection of reduced blood flow through the sinusoids of the liver. The HVPG is a good predictor of mortality, and thus reduction of HVPG to levels <10–12 mmHg is clinically desired (Ripoll et al. 2007) (reviewed in Bosch and Iwakiri 2018). FXR agonists, both OCA and the nonsteroidal Px-102, have demonstrated to reduce the HVPG in rat models of cirrhosis and portal hypertension through various mechanisms including endothelial nitric oxide synthase (eNOS)-mediated sinusoidal vasodilation and reduction of the vasoconstrictive factors endothelin-1 and p-Moesin (Schwabl et al. 2017; Verbeke et al. 2014).
4.5 Cancer
Hepatocellular carcinoma (HCC) often occurs on the basis of chronic liver inflammation or fibrosis. There is a growing body of evidence indicating that FXR is involved in carcinogenesis. Fxr −/− mice were found to spontaneously develop liver tumors as they age (Yang et al. 2007), while selective activation of intestinal FXR protected mice against development of HCC (Modica et al. 2008). In a diethylnitrosoamine (DEN)-mediated liver cancer mouse model, GW4064 was found to reduce the expression of oncoprotein gankyrinin preventing liver cancer development (Jiang et al. 2013). Furthermore, stable overexpression on FXR or activation by Px-102 led to transcriptional induction of N-myc downstream-regulated gene 2 (NDRG2) a tumor suppressor and reduced liver tumor growth in an orthotopic xenograft model in nude mice (Deuschle et al. 2012). In Abcb4 −/− mice, which are characterized by hepatic accumulation of BA and development of HCC, steroidal dual FXR/TGR5 agonist INT-767 administration significantly reduced the number and size of HCC nodules (Cariello et al. 2017). As FXR maintains BA pool size and composition within a physiological range and elevated BAs are considered as tumor-promoting factors in colorectal cancer development (Bernstein et al. 2005; Debruyne et al. 2001), an involvement for FXR was also implicated in the modulation of intestinal tumorigenesis. FXR mRNA expression was decreased in different colorectal adenoma and carcinoma cell lines (De Gottardi et al. 2004), and FXR deficiency led to significantly increased sizes and numbers of tumors in murine intestine tumorigenesis models APCmin mice and azoxymethane (AOM)-induced colon cancer (Maran et al. 2009). Moreover, activation of FXR induced a proapoptotic program in the differentiated normal colonic epithelium as well as transformed colonocytes and is thereby hypothesized to remove genetically altered cells, which may otherwise progress to complete neoplastic transformation (Modica et al. 2008). In human colon carcinomas tissues and human cell lines, FXR expression was markedly reduced and might be associated with an adverse prognosis (Lax et al. 2012). Furthermore, it was shown that early in the development of human colon carcinoma, FXR is silenced by DNA methylation and Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) signaling (Bailey et al. 2014). Together, these clues suggest that FXR plays a key role in the pathogenesis of different carcinomas and that restoration of FXR expression or FXR activation could prevent tumor development or progression. In contrast to this tumor-preventive role of FXR, several studies implicate the FGF19-FGFR4 signaling axis as a tumor promoter or at least as a marker of HCC (Zhou et al. 2014) and colon carcinoma. The expression of intestinal FGF19 is controlled by FXR. Fgf15 −/−mice showed less tumors and histological neoplastic lesions compared to wild-type mice, and the hepatocellular proliferation was reduced in Fgf15 −/− mice, which also expressed lower levels of the HCC marker alpha-fetoprotein (AFP) (Uriarte et al. 2015). In transgenic mice overexpressing human FGF19, increased hepatocellular proliferation was observed, and HCC development was evident within 12 months (Nicholes et al. 2002), while clinically it has been shown that FGF19 overexpression correlates with HCC progression and poorer progression (Miura et al. 2012). However, other studies have shown that it is not FGF19 but the overexpression of FGFR4 that is associated with poor clinical prognosis in HCC (French et al. 2012; Ho et al. 2009), prostate cancer (Murphy et al. 2010; Wang et al. 2004), cholangiocarcinoma (Xu et al. 2014), and breast cancer (reviewed in Lang and Teng 2019). Understanding the balance between pro- and anti-tumorigenic properties of FXR activation and unifying these findings in a consistent model how FXR impacts carcinogenesis require more preclinical and clinical validation. And upon refining our understanding, it will become clearer if we need FXR agonists or antagonists for cancer applications.
