Recent studies in preclinical species have shown that FGF21, an atypical endocrine hormone–like member of the FGF superfamily, can address multiple pathophysiologic elements of T2D. These include mitigation of hyperglycemia and insulin resistance, the preservation of insulin production and pancreatic β-cell function, amelioration of dyslipidemia and altered lipid storage, and improvement of intermediary metabolism substrate fluxes (1
). To our knowledge, no adverse effects of FGF21, including hypoglycemia at high doses, have yet been described in the literature. Thus, FGF21 has emerged as a promising candidate for the treatment of T2D.
Unfortunately, the short duration of circulation and action of native FGF21 undermines its therapeutic utility. Thus, the search for a means of prolonging the pharmacokinetics of FGF21 while maintaining its intrinsic activity led to the utilization of a novel, selective PEGylation strategy. The importance of such selectivity has been amply demonstrated, because conventional PEGylation of therapeutic proteins typically leads to the generation of heterogeneous mixtures, or less than optimal site selection (6
). As a result, the specific activity is often reduced relative to the native protein, thereby necessitating higher clinical doses that lead to increased concerns about increased immunogenicity, renal vacuolation, and treatment cost.
Homology modeling allowed directed selection of pAcF site incorporation predicted to be distant from the receptor binding regions of human FGF21. Functional characterization of the PEGylated variants using cell-based assays measuring induction of ERK phosphorylation and glucose uptake validated the structural model we had produced, demonstrating that mono-PEGylation of FGF21 with a linear PEG30 polymer at putative receptor binding sites caused large losses in protein potency, whereas PEGylation at sites distal to those involved in FGFR interactions maintained high potency. These findings indicated that site matters in prolonging time of action while maintaining specific activity. Recode methodology enables selection based on biological properties as opposed to conventional approaches rooted in the native placement of lysine. In addition, we showed that the same PEG30 polymer differentially improved the pharmacokinetic character of FGF21, when conjugated to different sites, perhaps due to differential absorption and proteolytic lability. These data collectively illustrated the pharmacological power in being unconstrained in site selection through biosynthesis with unique chemical functionality.
With no detectable weight change upon exposure to WT FGF21 or its PEGylated variants, db/db mice served as a good model to assess the direct metabolic actions of this therapy in a stringent rodent model of T2D. We found that BIW administration of the five most potent PEGylated FGF21 variants led to impressive glucose lowering with no sign of hypoglycemia during the 12-day study period. The rank ordering of their glucose-lowering potencies was similar to that observed in the pharmacokinetic study, with the best overall performance demonstrated by variants Q108 and R131. Such data provide strong evidence that FGF21 has direct antihyperglycemic activity and that site-specific mono-PEGylation of FGF21, as described in this work, provided molecular entities with superior pharmacological properties.
Accompanying the antihyperglycemic efficacy, treatment with PEGylated FGF21 analogs preserved pancreatic islet morphology, size, and number and improved glucose-dependent insulin secretion. These results are important because loss of islet function and mass are hallmarks of T2D (13
), and the ability to mitigate endocrine pancreas deterioration promises durable antidiabetic therapy.
PEGylated FGF21 variants enhanced insulin sensitivity, as was detectable by diminished fasting insulin and improved insulin tolerance as early as 12 h after a single administration and long before significant glucose-lowering efficacy was observed. Additionally, increased insulin sensitivity was manifested by potentiation of insulin-induced Akt phosphorylation in major metabolic tissues, including liver, skeletal muscle, and adipose, which suggests that these tissues mediate the observed FGF21-induced improvements in whole-body insulin sensitivity and glucose disposal.
Acute administration of PEG30-FGF21 resulted in a rapid diminution in DNL. Though modest in scale, this change probably contributed to the reduction in hepatic steatosis seen after chronic treatment with PEGylated FGF21, which in turn would fortify its aforementioned insulin-sensitizing activity. The inhibition of hepatic DNL by PEG30-FGF21 might also mediate the observed improvement in dyslipidemia.
Our work demonstrated that FGF21 can modify nutrient utilization based on the availability of fuel substrate. In particular, when provided to hyperglycemic db
mice, PEG30-FGF21 durably increased whole-body carbohydrate oxidative catabolism as indicated by an elevation in RER. Therefore, we proffer that PEGylated FGF21 enhances metabolic flexibility, i.e., the switch to insulin-stimulated glucose oxidation during periods of elevated circulating glucose, which has previously been shown to be impaired in T2D (14
). Alleviating such a metabolic derangement could aid in the mitigation of insulin resistance.
In summary, our findings demonstrate that site-specific PEGylation of FGF21 results in the generation of potent, long-acting molecules with highly desirable antidiabetic therapeutic profiles. Such uniquely modified proteins hold significant promise in addressing the complex syndrome of T2D.