Application of FGF4 and Fgfr1 binding ligands in diabetes

Abstract

Provided are fibroblast growth factor 4 (FGF4) and Fgfr1 binding ligands for treating metabolic diseases, a treatment method using the fibroblast growth factor 4 (FGF4) and Fgfr1 binding ligands, and use of the fibroblast growth factor 4 (FGF4) and Fgfr1 binding ligands in preparing medicines.

Description

Technical Field

[0001] The present invention relates to FGF4 and Fgfr1 binding ligands for treating metabolic diseases, a treatment method using the FGF4 and Fgfr1 binding ligands, and use of the FGF4 and Fgfr1 binding ligands in preparing medicines. Background Art

[0002] The fibroblast growth factor (FGF) family consists of 22 structurally related polypeptides that act in an autocrine, paracrine, or endocrine manner to mediate many key events in mammalian development, tissue homeostasis / repair, and body metabolism (Beenken and Mohammadi, 2009; Itoh and Ornitz, 2011; Ornitz, 2005). Over the past few decades, a large amount of evidence has shown that the endocrine members FGF19 and FGF21, as well as the paracrine member FGF1, have great potential for treating obesity and type 2 diabetes (T2D) (Degirolamo et al., 2016; Fu et al., 2004; Huang et al., 2017; Jonker et al., 2012; Kharinenkov et al., 2005; Lan et al., 2017; Suh et al., 2014). Notably, a single dose of FGF1 administered to the central nervous system (CNS) induces sustained diabetic remission in T2D rodents, in contrast to the transient effects seen following central administration of endocrine FGFs (Morton et al., 2013; Perry et al., 2015; Sarruf et al., 2010; Scarlett et al., 2016). However, the mechanism by which FGF1 induces this sustained effect remains unclear. FGF1 administration is associated with a transient reduction in food intake, stimulation of hepatic glucose uptake, and preservation of pancreatic β cells (Scarlett et al., 2019; Tennant et al., 2019). Studies have also suggested that monocytes, glia, and neuronal nets (PNNs) assembly in the arcuate nucleus and adjacent median eminence (ARC-ME) may play an important role in the sustained glucose-lowering effects of FGF1 (Alonge et al., 2020; Bentsen et al., 2020; Brown et al., 2019; Scarlett et al., 2016). For example, central administration of FGF1 rapidly induces activation of MAPK-ERK signaling in monocytes and leads to increased coverage of dendritic spines by processes from perisynaptic astrocytes in ARC neurons of ob / ob mice (Bentsen et al., 2020; Brown et al., 2019). Although not essential for initial anorexia and glucose reduction, melanocortin signaling and PNNs within the ARC are partially required for sustained effects (Alonge et al., 2020; Bentsen et al., 2020). However, the key receptors and cellular mechanisms that mediate this sustained effect remain unclear. Similarly, it is not known whether this amazing anti-diabetic activity is unique to FGF1 in the FGF family. These gaps in knowledge hinder the development of more effective FGF-based approaches to treat T2D.

[0003] FGF mediates a variety of biological functions by binding, dimerizing, and thereby activating seven subtypes of homologous FGF receptors (FGFRs), each of which has a unique spatiotemporal expression profile (Fon Tacer et al., 2010; Goetz and Mohammadi, 2013; Kurosu et al., 2007). It has recently been discovered that different FGFs can modulate the strength of FGFR dimerization to perform corresponding cellular functions in metabolic regulation or cell proliferation (Huang et al., 2017). Therefore, it is speculated that there may be other FGFs with similar or more significant anti-diabetic potential as FGF1.