5 Applications for Nonsteroidal FXR Agonists: Clinical Effects
In the past decade, NASH has emerged as a leading cause for chronic liver disease. NAFLD and NASH represent a complex spectrum of liver diseases, but its stage and severity can be characterized by histopathological determination of the degrees of (1) steatosis, (2) cytoskeletal damage (hepatocellular ballooning), (3) lobular inflammation, and (4) fibrosis (Bedossa 2017). As NAFLD is strongly associated with obesity and diabetes, it is considered to be the hepatic manifestation of the metabolic syndrome and of T2D (Hu et al. 2017; Loomba and Sanyal 2013). The majority of subjects with NAFLD are asymptomatic and are diagnosed incidentally. While patients with simple steatosis suffer no negative consequences in terms of life expectancy, the fraction that progress to NASH face a worse outcome (Torres et al. 2012). Despite growing prevalence, the factors influencing NAFLD development and subsequent progression to NASH, liver fibrosis, cirrhosis, and HCC are still not fully elucidated. For late-stage NASH patients, often the only therapy option left is liver transplantation leading to the assessment of NAFLD soon becoming the leading cause of liver transplantation worldwide (Byrne and Targher 2015).
As of today, there are no approved drugs for the treatment for NAFLD and NASH. But as a large number of emerging therapies are being evaluated in the clinic (extensively reviewed in Konerman et al. 2018), FXR agonists are among other nuclear receptor modulators such as thyroid hormone receptor beta or PPAR agonists, the most promising therapeutic agents for NASH (Schaap et al. 2014). Among other metabolic changes, NASH patients also have an altered BA profile. The strong impact on BA metabolism exerted by FXR agonists is also expected to have beneficial effects (Puri et al. 2018).
The most advanced FXR agonist in clinical development is OCA, a derivative of the natural ligand CDCA. The clinical trials with OCA have validated FXR as target and demonstrated the clinical potential of FXR agonists to treat hepatic steatosis, inflammation, and fibrosis while increasing insulin sensitivity (Mudaliar et al. 2013; Neuschwander-Tetri et al. 2015). Currently, two Phase III trials are ongoing evaluating the effects of OCA in non-cirrhotic NASH subjects (NCT02548351) and in adults with compensated cirrhosis due to NASH (NCT03439254). As OCA is also under clinical investigation for PBC and while it was consequently more broadly dosed in patients, it was found that hepatically impaired patients, in particular, bear the risk of developing severe side effects. This was attributed to a non-adjustment of the dose to liver function and could occur even when treated with very low doses of OCA once a week.Footnote 1 , Footnote 2 Following in OCAs footsteps, a number of nonsteroidal FXR agonists are in clinical development, promising a better safety profile and more controllable PK compared to the bile acid-like OCA. An overview of the clinical evaluation of different FXR agonists in NASH is shown in Table 1.
Most clinical trials evaluate liver histopathology, more specifically the NAFLD activity score (NAS) which covers the three qualities steatosis, inflammation and hepatocyte ballooning, and the degree of liver fibrosis, typically assessed by the METAVIR scale, as main outcomes.Footnote 3 Furthermore, current data suggest that different approaches may be beneficial in subgroups of patients with NASH. Since the phenotype of NASH develops in the context of different genetic predispositions and environmental exposures, it is most likely that no single therapy will reverse NASH in all patients. For this reason, companies are evaluating combination therapies pairing nonsteroidal FXR agonists with other compounds targeting different pathways in the NASH pathophysiology.
