FGF21-based pharmacotherapy--potential utility for metabolic disorders.

TEM-941; No. of Pages 9

Review

FGF21-based pharmacotherapy – potential utility for metabolic disorders Ruth E. Gimeno and David E. Moller Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285, USA

Currently available therapies for diabetes or obesity produce modest efficacy and are usually used in combination with agents targeting cardiovascular risk factors. Fibroblast growth factor 21 (FGF21) is a circulating protein with pleiotropic metabolic actions; pharmacological doses of FGF21 produce anti-diabetic, lipid-lowering, and weightreducing effects in rodents. Several potential benefits have translated to non-human primates and obese humans with type 2 diabetes (T2D). Accumulating results point to a specific receptor complex and actions in adipose tissue, liver, and brain; several pathways lead to enhanced fatty acid oxidation, increased insulin sensitivity, and augmented energy expenditure. A range of strategies are being explored to derive potent, safe, and convenient therapies which could potentially represent novel approaches to prevent and treat a variety of metabolic disorders.

of thermogenesis and fatty acid oxidation (see below). The magnitude of FGF21-induced weight loss is significantly less in leptin-deficient mice [2], suggesting a possible requirement for a functional leptin axis. The importance of FGF21 as a metabolic regulator has been confirmed in studies in non-human primates. In diabetic rhesus monkeys, FGF21 lowered fasting glucose, insulin, and plasma fructosamine to near-normal levels over a period of 6 weeks [8]. FGF21 had profound effects on serum lipids, including substantive reductions in triglycerides, total cholesterol, and low-density lipoprotein (LDL)-cholesterol, whereas high-density lipoprotein (HDL)-cholesterol levels were increased. Relative to mouse models, weight loss was modest with a cumulative 4% effect at the end of treatment [8]. Similar metabolic effects on weight and lipids have been reported in a separate study using obese, nondiabetic rhesus monkeys [6].

Discovery and characterization of FGF21 as a metabolic regulator FGF21 was identified as a potential metabolic factor in a screen for proteins that induce glucose uptake in adipocytes [1]. Subsequent in vivo evaluation of recombinant FGF21 uncovered a wide variety of metabolic effects. In mouse models of diabetes and obesity, FGF21 administration lowers glucose and improves insulin sensitivity, decreases body weight, and lowers serum triglyceride and cholesterol as well as hepatic triglyceride levels [1–6]. The effects of FGF21 on glucose are observed as early as 1 h after injection, are independent of weight changes, and plateau at lower doses compared to the weight changes [2,4,5]. Clamp studies show improved hepatic, and in some settings peripheral, insulin sensitivity [3,4,7], and no hypoglycemia has been observed following FGF21 administration [1,6]. FGF21-induced weight loss can be substantial, reaching up to 20% in diet-induced obese mice after as little as 14 days of treatment [2,3]. This weight loss is due to increased energy expenditure and is not associated with decreased food intake. FGF21 administration increases core body temperature and decreases the respiratory quotient [2], whereas thyroid hormone levels and plasma adrenalin levels are not affected [2], consistent with local activation

FGF21 receptors and FGF21-induced signaling The metabolic effects of FGF21 observed in animal models prompted efforts to characterize its receptor complex and downstream signaling pathways. It is now clear that FGF21 requires the cofactor b-Klotho (KLB) for signaling both in vitro [9,10] and in vivo [11,12]. The C-terminus of FGF21 binds with high affinity to KLB [13], which is present in a complex with FGF receptors (FGFRs) on the surface of cells [10] (Figure 1). Binding of FGF21 to KLB enables its interaction with the FGFR, thus activating receptor autophosphorylation and signaling. Although FGF21 can activate multiple FGFR isoforms in vitro, it is believed that FGFR1c is the primary receptor mediating activity in vivo [14–16]. Binding of FGF21 to the FGFR1–KLB complex induces rapid phosphorylation of the FGF receptor substrate 2 (FRS2), the kinases ERK1/2 (extracellular signal-regulated kinase 1 and 2), GSK3 (glycogen synthase kinase 3), AKT (protein kinase B), p70S6K, and Raf, the phosphatase SHP2 (src-homology domain-2-containing phosphatase 2), and the transcription factor STAT3 (signal transducer and activator of transcription 3), as well as a rapid increase in intracellular calcium. [1,10,17]. These events induce transcription of early response genes, including c-fos and EGR1 (early growth response 1) [18,19], and numerous gene expression changes in adipocytes [20–22].