[0004] Fgfr1 is abundantly expressed in white adipose tissue and the central nervous system (CNS) (Fon Tacer et al., 2010). Previous studies have shown that Fgfr1 is a key receptor target mediating FGF metabolic regulation in white adipose tissue (Adams et al., 2012; Ye et al., 2016). Fgfr1 in the CNS is also considered to be a receptor target mediating FGF19 and FGF21 metabolic regulation (Lan et al., 2017). However, whether Fgfr1 mediates the sustained hypoglycemic effect of FGF1 in the CNS remains unclear. Glucose sensing neurons (GSN) include glucose-excited neurons (GE) and glucose-inhibitory neurons (GI). GSNs have been found to sense changes in extracellular glucose levels and provide feedback to regulate blood glucose (Anand et al., 1964; Marty et al., 2007; Oomura et al., 1969; Steinbusch et al., 2015). Therefore, based on the important roles of Fgfr1 and GSN in metabolic regulation, we hypothesized that FGF may exert its sustained anti-hyperglycemic effect by acting on GSN and activating Fgfr1 on related cells.

[0005] In this study, multiple representative members of the FGF paracrine family were screened to test their anti-hyperglycemic effects in the db / db model of type 2 diabetes mice, and it was found that central administration of FGF4 could sustainably alleviate hyperglycemia. It is worth noting that FGF4 has significantly better anti-diabetic efficacy than FGF1 in diet-induced obese mice. Mechanistic studies have shown that FGF4 needs to mediate its effect of reducing glucose levels in T2D rodent models through Fgfr1 and GSN in the hypothalamus. Acute perfusion of FGF4 inhibits GI neurons, while long-term administration of FGF4 re-sensitizes GI neurons and GE neurons and resets the ratio of GI neurons to GE neurons. It is worth noting that non-invasive nasal administration of FGF4 can induce sustained diabetic remission. Therefore, the data indicate that FGF4 is a new biologic with high translational potential and can achieve durable remission of diabetes. Summary of the invention

[0006] The present invention relates to Fgfr1 ligands (such as FGF4) for treating metabolic diseases, treatment methods using Fgfr1 ligands, and uses of Fgfr1 ligands in preparing drugs.

[0007] In a first aspect, the present invention provides a method for treating a metabolic disease, comprising the step of administering a therapeutically effective amount of a Fgfr1 ligand to a patient in need thereof.

[0008] In a second aspect, the present invention provides a Fgfr1 ligand for treating metabolic diseases.

[0009] In a third aspect, the present invention provides a use of an Fgfr1 ligand in the preparation of a medicament for treating metabolic diseases.

[0010] Preferably, the Fgfr1 ligand is FGF4.

[0011] Preferably, the metabolic disease is selected from obesity, diabetes, insulin resistance, hyperinsulinemia, glucose intolerance and hyperglycemia. In an embodiment, the diabetes is type 1 diabetes or type 2 diabetes, in particular type 2 diabetes.

[0012] The Fgfr1 ligand is isolated. The isolated Fgfr1 ligand can be included in a pharmaceutical composition. In an embodiment, FGF4 is the only active ingredient in the pharmaceutical composition.

[0013] Preferably, the Fgfr1 ligand or the pharmaceutical composition comprising the Fgfr1 ligand is administered intracerebrally or intranasally. Therefore, the pharmaceutical composition is a pharmaceutical composition suitable for intracerebral or intranasal administration, and the drug of the third aspect is a drug suitable for intracerebral or intranasal administration.

[0014] Preferably, the intracerebral administration is chronic intracerebral administration.

[0015] The pharmaceutical composition may further comprise a pharmaceutically acceptable dispensing agent, such as a carrier, an adjuvant, a solubilizer, a stabilizer and / or an antioxidant. In an embodiment, the pharmaceutical composition further comprises a liposome suitable for intranasal administration.

[0016] In an embodiment, the frequency of intracerebral administration is less than once every 2 weeks, preferably less than once every 5 weeks, such as once every 6 to 16 weeks.

[0017] In another embodiment, the frequency of intranasal administration is less than once every 2 days, preferably less than once every 5 days, such as once every 6 to 9 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1It was shown that a single injection of FGF4 into the CNS alleviated hyperglycemia in diabetic mice.