The FLINT trial represents the first finished Phase II trial for NASH. This multicenter, randomized trial evaluated 72 weeks of OCA treatment (25 mg) versus placebo in patients with non-cirrhotic NASH (Neuschwander-Tetri et al. 2015). Here a total dose of 25 mg OCA daily for 72 weeks resulted in improvements in the composite NAS and fibrosis, while severe pruritus reported in 33 of 141 (23%) OCA-treated patients (p < 0.0001 vs placebo), and an increase in total cholesterol and LDL cholesterol and a modest decrease in HDL cholesterol (changes peaked at 12 weeks of treatment and then decreased slowly and stabilized) were discerned as possible therapy-limiting side effects.
At the beginning of 2019, pharma companies Gilead Sciences, Novartis, and Enyo Pharma were conducting Phase II trials in NASH patients testing nonsteroidal FXR agonists cilofexor, nidufexor, tropifexor, and EYP001.
First data for cilofexor were reported from a Phase II study with 140 NASH patients, treated with cilofexor at 100 or 30 mg or placebo orally, once daily for 24 weeks (Patel et al. 2018). At 24 weeks, a significant decrease in hepatic steatosis of at least 30% assessed by magnetic resonance imaging-proton density fat fraction (MRI-PDFF) was observed in 38.9% of patients treated with cilofexor 100 mg (p = 0.011 vs placebo), 14% treated with cilofexor 30 mg (p = 0.87), and 12.5% treated with placebo. Further significant observations in the cilofexor-treated patients were improvements in liver biochemistry (serum gamma-glutamyltransferase (GGT)) and response pharmacodynamic markers (serum 7α-hydroxy-4-cholesten-3-one (C4) and BAs). Cilofexor was generally well tolerated, and changes in lipid profile and glycemic parameters did not differ between cilofexor and placebo-treated patients. However, moderate to severe pruritus occurred in 14% of patients in the cilofexor 100 mg arm compared to 4% in the cilofexor 30 mg and placebo arms.
A separate Phase II study (ATLAS) is investigating treatment with cilofexor alone or in combination with investigational NASH drugs acetyl-CoA carboxylase (ACC) inhibitor firsocostat (GS-0976) and apoptosis signal-regulating kinase 1 (ASK-1) inhibitor selonsertib, in patients with advanced fibrosis due to NASH. This randomized, double-blind 52-week trial will evaluate improvement in fibrosis without worsening of NASH, adverse events, and plasma laboratory abnormalities. First data from a proof-of-concept study using cilofexor were presented at the International Liver Congress 2018 (Lawitz et al. 2018). The study also included combination treatment with selonsertib and firsocostat. A 12-week treatment regime was evaluated for safety and efficacy in patients with NASH and F2-F3 fibrosis. MRI-PDFF evaluation showed a median relative change to baseline −15.6% for cilofexor and beneficial effects on liver biochemistry (ALT –29.7%, GGT –19.3%). Fibrosis was not evaluated.
Further interim results were reported on a Phase II study assessing several doses of tropifexor for safety, tolerability, and efficacy in NASH patients (FLIGHT-FXR) after 12 weeks of therapy (Sanyal et al. 2018). At 12 weeks, a significant decrease in hepatic steatosis of at least 5% assessed by MRI-PDFF was observed in 33.3% of patients treated with 90 μg, 27.8% treated with 60 μg, and 14.6% treated with placebo. Furthermore, a dose-response decrease in GGT levels was observed as well as increases in FGF19. Adverse events and pruritus were reported to be comparable between 90 μg tropifexor arm and placebo. However a mild dose-related increase in LDLc and decrease in HDLc were observed in the 60 and 90 μg arms. As reported for the cholesterol changes during the FLINT trial (Neuschwander-Tetri et al. 2015), these effects were more prominent after initiation of treatment (2 weeks) and then slowly declined.