Corresponding authors: Gimeno, R.E. ([email protected]); Moller, D.E. ([email protected]). 1043-2760/$ – see front matter ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tem.2014.03.001

Tissues and pathways mediating the metabolic effects of FGF21 administration Gene expression in mice The metabolic effects of exogenously administered FGF21 and its potential target tissues are summarized in Trends in Endocrinology and Metabolism xx (2014) 1–9

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Trends in Endocrinology and Metabolism xxx xxxx, Vol. xxx, No. x

FGF21

C N

β-Klotho

FGFR1c

TRENDS in Endocrinology & Metabolism

Figure 1. Components of the fibroblast growth factor 21 (FGF21) signaling complex. Hypothetical structural representation of extracellular domains of b-Klotho (green), FGF21 (orange), and the extracellular domains of the FGF receptor FGFR1c (blue). FGF21 binds with high affinity (15 nM) to the extracellular domain of b-Klotho, a single membrane-spanning protein related to b-glucosidase [10,80]. The extracellular domain of b-Klotho interacts with the FGFR1c extracellular domain to form a receptor complex on the surface of cells [10,81]. Binding to b-Klotho allows FGF21, which by itself has very low affinity for the FGFR, to interact with the D2–D3 domain of FGFR1c to induce receptor dimerization, autophosphorylation, and downstream signaling events through its intracellular tyrosine kinase domain [13]. The C-terminal domain of FGF21 is required for the interaction with b-Klotho, whereas the N-terminal domain is necessary for FGFR1 activation [80]. Structural modeling was performed using the crystal structures of b-glucosidase (PDB ID: 3OJV), FGFR structures (PDB ID: 2CR3 and 2JFE), and FGF19 (PDB ID: 2PWA) as templates. Membrane-spanning domains are represented at the bottom; the intracellular tyrosine kinase domain of FGFR1c is not depicted.

Figure 2. initial clues to target tissues and more specific in vivo mechanisms were evident from gene expression changes following FGF21 administration. Within 2 h of FGF21 injection, the glucose transporter GLUT1 is upregulated in white adipose tissue [1,20], and several target genes in the liver are induced within 1–4 h [18,23]. Three days of FGF21 treatment induces a prominent thermogenic gene expression signature in white and brown adipose tissue, and this is accompanied by histological changes consistent with browning of white adipose tissue [21,24]. Upon chronic administration, FGF21 causes patterns of gene expression changes consistent with decreased de novo lipogenesis and altered cholesterol and bile acid metabolism in the liver, increased lipogenesis and lipolysis in white adipose tissue, as well as increased thermogenesis in brown and white adipose tissue [2,22,24]. Liver and adipose leptin receptor transcripts are upregulated by exogenous FGF21 [2], consistent with a possible leptinsensitizing action of FGF21 [25]. In vivo role of KLB and adipose tissue in the pharmacologic actions of FGF21 Although FGFRs are widely expressed, the FGF21 cofactor, KLB, is present in only a small number of rodent tissues, notably adipose tissue, liver and pancreas, but also in the hypothalamus and the brainstem [11,12,18,26]. The key role 2

of KLB was confirmed by showing that the metabolic actions of exogenous FGF21 are completely abolished in mice with a whole-body KLB deletion [11,12]. Tissue-specific knockout studies of FGF21 receptor components identified adipose as a key target tissue for the metabolic actions of FGF21: the insulin-sensitizing effects of chronic FGF21 overexpression were abrogated in mice lacking KLB in adipose tissue [12]. Similarly, deletion of FGFR1 in adipose tissue abolished the insulin-sensitizing effects of acute or chronic FGF21 administration, and eliminated the majority of the weight loss and circulating or hepatic lipid changes [19,27]. This genetic model was also resistant to the majority of FGF21-induced gene expression changes in liver [19]; thus, the profound effects of FGF21 on hepatic metabolism are likely secondary to its action in adipocytes. Interestingly, FGF21 induces thermogenic gene expression, oxygen consumption, and heat production in human adipocytes in vitro [28], suggesting that the potent effects of FGF21 on body weight in vivo may be due to its direct action on adipocytes as a ‘browning factor’. Adiponectin as a downstream effector of FGF21 Adiponectin is an adipocyte-derived factor that improves insulin sensitivity in liver and muscle [29]; it has recently been shown to be an important mediator of FGF21 action [30,31]. Adiponectin is rapidly released from adipocytes