[0019] (A) Representative FGFs from each subfamily.

[0020] (BC) Changes in blood glucose (B) and body weight (C) after a single icv administration of paracrine FGF (3.0 μg each) to db / db mice.

[0021] (DE) Changes in blood glucose (left) and area under the curve (AUC) (right) in db / db mice subjected to ipGTT 7 days (D) or 50 days (E) after icv FGF1 or FGF4 (3.0 μg), as indicated.

[0022] (F) Intravenous injection 18 Representative micro-PET / CT images (left) and quantitative analysis (right) were performed 30 min after F-FDG in db / db mice that had received icv FGF1 or FGF4 (3.0 μg) for the previous 3 days.

[0023] (G) Expression of mRNA encoding glucose-regulating enzymes in the liver of db / db mice 7 days after icv FGF4 (3.0 μg).

[0024] (H) Glycogen staining in liver and skeletal muscle of db / db mice 7 days after icv FGF4 (3.0 μg) using periodic acid-Schiff (PAS). Scale bar, 100 μm.

[0025] (I) Changes in blood glucose after a single icv injection of FGF4 (3.0 μg) in DIO mice.

[0026] (J) Changes in blood glucose (left) and AUC (right) in DIO mice subjected to ipGTT 7 days after icv FGF4 (3.0 μg).

[0027] (K) Changes in body weight after a single icv FGF4 (3.0 μg) in DIO mice.

[0028] (LM) Changes in body activity (L) and thermogenesis (M) in DIO mice 7 days after icv FGF4 (3.0 μg).

[0029] Data are shown as mean ± sem, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 compared with the corresponding control group; #p ≤ 0.05; ###p ≤ 0.001 compared with the FGF1 group, ns, not significant.

[0030] Figure 2It was shown that Fgfr1 in hypothalamic neurons is required for the anti-hyperglycemic effect of FGF4.

[0031] (AC) Fgfr1 expression in the hypothalamus by mRNA in situ hybridization. Scale bars, 1 mm (A) and 500 μm (B, C).

[0032] (D) For the treatment of Fgfr1 flox / flox Protocol for generating hypothalamic Fgfr1 knockout in mice.

[0033] (E) Hypothalamic expression of AAV8-CMV-CreGFP. Scale bar, 200 μm.

[0034] (F)Fgfr1 CMV-Cre The blood glucose of mice increased significantly. Virus was injected at t=0.

[0035] (G) In Fgfr1 CMV-Cre Blockade of FGF4 has anti-hyperglycemic effects in mice.

[0036] (H) Designed to express AAV8-hSyn-Cre into Fgfr1 flox / flox To knock out Fgfr1 in hypothalamic neurons in mice.

[0037] (IJ) Fgfr1 expression under different dietary conditions (normal chow or HFD) hSyn-Cre Changes in blood glucose (I) and body weight (J) of mice and GFP controls.

[0038] (KN)Fgfr1 hSyn-Cre Changes in blood glucose (K, L) and body weight (M, N) after icv FGF4 (3.0 μg) in GFP mice (K, M) and GFP control (L, N) mice.

[0039] (OP) Changes in blood glucose (O) and body weight (P) after injection of FGF4 (1.5 μg) into VMH.

[0040] (Q) Changes in blood glucose (left) and AUC (right) in DIO mice subjected to ipGTT 24 h after injection of FGF4 (1.5 μg) into the VMH.

[0041] VMH, ventral hypothalamus; DMH, dorsal hypothalamus; LH, lateral hypothalamus; ARC, arcuate nucleus of the hypothalamus; 3V, third ventricle. Data are shown as mean ± sem, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 compared with the corresponding control group, ns, not significant.

[0042] Figure 3It was shown that FGF4 restored the function of VMH GSN in diabetic mice.

[0043] (AB) Representative firing rate changes associated with VMH GE neurons (A) and GI neurons (B) from wt, db / db, and db+FGF4 mice in response to low glucose (0.1 mM). db+FGF4 represents db / db mice injected with FGF4 (3.0 μg, 7 to 10 days post icv).