Tropifexor is also under investigation in a randomized, double-blind combination trial (TANDEM) with cenicriviroc, an investigational C-C chemokine receptor-type (CCR) 2/5 inhibitor (Stringer 2019). This combination therapy headed by Novartis should address metabolic, anti-inflammatory, and anti-fibrotic pathways involved in NASH. The 48-week trial will assess improvement in liver histology and occurrence of adverse events and serious adverse events in single and combination arms in approximately 200 patients with NASH and liver fibrosis.
Figure 9 compares data from Phase II trials on hepatic steatosis reduction. Due to profound differences in the individual trial designs, a full evaluation whether nonsteroidal FXR agonists are indeed superior to bile acid-like structures like OCA is not possible. The same caveat is true for the comparison of possibly dose-limiting side effects shown in Table 2 as no direct comparative clinical data are available.
However, the available evidence from clinical trials with nonsteroidal FXR agonists suggests that these class of substances may hold the potential to bring forth better FXR-targeting drugs with improved pharmacological actions and reduced adverse effects in particular in terms of cholesterol metabolism and pruritus. In conclusion, currently ongoing Phase II trials with nonsteroidal FXR agonist will have to prove if this class of substances is indeed superior in terms of observed side effects while retaining efficacy. This is of utmost importance, since NASH is (1) a largely asymptomatic disease and (2) future treatment will probably be long-term or even lifelong. Drugs with an onerous side effect profile are not acceptable, and potential treatment options shall not adversely impact cardiovascular risk, in particular, as this is the most common cause of death in patients with NASH.
As a consequence of the general hepatoprotective role of FXR as a master regulator of BAs, glucose, and lipid homeostasis, FXR also has been suggested as a promising pharmacological target in cholestatic liver diseases (recently reviewed in Goldstein and Levy 2018). PSC and PBC are the most common immune-mediated chronic cholestatic liver diseases leading to cirrhosis and liver failure. In particular for PSC there is an unmet need for effective medical treatments; as of today the only curative therapy is liver transplantation reserved for those with end-stage liver disease (Goldstein and Levy 2018). The first-line treatment for PBC is ursodeoxycholic acid (UDCA); it has choleretic and immunomodulatory properties and stimulates biliary bicarbonate secretion (Copaci et al. 2005). While UDCA is very well tolerated and can ultimately improve survival free of liver transplantation, the treatment offers an unfavorable response rate as approximately 40% of patients do not respond to UDCA and are at risk for progression (Corpechot et al. 2008).
The rationale for the use of FXR agonists in cholestatic liver diseases is that FXR activation is postulated to reduce toxic bile production and induce secretion through FXR-mediated pathways. Activation of FXR inhibits CYP7A1 (the rate limiting step of bile acid synthesis) directly, through translational activation of SHP, as well as indirectly, through the release of FGF19, which binds to FGFR4 on hepatocytes and leads to additional CYP7A1 inhibition (Inagaki et al. 2005; Rizzo et al. 2005). FXR upregulates (bile salt) transporters BSEP, MDR2/3, MRP2, and OSTα/β, further decreasing hepatocellular bile concentrations through increased canalicular secretion (Ananthanarayanan et al. 2001; Boyer et al. 2006; Kast et al. 2002). These effects should result in a limited accumulation of toxic BAs within the hepatocyte, thus reducing liver injury and potentially ameliorating inflammation of bile ducts.