Review


Glucose

Liver

Insulin

Free fay acids Growth hormone response Lepn sensivity

TG/Cholesterol

Pancreas Body weight Energy expenditure

FGFR1

C

Adiponecn

β-Cell protecon Insulin synthesis

WAT/BAT

N

FGF21

Bone Chondrocytes Growth hormone response

KLB

SCN/DVC

Female ferlity

SCN/DVC Corsol Altered hepac gene expression

CNS

icv Food intake Energy expenditure Hepac glucose producon TRENDS in Endocrinology & Metabolism

Figure 2. Metabolic effects of exogenously administered fibroblast growth factor 21 (FGF21). FGF21 acts directly on adipose tissue to reduce glucose and increase hepatic, and possibly peripheral, insulin sensitivity, decrease body weight, and decrease serum lipids [19]. The insulin-sensitizing effects of FGF21 require adiponectin, whereas the bodyweight-decreasing effects are likely mediated by induction of thermogenesis in brown adipose tissue (BAT) and ‘browned’ white adipose tissue (WAT) [24,30,31]. FGF21 also has direct effects on pancreatic islets [35] and growth plate chondrocytes [48]. Administration of FGF21 directly into the brain has been shown to affect hepatic glucose production, but not body weight, due to a counterbalance of effects on food intake and body weight [38]. Peripherally injected FGF21 can also increase serum cortisol levels and decrease female fertility; both of these effects are thought to be mediated by FGF21 acting on neurons in the hypothalamus (suprachiasmatic nucleus, SCN) or brainstem (dorsal vagal complex, DVC) [26,42]. Abbreviations: FGFR1, FGF receptor 1; icv, intracerebroventricular; KLB, b-Klotho; P, phosphorylation; TG, triglycerides.

in response to FGF21, and is elevated by FGF21 administration in mice and monkeys [1,8,30,31]. Mice lacking adiponectin are resistant to the glucose-lowering and insulin-sensitizing effects of exogenous FGF21, while retaining the majority of its weight-reducing effects, and possibly some of its lipid-lowering effects [30,31]. Thus, at least some of the pleiotropic actions of FGF21 in vivo require adiponectin. Interplay between peroxisome proliferator receptor g (PPARg) and FGF21 FGF21 interacts with the PPARg pathway in a complex manner. PPARg agonists, such as rosiglitazone, potentiate the effects of FGF21 in adipocytes in vitro, likely by inducing the expression of KLB [17]. Rosiglitazone can also upregulate FGF21 expression in adipose tissue, resulting in increased local, and in some studies, systemic FGF21 levels [32,33]. Conversely, FGF21 induces PPARg expression in adipocytes in vitro [17] and preadipocytes from FGF21 knockout mice show a defective response to rosiglitazone [33]. In one recent study, rosiglitazone failed to lower glucose in FGF21 null mice, whereas its lipid- and insulin-lowering effects were largely maintained [33]; FGF21 knockout mice also avoided the increase in body weight and fluid mass observed upon rosiglitazone treatment. However, a second study using an independent line of FGF21 knockout mice failed to reproduce these findings [34]. Reasons for these discrepant results require further investigation.

FGF21 action in the pancreas FGF21 is most highly expressed in the pancreas [34], but its pharmacological actions in this tissue are not well characterized. In vitro, using rat islets or b cell lines, FGF21 induces FGFR signaling and insulin gene expression, and protects b cells from stress-induced apoptosis [35]. Although FGF21 does not augment insulin secretion from normal islets, it can potentiate glucose-stimulated insulin secretion in islets from diabetic mice, suggesting that FGF21-treatment might alleviate b cell dysfunction [35]. In vivo, FGF21 exerts b cell protective effects in db/db mice [35–37]; it is unclear whether this is secondary to improved insulin sensitivity or represents a direct effect. Because FGF21 decreases glucagon levels in both rodents and monkeys [1,8], it is tempting to speculate that FGF21 may have effects on a cells as well. To date, no studies have been conducted in human islets. FGF21 action in the brain Some of the functions of FGF21 may be mediated through its direct actions on the brain. Intracerebroventricular infusion of FGF21 in rats increases energy expenditure, food intake, and insulin sensitivity [38]. FGF21 is also detectable in the cerebrospinal fluid in man [39], and peripherally administered FGF21 can enter the brain via passive diffusion, although at a low rate [40]. Although no neuronal sites of expression of FGF21 have been identified in vivo, FGF21 can be highly induced in rat neuronal cultures [41]. Recent studies suggest that FGF21-signaling 3