[0044] (C) Relative percentages of GSN cell types in wt, db / db, and db+FGF4 mice. INS, glucose insensitive.

[0045] (D-F) Changes in mean firing rate (D), peak firing rate (E), and peak time (F) of GE neurons in response to 0.1 mM glucose.

[0046] (GI) Mean firing rate change (G), peak firing rate change (H), and peak time (I) of GI neurons in response to 0.1 mM glucose.

[0047] (J) Experimental design to test the acute effects of FGF4 on neurons of db / db mice.

[0048] (K) Representative traces showing inhibition of GI neurons by FGF4 infusion.

[0049] (L) Mean firing rate changes associated with GI neurons and their percentage perfused by FGF4.

[0050] (M) Experimental design to test the effect of blocking Fgfr1 by PD166866.

[0051] (N) Representative traces showing that D166866 inhibits FGF4-repressed GI neurons.

[0052] (O) Mean firing rate changes and percentages in GI neurons treated with FGF4 plus PD166866.

[0053] (P) Central administration of GI inhibitors lowers blood glucose in db / db mice.

[0054] (Q) Experimental design to test the acute effects of FGF4 on neurons in DIO mice.

[0055] (R) Representative traces showing that FGF4 infusion inhibits GI neurons.

[0056] (S) Changes in mean firing rate of GI neurons and their percentage perfused by FGF4.

[0057] (T) Experimental design to test the effects of PD166866 on GI neurons stimulated or inhibited by FGF4.

[0058] (U) Fgfr1 inhibitor partially abolished the effect of FGF4 on GI neurons.

[0059] (V) Mean firing rate changes and percentages in FGF4-inhibited GI neurons treated with PD166866.

[0060] Data are shown as mean ± sem, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 compared with the corresponding groups.

[0061] Figure 4 Nose-to-brain delivery of FGF4 was shown to alleviate hyperglycemia.

[0062] (A) Representative images of liposome nanoparticles loaded with gelatin solution (control) and FGF4-gelatin solution (FGF4). Scale bar, 100 nm.

[0063] (B) Distribution of liposome nanoparticle diameters.

[0064] (CD) Changes in blood glucose (C) and body weight (D) in db / db mice after intranasal delivery of FGF4 (30 μg).

[0065] (EF) Changes in blood glucose (left) and AUC (right) in db / db mice subjected to ipGTT (E) or ipITT (F) 24 h after intranasal FGF4 delivery.

[0066] (G) Blood glucose in db / db mice after repeated intranasal delivery of FGF4.

[0067] Data are shown as mean ± sem, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 compared with the corresponding control group.

[0068] Figure 5 Shown with Figure 1 Related, phenotypes in wt and db / db mice following FGF injection.

[0069] (A) There was no significant change in blood glucose after a single icv administration of endocrine FGF21 (3.0 μg) in db / db mice.

[0070] (BC) Changes in blood glucose (B) and body weight (C) in response to increasing doses of FGF4.

[0071] (D) Changes in blood glucose (left) and AUC (right) in db / db mice subjected to ipGTT 7 days after the indicated doses of icv FGF4 (3.0 μg).

[0072] (E) Glucose levels in db / db mice did not decrease after ip injection of FGF4 (3.0 μg).

[0073] (F) Transient decrease in blood glucose after ip injection of FGF4 (30 μg).

[0074] (G) Changes in blood glucose after icv FGF4 (3.0 μg) in wt mice.

[0075] (H) Changes in blood glucose (left) and area under the curve (AUC) (right) in wt mice subjected to ipGTT 7 days after icv FGF4 (3.0 μg).

[0076] (I) Changes in body weight after a single icv administration of FGF4 (3.0 μg) to wt mice.