OCA was granted conditional approval in May 2016 for treatment of PBC patients that were exhibiting an inadequate response to UDCA. It should be prescribed either in conjunction with UDCA or as single therapy if UDCA is not tolerated. This approval was based on results of the POISE trial, where 216 patients with PBC and an inadequate response to UDCA received either placebo, 5 mg, or 5 mg titrated to 10 mg OCA. After 12 months of treatment, 47% in the 5 mg OCA group and 46% in the 5–10 mg OCA group compared to 10% in the placebo group met the primary endpoint (serum alkaline phosphatase (ALP) <1.67 × ULN with a reduction of ≥15% from baseline, normal total bilirubin) (Nevens et al. 2016). The most common side effect of OCA was a dose-dependent development of itching, and use of OCA was also associated with a dose-dependent reduction in HDLc. While OCA is promising in terms of its ability to significantly decrease ALP levels in PBC patients and possibly improve survival free of liver transplantation, a follow-up with studies is necessary to examine the long-term effects of OCA therapy and validate its suitability for patients with more advanced liver disease.
In regard to the use of OCA in PSC, the AESOP trial, a Phase II trial for dose-finding and evaluation of the efficacy and safety of OCA, looked at the effect of 24 weeks OCA treatment compared to placebo in 77 patients with PSC. The primary endpoint of the AESOP trial was the mean change in serum ALP levels. A statistically significant decrease in baseline ALP of 22% in both the low-dose (1.5–3 mg) and high-dose (5–10 mg) groups was observed (Kowdley et al. 2018; Larusso et al. 2018), and a long-term extension phase is ongoing. While encouraging, further trials are needed to assess if these results translate into a clinically significant endpoint such as increased time to transplantation or death.
Nevertheless, the efficacy demonstrated by OCA in the treatment of PBC has established FXR as a valuable therapeutic target, and different nonsteroidal FXR agonists are currently tested in PBC and PSC trials. Trials for PBC and PSC are summarized in Table 3.
Currently, three other FXR agonists are undergoing Phase II testing for PBC. These novel molecules include cilofexor and tropifexor and the steroidal FXR agonist EDP-305 by Enanta Pharmaceuticals (ClinicalTrials.gov identifiers: NCT03394924, not covered here).
For cilofexor, a Phase II, double-blind, placebo-controlled study evaluating the safety and tolerability of 30 mg and 100 mg cilofexor for 12 weeks in patients with PBC without cirrhosis is currently ongoing. Primary study outcomes include the incidence of treatment-emergent adverse events (AE) and serious adverse events (SAE) and laboratory abnormalities. Furthermore, a proof-of-concept Phase II trial assessing cilofexor in patients with PSC in an open-label fashion is also under investigation. This study enrolled 52 non-cirrhotic patients with PSC who received either 100 mg cilofexor, 30 mg cilofexor, or placebo orally once daily for 12 weeks. Dose-dependent reductions in liver biochemistry were observed, and after 12 weeks of treatment, 100 mg cilofexor led to significant improvements in liver biochemistry parameters, serum ALP (−21%; p = 0.029 vs placebo), GGT (−30%; p < 0.001), alanine aminotransferase (ALT) (−49%; p = 0.009), and aspartate aminotransferase (AST) (−42%; p = 0.019). In both groups treated with cilofexor, reduced serum levels of C4 were reduced compared with placebo (−23.2% in the 100 mg group, p = 0.21; and −30.5% in the 30 mg group, p = 0.024). Reductions in serum BAs were greatest with the 100 mg dose. Cilofexor was well tolerated, and the incidence of grade 2 or 3 pruritus was lower with cilofexor 100 mg (13.6%) and 30 mg (20%) compared with placebo (40%). There were no elevations in serum lipids (Trauner et al. 2019).
Tropifexor is also currently evaluated in a multipart, double-blind, placebo-controlled Phase II study to assess the safety, tolerability, and efficacy in patients with PBC. Part 1 includes a 28-week treatment and part 2 a 12-week treatment period with tropifexor. The primary study outcomes include safety and tolerability as well as changes in markers of cholestasis compared to baseline, while secondary objectives include evaluation of disease-specific quality of life and pharmacokinetics.