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in the hypothalamus and/or brainstem is required for several non-metabolic effects of overexpressed FGF21, including female infertility, short growth, altered daily wheel-running behavior, and an altered plasma corticosterone/adrenocorticotropic hormone (ACTH) serum ratio [26,42]. By contrast, insulin-sensitizing effects of exogenous FGF21 were preserved when KLB was selectively deleted in neurons [12]. Physiological functions of endogenous FGF21 Pharmacological or overexpression studies achieve circulating levels of FGF21 which significantly exceed endogenous levels. A crucial issue to be addressed concerns the physiologic role of endogenous FGF21. FGF21 is primarily expressed in pancreas, liver and adipose tissue [34]; it is still unclear to what extent these tissues contribute to the circulating levels of FGF21. Another important question is the relative importance of FGF21 as an autocrine/paracrine versus endocrine factor. Different strains of FGF21 knockout mice show variable and subtle effects on body weight, fat mass, glucose, and lipids [23,33,34,43,44], suggesting that FGF21 does not play a major role in metabolic regulation under normal physiologic conditions. Similarly, FGF21 null mice show normal growth and development, and normal fertility [43,44]. FGF21 as a starvation hormone Early studies in mice showed that hepatic FGF21 is strongly induced following prolonged fasting or by a ketogenic diet; this induction is mediated by PPARa, a nuclear hormone receptor that coordinates transcriptional changes leading to increased lipid oxidation [45,46]. FGF21 transgenic mice show phenotypes reminiscent of chronic starvation, including reduced linear growth [47], female infertility [42], and induction of torpor upon prolonged fasting [45]. The growth retardation of FGF21 transgenic mice has been linked to inhibition of growth hormone (GH) signaling [47,48]; similar to GH-resistant mice [49], FGF21 transgenic mice also have an increased lifespan [50]. In mice, hepatic FGF21 is required for the metabolic adaptation to the ketogenic diet [43], or prolonged starvation [47], consistent with a role for FGF21 as a starvation hormone. FGF21 and stress responses More recently, a broader view of FGF21 as a stress-induced factor has emerged [51]. In rodents [24,33,45,46,52,53] as

well as humans (see below), FGF21 expression and serum levels are increased by a variety of metabolic stressors – including cold, exercise, nutrient deprivation, and nutrient overload. Endoplasmic reticulum stress secondary to abnormalities in lipid metabolism [54,55] or mitochondrial dysfunction results in profound increases in serum FGF21 levels [54] and, in some cases, this increase in FGF21 represents a beneficial, adaptive metabolic response [43,54]. Interestingly, FGF21 has also been shown to be upregulated in response to pancreatic injury and, when overexpressed, it can ameliorate the severity of cerulenininduced pancreatitis in mice [56]. Are there connections to human disease? As described above, the discovery and characterization of FGF21 as a potential metabolic regulator was based largely on studies conducted in preclinical model systems. To date, no clear genetic associations between known components of the FGF21 pathway and inherited human disorders (including polygenic forms of T2D) have been reported. Although the potential role(s) of FGF21 biology in human disease are not well characterized, some important clues – based largely on measurements of circulating FGF21 levels – are emerging (Table 1). Plasma levels of FGF21 are detectable in humans using several assay techniques [57,58]. Importantly, the very broad range of fasting levels (0.05–5.5 ng/ml) reported in healthy subjects appears to indicate a high degree of interindividual variation. Surprisingly, average FGF21 concentrations are also moderately (20–50%) higher in patients with obesity or T2D, compared with lean healthy subjects [59–62]. Moreover, higher FGF21 concentrations appear to be an independent predictor of T2D and metabolic syndrome [61,62]. By contrast, patients with T1D reportedly have lower plasma FGF21 levels [60]. A striking increase in circulating FGF21 levels as well as hepatic expression of FGF21 mRNA has also been reported in patients with non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH) [59]. In addition to the disease associations noted above, circulating FGF21 levels are reportedly altered in response to several other perturbations (Table 1). Unlike rodents where short-term fasting results in substantial increases in plasma levels, circulating FGF21 is either not increased or is only moderately elevated during more prolonged fasting in humans [57,63,64]. Because PPARa activation

Table 1. Regulation of FGF21 levels in humans Context Healthy human subjects Obesity and T2D T1D NAFLD, NASH Fasting and PPARa activation Circadian regulation Overfeeding and hyperinsulinemia Fatty acids 4

Major finding(s) Broad range of fasting plasma concentrations (0.05–5.5 ng/ml) Moderately increased concentrations; higher levels predict diabetes and metabolic syndrome Lower average concentrations Markedly elevated plasma concentrations and liver mRNA (in patients with NAFLD > NASH) Modest increase in levels after prolonged fasting or during treatment with fenofibrate or GW590735 (selective PPARa agonist) Diurnal regulation during fasting (peak levels at night) Modest (and transient) increase with high-fat overfeeding. Increased FGF21 mRNA in skeletal muscle after acute hyperinsulinemia. Slightly increased levels during lipid infusion (healthy subjects).