[0077] (JO) Changes in plasma hormones (J), food intake (K), body fat content (L), physical activity level (M), respiratory exchange ratio (RER, N), and heat production (O) in db / db mice on the indicated days after icv FGF4 (3.0 μg) injection. 12-light-dark photoperiod is indicated.

[0078] (P) Changes in blood glucose (left) and AUC (right) in db / db mice subjected to ipITT 10 days after icv FGF4 (3.0 μg).

[0079] (Q) Representative micro-PET / CT images and intravenous injection 18 Quantitative analysis at 15 min (left) and 60 min (right) after F-FDG was performed in db / db mice that had received icv FGF1 or FGF4 (3.0 μg) 3 days previously.

[0080] Data are shown as mean ± sem, compared with the corresponding control group, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; compared with the FGF4 (0.3 μg) group, ###p ≤ 0.001, ns, not significant.

[0081] Figure 6 Shown with Figure 1 Related, phenotypes in DIO mice after FGF injection.

[0082] (AB) Changes in blood glucose (A) and body weight (B) after a single injection of FGF1 (3.0 μg) in DIO mice.

[0083] (C) Changes in blood glucose (left) and AUC (right) in DIO mice subjected to ipITT 7 days after icv FGF4 (3.0 μg).

[0084] (DF) Changes in body fat content (D), food intake (E), and RER (F) after a single icv FGF4 (3.0 μg) in DIO mice. Days after injection and 12-h light-dark cycle are indicated.

[0085] (G) Transient reduction in blood glucose after ip injection of 30 μg FGF4 in DIO mice.

[0086] Data are shown as mean ± sem, *p ≤ 0.05; ***p ≤ 0.001 compared with the corresponding control group.

[0087] Figure 7 Shown with Figure 2 Related, hypothalamic Fgfr1 is required for FGF4-mediated glucose-lowering effects.

[0088] (A) Fgfr1 is expressed in the hypothalamus. Scale bars, 1 mm (left); 100 μm (right).

[0089] (B) Fgfr1 + The proportion of cells is expressed as the number of Fgfr1+ cells divided by all cells from wt mice (Dapi+).

[0090] (C) Relative expression of Fgfr1 in different hypothalamic regions normalized to VMH (n=8 slides from 5 mice).

[0091] (D) For the treatment of Fgfr1 flox / flox Protocol for generating hypothalamic Fgfr1 knockout in mice.

[0092] (E) AAV8-CMV-CreGFP expression in different brain regions, brain region B, hypothalamus.

[0093] (F)GFP + Cells and all cells from the indicated hypothalamic nuclei (Dapi + ) ratio.

[0094] (G) Reduction of Fgfr1 mRNA after CMV-Cre expression.

[0095] (H) Body weight changes of CMV-GFP and CMV-Cre mice.

[0096] (I) Changes in blood glucose (left) and AUC (right) in CMV-Cre mice subjected to ipGTT 6 weeks after virus injection.

[0097] (J) Changes in body weight of CMV-cre mice after icv FGF4 (3.0 μg).

[0098] (KL) Changes in blood glucose (K) and body weight (L) in CMV-GFP mice after icv FGF4 (3.0 μg).

[0099] Data are shown as mean ± sem, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 compared with the corresponding control group.

[0100] Figure 8 Shown in Lepr + Deletion of Fgfr1 in either cell line or astrocytes did not abrogate the hypoglycemic effect of FGF4.

[0101] (AB) Changes in body weight (A) and blood glucose (B) in Lepr:CKO mice.

[0102] (CD) Changes in blood glucose (left) and AUC (right) in Lepr:CKO mice (14 weeks old) subjected to ipGTT (C) or ipITT (D).

[0103] (E) Body weight changes of Lepr:CKO mice under HFD.

[0104] (F) Blood glucose of Lepr:CKO mice at week 12 on HFD.

[0105] (GH) Changes in blood glucose (G) and body weight (H) in Lepr:CKO mice after icv FGF4 (3.0 μg).