In a proof-of-concept study, treatment with OCA stimulated FGF19 release and decreased BA synthesis, producing clinical benefit in patients with bile acid diarrhea (BAD) after 2 weeks of treatment (stool frequency (24%; p = 0.03), stool form (14%; p = 0.05), and diarrhea index (34%; p = 0.005)) (Walters et al. 2015). Treatment was reported as well tolerated, and adverse effects included a change in lipids (increase in LDLc), mild headache in 11% of patients, and no reports of pruritus. BAD is a common cause of chronic diarrhea, occurring as a primary condition or secondary to ileal disease or resection. Many patients have reduced levels of the ileal hormone FGF19, which acts as an inhibitory regulator of hepatic BA synthesis and secreted in response to FXR activation in the intestine. In primary BAD, impaired FGF19 production results in increased CYP7A1 expression and enhanced hepatic synthesis of BAs. In turn, this results in a larger BA pool with increased colonic delivery (Keely and Walters 2016). This proof-of-concept study supports a future role for FXR agonists in the treatment of BAD. Recently a double-blind, randomized, placebo-controlled crossover multiple-dose study of tropifexor to assess safety, tolerability, and efficacy in patients with primary BAD was concluded and is under evaluation. Still, for a more definitive assessment of long-term efficacy, further studies with larger numbers of patients are required both in patients with primary and secondary BAD.
Furthermore, as FXR controls the expression of NTCP, FXR agonism could be a viable principle to address viral hepatitis infections. NTCP has been demonstrated to be a functional receptor for HBV, mediating viral entry and consequent infection (Yan et al. 2012). By repressing NTCP, FXR agonism may block HBV entry and infection. Furthermore, FXR agonism could also directly inhibit HBV mRNA, DNA, and protein production and reduced covalently closed circular DNA pool size (Radreau et al. 2016). In cell culture, an enhanced effect was observed when combined with antiviral treatments entecavir or tenofovir (Joly et al. 2017). EYP001 is therefore explored as therapeutic option for HBV in a Phase Ib trial.
6 How to Differentiate Diverse FXR Agonists from Each Other?
In the previous section, we discussed the huge and diverse clinical potential of FXR agonists in various clinical indications, in general. The pioneering frontrunner FXR agonist in most applications except for HBV was OCA which represents a modified bile acid. There is a discussion in the literature that nonsteroidal FXR agonists might be superior to bile acid-type ones (Verbeke et al. 2017), and in a head-to-head comparison between Px-102 and OCA in a CCl4-induced rat model of cirrhosis and portal hypertension, the nonsteroidal FXR agonists performed better indeed, but this could be a simple function of better in vivo potency (Schwabl et al. 2017). The key liabilities of OCA are the induction of pruritus in NASH patients, and in PBC patients, OCA even exacerbates the already existing disease-related pruritus. This is an adverse effect which directly affects the quality of patients’ lives, while the changes in lipoprotein cholesterol toward a worse atherogenic index (defined as the ratio of HDLc to LDLc) pose more a long-term threat. A third issue of OCA is its uncontrolled PK since OCA as every bile acid is massively taurine- and glycine-conjugated in humans and in fact upon chronic dosing, these conjugated forms represent 90–95% of the plasma detectable metabolites of OCA. Genetic variations in the transporters involved in this enterohepatic cycling as well as food interactions and other individual variations may be responsible for the effect that the same dose of OCA, e.g., 10 mg daily, may result in substantially different plasma and FGF19 levels as a pharmacodynamics readout of FXR activation and this may give rise to over- and under-responders in the same dosing group (Marschall et al. 2012). Such effects can clearly be overcome by synthetic FXR agonists since it was shown that, e.g., Px-102 yielded a more drug-like PK behavior in Phase I healthy volunteers with acceptable low individual variations.