Refs [57] [59,61,62] [60] [59] [57,64] [63] [65,67] [66]


Review


was implicated as a fasting-related mechanism for the regulation of FGF21 in rodents, this hypothesis has also been addressed in humans; indeed, modest increases have been observed during treatment with two distinct PPARa agonists [57,64]. Perhaps paradoxically, FGF21 levels are also (at least transiently) increased during overfeeding [65]. Small changes in FGF21 levels and gene expression have also been observed in response to lipid and insulin infusions (Table 1) [66,67]. FGF21-based therapeutics – potential approaches Significant effort is being expended to identify therapeutic agents that mimic the potential beneficial effects of native FGF21, while addressing its shortcomings, namely poor biophysical properties and a short half-life in vivo (Table 2). An analog, LY2405319, was designed to achieve improved biophysical properties along with biological effects that are indistinguishable from native FGF21 in vitro. LY2405319 shows glucose-, bodyweight-, and lipid-lowering activity in diabetic rodent or monkey models similar to native FGF21 [68,69]. This molecule was suitable for larger-scale production, allowing its effects in man to be investigated (see below).

Several groups have focused on the development of other, long-acting FGF21 analogs. These efforts include Fc–FGF21 fusion proteins [5], FGF21 analogs covalently coupled to an antibody [37], pegylated FGF21 molecules [36,70], and FGF21 fused to an albumin-binding domain of fibronectin [71]. Although all of these molecules show extended time-action in at least some animal models, only one, Fc–FGF21, has been optimized to prevent aggregation and proteolytic degradation of the FGF21 portion. Pharmacokinetic studies performed with the other analogs did not evaluate whether the FGF21 portion of the molecules remained intact and active. It is likely that several molecules have been examined in clinical studies (Table 2); however, results have only been reported with LY2405319 to date. Alternative approaches to MN note: directly activate the FGF21 receptor complex may also provide a means to mimic FGF21 pharmacology. The large receptor–ligand interaction interface makes the development of small-molecule agonists of the FGF21 receptor complex very challenging; however, discovery of agonist antibodies that act by dimerizing the receptor complex has proved feasible. Antibodies that target the FGFR1 receptor mimic many of

Table 2. FGF21 analogs with potential therapeutic utility Name

Company

Description

Features

FGF21

Not applicable

Native FGF21

LY2405319

Lilly

FGF21 DHPIP, L118C, A134C, S167A

Fc-FGF21

Amgen

Fc (IgG1)-FGF21 (L98R,P171G)

PEG-FGF21

Amgen

PEG20 mono- and dual-modified FGF21 analogs

ARX-618 related molecules

Ambrx/Merck

PEG30-FGF21 (Q108pAcF)

ARX-618 FGF21-PKE Adnectin

Ambrx/BMS a BMS

Not disclosed FGF21-adnectin fusion protein

CVX-343

Pfizer

mimAb1

Amgen

FGF21 (DH1, A129C) covalently linked to an IgG1k antibody (CVX200) Fully human antibody with high affinity for KLB

C3201-HSA

Amgen

HSA-coupled avimer combining subunits with high affinity for FGFR1c and KLB

R1Mab

Genzyme

Fully human antibody with high affinity for FGFR1c/b

Glucose-, weight-, and lipid-lowering in mouse and monkey models of diabetes and obesity. T1/2 = 0.5–2 h Aggregation-resistant FGF21 analog with improved physical stability. In vitro and in vivo properties similar to native FGF21 in mice and monkeys. T1/2 = 1.5–3 h Aggregation-and degradation-resistant FGF21 analog with long duration of action. In vitro and in vivo properties similar to native FGF21 in mice and monkeys. T1/ 2 = 12–30 h Pegylated FGF21 analogs with in vitro and in vivo properties similar to native FGF21 in mice. Predicted to be long-acting FGF21 analog with long time of action. In vitro and in vivo properties similar to native FGF21 in mice. T1/2 (rats) = 22 h Not disclosed FGF21 analog with glucose-lowering similar to native FGF21 in mice and long timeaction. T1/2 (monkeys) = 96 h FGF21 analog with glucose-lowering similar to native FGF21 in mice and long timeaction. T1/2 (mice, rats, monkeys) = 28–65 h FGF21 mimetic which specifically activates the FGFR1c/KLB complex. Efficacy similar to Fc-FGF21 in monkeys, but extended timeaction. T1/2 (monkeys) = 11 days FGF21 mimetic which specifically activates the FGFR1c/KLB complex. Efficacy similar to Fc-FGF21 in monkeys, but shorter timeaction due to antibody formation. T1/2 (monkeys) = 50 h FGF21 mimetic which activates FGFR1c/b in the absence of KLB. Activities consistent with FGF21 and FGF23 in mice and monkeys

Development status Not suitable for clinical development Phase I, discontinued

Refs [1,6,8,58,68]

[68,69]

Not disclosed

[5,6,58]

Not disclosed

[70]

Alliance discontinued

[36,82]

Phase I Dropped from pipeline

[71]

Not disclosed

[37]

Not disclosed

[27]

Not disclosed

[73]

Not disclosed

[72]

a

Ambrx pipeline: http://ambrx.com/pipeline; BMS pipeline (as of June 30, 2013): http://www.bms.com/research/pipeline

5


Review the effects of FGF21 in rodents [72]. Because these antibodies do not depend on KLB for signaling, inappropriate activation of FGFR1 has been observed [72], representing a significant safety concern. Antibodies targeting KLB itself provide a more specific way to activate the FGF21 receptor complex. mimAb1 is a monoclonal antibody that recognizes primate KLB with high affinity, promotes binding of KLB to FGFR1c, and activates signaling specifically through the FGFR1c/KLB complex [27]. This antibody has effects similar to FGF21 in obese cynomolgus monkeys and achieves longer duration of action than Fc-FGF21 [27]. Specific activation of the KLB/FGFR1c complex has also been achieved by engineering novel proteins linking sequences that bind with high affinity to FGFR1c and KLB (avimers). When linked to a moiety that provides half-life extension, one such avimer, C3201– HSA (C3201 linked to human serum albumin), showed in vitro and in vivo activity similar to FGF21 [73]. However, immunogenicity of C3201HSA limited its duration of action in monkeys, with loss of the pharmacodynamic response after 2 weeks of treatment [73]. It may also be possible to mimic FGF21 action by increasing endogenous FGF21 levels either through increasing synthesis and secretion or by blocking degradation. Our incomplete understanding of which tissues and factors contribute to circulating FGF21 levels, the role of circulating versus local FGF21 in different tissues, as well as the proteins involved in FGF21 degradation, make the search for therapeutics that act as FGF21 secretagogues challenging. Nevertheless, it has recently been reported that oxyntomodulin exerts some of its bodyweight-reducing effects in mice by activating FGF21 transcription and secretion in the liver through a glucagon receptor-dependent pathway [74]; interestingly, increases in serum FGF21 levels in response to glucagon have also been demonstrated in man [74,75]. Because FGF21 is upregulated under conditions that induce ER stress and mitochondrial dysfunction, any molecules that increase FGF21 serum levels will need to be carefully evaluated for negative side effects. Initial human proof-of-concept The pharmacologic effects of exogenous FGF21 in humans have only recently begun to be explored. We recently tested the clinical effects of LY2405319, an analog of human FGF21. Initial Phase I studies indicated that subcutaneous injections of LY2405319 were well tolerated across a broad dose range (0.6–30 mg) and produced systemic drug levels that were in the range achieved in preclinical animal models [68]. A subsequent 28 day Phase IB study was conducted in obese subjects with T2D [76]. Forty-six subjects were randomized and received either daily placebo injections or one of three dose levels (3, 10, 20 mg daily) of LY2405319. Eight subjects discontinued therapy – three because of adverse effects (one was a case of systemic hypersensitivity that was attributed to the study drug). The effects of LY2405319 on lipids and lipoproteins were substantial; these included reductions in total and LDLcholesterol (up to –29%), rapid and robust decreases in mean fasting triglycerides (up to –46%), and increases in HDL-cholesterol (up to 20%). In addition, modest but 6