The basis of the lipoprotein cholesterol changes and of the pruritogenic effects is not well understood. Some publications related the pruritus to activation of TGR5, the other, GPCR-type bile acid receptor (Alemi et al. 2013; Lieu et al. 2014). Others make the increased levels of autotaxin, a phospholipase that releases lysophosphatidic acid (LPA) from circulating phospholipid substrates, responsible for bile acid mediated pruritus, in general (Kremer et al. 2010; Oude Elferink et al. 2011). Some research groups have described OCA as a submicromolar activator of TGR5 which might be one explanation (Fiorucci et al. 2014) of its pruritogenic potential but it cannot be the sole one since pruritus was also described as a mild side effect of cilofexor in the first Phase II study. Thus it is difficult to ascribe this adverse effect to a certain structural or molecular feature of a FXR agonist, and it needs to be demonstrated by future clinical study results how synthetic FXR agonists compare to steroidal ones in this regard.
Another point of differentiation very controversially discussed is the ratio of intestinal- versus liver-specific FXR activation. Fang et al. (2015) have shown that selective pharmacological activation of FXR by fexaramine, which has very limited bioavailability, is sufficient to elicit several of the aforementioned beneficial metabolic effects. Modica et al. point into the same direction by having shown that constitutive FXR expression just in intestines of mice is sufficient to yield potent anti-cholestatic effects (Modica et al. 2012). However, there are other publications that claim that antagonizing or inhibiting FXR in the intestine is what is needed to elicit desirable effects (Jiang et al. 2015; Kim et al. 2007) and that liver-selective FXR activation yields potent lipid reducing effects (Schmitt et al. 2015). The groups of Frank Gonzalez and John Chiang, the former responsible for promoting the idea that FXR antagonism is beneficial rather than agonism, very recently surprised by the finding that fexaramine indeed elicits adipose tissue browning and improves insulin sensitivity using a formerly not described pathway: intestinal FXR activation by fexaramine increases taurolithocholic acid and TGR5 as its receptor and that has an impact of the gut microbiome (Pathak et al. 2018). Pathak et al. ultimately make the modified gut microbiome responsible for the beneficial changes since the effects are gone when antibiotics are given to the mice.
The publication by Houten et al. (2007) sheds a light on the question of which tissue is physiologically more relevant in terms of FXR activation by endogenous ligands. Houten et al. have generated a transgenic mouse that harbors a luciferase under control of a FXR-responsive promoter. In this FXR-sensitive reporter mouse, only ileal FXR is active under normal physiological conditions. Liver FXR only becomes activated under cholestatic conditions of bile acid overflow, in this case simulated by bile duct ligation. Even GW4064 generates mainly an intestinal FXR signal although GW4064 is widely published to exert various beneficial metabolic effects.
Intestinal FXR induces FGF15 (in rodents) or FGF19 (in primates and humans) which circulates to the liver through the portal circulation where it can bind to FGFR4, a receptor tyrosine kinase. Activated FGFR4 induces signaling via the extracellular signal-regulated protein kinase (ERK) pathway to control glucose and glycogen synthesis. It also controls the activity of SHP to repress transcription of CYP7A1, the key pacemaker enzyme for the conversion of cholesterol into bile acids (Inagaki et al. 2005), probably by a c-Jun N-terminal kinase (JNK)-mediated pathway (Holt et al. 2003). Kong et al. have demonstrated that in mice, intestinal FXR activation via FGF signaling is sufficient to suppress transcription of both key enzymes, CYP7A1 for overall bile acid synthesis and CYP8B1 which introduces a third hydroxy group into the bile acid steroid nucleus, thus controlling the hydrophobicity of the bile acid pool (Kong et al. 2012). Liver FXR activation results in upregulation of SHP which acts as a suppressor of CYP7A1 on the promoter of this gene (Goodwin et al. 2000; Lu et al. 2000); however, Kong et al. have found that liver FXR exerts stronger repressive control on CYP8B1 than on CYP7A1, whereas the intestinal FXR-FGF-FGFR4 axis has a stronger effect on CYP7A1 compared to CYP8B1. These data were mainly generated in mice, but if they could be extrapolated to humans, it had important functional meanings. De Boer et al. (2017) showed in an elegant study that FGF15 signaling plus liver FXR activation by Px-102 in mice changed the hydrophobicity of the bile acid pool and that this change led to a massive increase of transintestinal cholesterol excretion (TICE) which finally resulted in plasma cholesterol lowering affecting LDL as well as HDL cholesterol. The compound used in this study, Px-102, however, is also very active in the liver, and its effects on liver CYP8B1 suppression are likely responsible for changing the hydrophobicity of the bile acid pool. If one compares the changes in the bile acid pool composition between Px-102-treated mice (de Boer et al. 2017) with the pattern from fexaramine-treated mice (Fang et al. 2015), it becomes obvious that the intestinally restricted FXR agonist fexaramine induces by far less changes in the hydrophobicity of the bile acid pool also with only little increase in tauro-beta muricholic acid compared to Px-102. The fexaramine-treated mice show many beneficial metabolic effects but only little cholesterol lowering, whereas Px-102 led to a massive increase in fecal neutral sterol excretion and thus plasma cholesterol reductions (see also Hambruch et al. 2012).