significant (vs baseline) decreases in body weight were observed. Although mean fasting insulin levels were suppressed from baseline, suggesting an insulin-sensitizing effect of LY2405319, fasting glucose levels were not significantly affected (a dose-dependent trend towards glucose lowering was apparent). Mechanistic insights were evident from the analysis of several other measured parameters. These included suppression of circulating apolipoprotein III levels – a protein known to attenuate triglyceride clearance [77]; increased total as well as high molecular weight adiponectin was also evident, suggesting a role for adiponectin in insulin sensitization, as also seen in animal studies reviewed above. A prominent increase in b-hydroxybutyrate levels was also measured; higher levels of this ketone are consistent with an increase in systemic fat oxidation and may also reflect mechanism(s) for triglyceride lowering and weight loss via augmented energy expenditure. Overall, the effects of LY2405319 in humans were similar to the pharmacologic effects of FGF21 and FGF21 mimetic molecules in rodent and non-human primate studies. However, the absence of clinically meaningful effects on glucose levels was unexpected. What are the potential explanations? Although the intrinsic potency of LY2405319 is similar to that of native FGF21, it may not be potent enough to exert strong effects on hyperglycemia in patients with established diabetes. Because significant effects on insulinemia were only apparent at the highest dose, it is also possible that greater degrees of insulin sensitization may require higher doses and/or more potent analogs. This clinical study was also only 28 days in duration, and it is plausible that longer-term therapy (e.g., 3–6 months) with FGF21-derived molecules will be needed to ameliorate hyperglycemia in humans with longstanding diabetes. Heterogeneity in response to this mechanism of action is also a likely factor. Finally, it is worth noting that the half life of LY2405319 in humans is relatively short, thus daily dosing may not have produced full 24 h coverage, which might have been required to achieve glycemic efficacy. Endogenous FGF21 levels were not measured in this clinical study; we recommend that future clinical studies incorporate this measurement because it might represent underlying differences in responsiveness to modulating the FGF21 pathway. On balance, clinical results obtained with LY2405319 are encouraging because meaningful effects on several metabolic parameters that represent comorbid conditions associated with T2D were demonstrated in a small number of subjects treated for a short period of time. To date there are no other reports describing therapeutic effects of an FGF21 analog or mimetic in humans. However, close examination of the patent literature and ongoing clinical trials (http://www.clintrials.gov) in patients with diabetes, obesity and dyslipidemia strongly suggests that more human clinical results with FGF21-derived therapies will be forthcoming soon. Potential concerns Safety is paramount in the development of new agents for metabolic disorders. Although our clinical study with LY2405319 did not identify obvious safety concerns (apart


Review from immune-mediated effects of the molecule), ongoing investigation of the biological functions of FGF21 in rodents has identified areas of concern that need to be taken into consideration. Mitogenicity represents a theoretical concern. However, unlike canonical FGFs, FGF21 does not induce cell proliferation in vitro [1], and mice chronically overexpressing FGF21 do not show any evidence of increased cell proliferation or tumor formation [1,50]. As noted above, FGF21 can inhibit GH signaling in the liver, leading to increased serum insulin-like growth factor I (IGF1) and GH levels [47]. More recently, FGF21 was also shown to antagonize the effects of GH on chondrocytes [48], raising the possibility that administration of FGF21 could interfere with skeletal growth or healing. Although 2 week treatment with recombinant FGF21 does not appear sufficient to induce systemic GH resistance in mice [2], the effects of long-term treatment with FGF21 in higher species remain unknown. Additional safety concerns were raised by recent studies showing decreased bone mineral density [78], increased plasma corticosterone levels [26], and decreased female fertility [42] in mice administered native FGF21 by peripheral infusion for 1–2 weeks. The effects of FGF21 on bone are reminiscent of the effects observed with PPARg agonists [78], and have been suggested to reflect FGF21 potential actions in mediating downstream effects of PPARg [42]. Given the extensive preclinical and clinical experience with thiazolidinedione drugs which are PPARg agonists, it should be feasible to assess this risk in further preclinical and clinical studies. Interestingly, bone mineral density is positively correlated with serum FGF21 levels in man [79]. Increased corticosterone levels and decreased fertility are thought to be manifestations of the starvation response; given the attenuated nature of this response in humans (see above), these effects might not be manifested in clinical use; however, it will be important to further evaluate this risk as well. Despite the concerns noted above and any new concerns that may emerge, it is reassuring that mice chronically overexpressing FGF21 do not exhibit any obvious pathologies as they age, and in fact have an increase in lifespan [50]. Concluding remarks and future perspectives Despite recent advances in healthcare delivery for patients with metabolic disease, many patients remain at risk of developing debilitating cardiovascular (CV) and microvascular complications. A compelling need for better therapies is evident, and disease-modifying approaches that could directly address aspects of underlying pathophysiology are particularly desirable. These might include new agents with insulin-sensitizing activity along with mechanisms that can potentiate lipid oxidation and alleviate tissue ‘lipotoxicity’ as well as increase energy expenditure (e.g., via brown fat activation). Accumulating evidence – reviewed here – points to FGF21 as a major regulator of metabolism and as a pharmacologic approach with the potential to spawn new therapeutic agents. Preclinical data from animal models and emerging clinical efficacy results suggest that FGF21 or FGF21 mimetic molecules have marked insulin-sensitizing actions, can produce potentially beneficial effects on


surrogate lipid markers of cardiovascular risk and can reduce excess fat mass via the induction of peripheral energy expenditure. Despite these apparently attractive features, there are many unanswered questions and challenges which lie ahead. The ability to discover and develop optimized analogs has been a major challenge owing to poor stability, selfaggregation, immunogenicity, and low yields in recombinant protein expression systems that are evident with native FGF21. Longer-acting molecules are also difficult to engineer despite being desirable from patient convenience and therapeutic adherence perspectives. Alternative approaches are being actively pursued; however, it is still ‘early days’. Further efforts to discover small molecule (orally bioavailable) approaches to enhance FGF21 production or mimic/augment its actions are clearly warranted. With regard to potential benefits, it is uncertain if monotherapy with FGF21-derived therapeutics will be sufficient to produce clinically meaningful improvements in hyperglycemia or more robust and durable weight-loss effects in typical obese patients with T2D. Thus, studies employing other (and potentially more potent) molecules, larger numbers of patients, and longer treatment periods are obviously required. In addition, important mechanistic questions remain to be addressed in translational research studies. For example, effects on metabolic rate versus food intake and appetite, as well as the potential effects of FGF21-based therapies in the context of food-restricted diets, should be examined. In addition, it will be important to determine if activation of brown adipose tissue could be detected in humans – and whether this might form the basis of identifying patients who are more likely to experience substantial weight loss. Translational clinical studies which employ glucose-clamp methods will also be necessary to assess the relative effects of FGF21 on peripheral insulin sensitivity, hepatic glucose metabolism, and pancreatic b cell function. The effects of LY2405319 on circulating lipids and apolipoproteins in human subjects are encouraging. However, many crucial questions remain. First and foremost is the issue of aggregate CV risk. Will FGF21-based therapy prevent recurrent CV events in patients with preexisting disease and on a background of therapy with statins? To date there are no clear data from animal models which suggest beneficial (or adverse) effects on atherosclerosis; thus more preclinical, as well as clinical, studies are needed. Short of longer-term CV outcome studies, assessments of HDL function, vascular reactivity, and vascular imaging in humans may be worth considering. As noted above, there are several emerging safety concerns that will need to be addressed carefully before longterm clinical studies employing FGF21-derived molecules are undertaken. The potential adverse effects of FGF21 on bone turnover are chief among these. To date, adverse effects on bone have only been reported in rodents. Therefore, long-term toxicology studies which employ both rodent and non-rodent species will be needed, together with an assessment of bone biomarkers and bone mineral density in future clinical trials. Given the broad range of endogenous FGF21 levels reported in humans, and the potential for FGF21 ‘resistance’ 7


Review in a proportion of patients with metabolic disease who have higher circulating levels, it will be crucial to determine parameters that can be used to guide dose selection and to predict greater versus lesser responsiveness to metabolic actions. FGF21-derived therapies might ultimately be more appropriate for discrete metabolic conditions rather than for broader use in patients with obesity and T2D. Among these, we suggest that patients with the following disorders be considered for future study: familial hypercholesterolemia, extreme forms of hypertriglyceridemia, NAFLD and NASH, and morbid obesity. Finally, we believe that utility of FGF21 as a therapeutic approach to metabolic conditions may well require optimizing its use with agents acting via other – complementary – mechanisms. There is the potential for true synergy if the right combination of therapies is used – in the right patients, at the right doses, and at the right time. Acknowledgments We would like to thank Craig Dickinson for modeling the FGF21 signaling complex and Andrew Adams and Alexei Kharitonenkov for critical reading of this manuscript.

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9

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