In essence we tended to follow the hypothesis from Fang et al. that intestinal FXR activation is sufficient to elicit most of the desired metabolic effects but avoids that undesired changes in BA pool composition which are likely responsible for the HDL cholesterol-lowering effects. For Px-102, an FXR agonist found to be very active in the intestine as well as in the liver compared to GW4064 (Houten et al. 2007) and OCA, it was shown that liver FXR activation also leads to upregulation of SR-BI as the scavenger receptor for HDLc and that this is one contributor to the apparent plasma HDLc lowering on top of the cholesterol losses through intestinal excretion (Hambruch et al. 2012). Thus it was a reasonable assumption that more intestinally restricted FXR agonist could be void of the BA-induced side effects, potentially even of the pruritogenic effects if they were really BA related, but that such a compound would still retain most of the beneficial metabolic potential, in particular for the treatment of NASH.
Thus we have developed cilofexor which is in Phase IIb in clinical development in NASH, PBC, and PSC. Cilofexor, due to its specific tissue distribution and physicochemical properties, yields an intestinally biased FXR agonist which is still bioavailable but mostly lacks transcriptional activity in the liver. The detailed mechanisms how this can be achieved will be published soon (manuscript in preparation). By now it can be stated that cilofexor yields less HDLc-lowering effects as determined by a 21-day Phase I study in healthy human volunteers (Myers et al. 2018) and that it yields decent antisteatotic effects with only little pruritogenic potential compared to OCA in a first Phase II clinical trial in NASH patients. If such an intestinally biased “designer” FXR agonist is really superior to other unbiased but very potent nonsteroidal FXR agonists such as tropifexor, it needs to be proven in large-scale clinical studies which carefully evaluate the therapeutic index between the beneficial liver-protective effects and the adverse effects in terms of pruritus, undesired HDLc to LDLc changes, and the potential liver proliferation and cancerogenicity in animal studies.
Notes
- 1.
FDA Drug Safety Communication. https://www.fda.gov/Drugs/DrugSafety/ucm594941.htm.
- 2.
FDA Adverse Event Reporting System (FAERS) Public Dashboard. https://fis.fda.gov/sense/app/777e9f4d-0cf8-448e8068-f564c31baa25/sheet/45beeb74-30ab-46be-8267-5756582633b4/state/analysis.
- 3.
FDA Noncirrhotic Nonalcoholic Steatohepatitis With Liver Fibrosis: Developing Drugs for Treatment-Guidance for Industry. https://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM627376.pdf.
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Gege, C., Hambruch, E., Hambruch, N., Kinzel, O., Kremoser, C. (2019). Nonsteroidal FXR Ligands: Current Status and Clinical Applications. In: Fiorucci, S., Distrutti, E. (eds) Bile Acids and Their Receptors. Handbook of Experimental Pharmacology, vol 256. Springer, Cham. https://doi.org/10.1007/164_2019_232
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