[0106] (IJ) Body weight changes of Gfap:CKO mice on a chow diet (I) or HFD (J).

[0107] (K) Blood glucose of Gfap:CKO mice at week 12 on HFD.

[0108] (LM) Changes in blood glucose (L) and body weight (M) in Gfap:CKO mice after icv FGF4 (3.0 μg).

[0109] Data are shown as mean ± sem, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns, not significant compared with the corresponding control group.

[0110] Fig. 9 Shown with Figure 2 Related, delayed phenotypes of Fgfr1 in hypothalamic neurons.

[0111] (A) By injecting AAV8-hSyn-CreGFP into Fgfr1 flox / flox We engineered mice to knock out Fgfr1 in hypothalamic neurons.

[0112] (BC) Changes in body weight (B) and blood glucose (C) in syn-GFP and syn-cre mice.

[0113] (D) AAV8-hSyn-CreGFP expression in different brain regions, brain region B, hypothalamus.

[0114] (E) Expression of Fgfr1 in Syn-GFP and Syn-Cre mice.

[0115] (F) Decreased Fgfr1 expression level after Syn-Cre expression.

[0116] Data are shown as mean ± sem, **p ≤ 0.01; ***p ≤ 0.001 compared with the corresponding control group.

[0117] Fig.10 Shown with Figure 3 Related, the effects of FGF4 on VMH GE neurons from db / db mice.

[0118] (A) Experimental design used to test the acute effects of FGF4 on neurons in db / db mice.

[0119] (BC) Representative traces showing excitation (B) or inhibition (C) of GE neurons perfused with FGF4.

[0120] (D) Changes in mean firing rate associated with VMH GE neurons and their percentage perfused by FGF4.

[0121] (E) Schematic diagram showing the experimental design to test the effect of the Fgfr1 inhibitor PD166866.

[0122] (FG) Changes in mean firing rate in FGF4-excited (F) or FGF4-inhibited (G) GE neurons treated with PD166866.

[0123] ACSF, artificial cerebrospinal fluid. Data are shown as mean ± sem.

[0124] Fig.11 Shown with Figure 3 Related, effects of FGF4 on VMH GE neurons from DIO mice.

[0125] (A) Experimental design to test the response of GSN to high (5.0 mM) and low (0.1 mM) glucose.

[0126] (BD) Mean firing rate changes associated with GE (B) neurons, GI (C) neurons, and INS (D) neurons treated with 5.0 mM glucose and 0.1 mM glucose.

[0127] (E) Experimental design used to test the acute effects of FGF4 on brain slices.

[0128] (F) Representative traces showing excitation, inhibition, and insensitivity of GE neurons perfused with FGF4.

[0129] (G) Changes in mean firing rate associated with GE neurons and their percentage perfused by FGF4.

[0130] (H) Experimental design used to test the effect of the Fgfr1 inhibitor PD166866 on GE neurons.

[0131] (I) PD166866 partially blocked FGF4-excited and -inhibited GE neurons.

[0132] (J) Mean firing rate changes and percentages of FGF4-elicited GE neurons treated with PD166866.

[0133] Data are shown as mean ± s.e.m. DETAILED DESCRIPTION

[0134] As used herein, the terms "a" or "an" mean one or more, unless expressly stated otherwise.

[0135] The term "isolated" molecule refers to a molecule that is substantially free of any other contaminating molecules or other contaminants found in its natural environment and that would interfere with its use in production or its therapeutic, diagnostic, preventive or research use. Thus, typically the isolated polypeptide of the present invention is a purified polypeptide and pathogen-free and pyrogen-free, having at least 80% purity, preferably at least 90% purity, more preferably at least 95% purity, and in particular pharmaceutical purity, i.e. at least 98% purity. Preferably, the isolated polypeptide of the present invention may be substantially free of other polypeptides, in particular polypeptides from animals.

[0136] The term "FGF4" - an abbreviation for fibroblast growth factor 4 - means full-length FGF4 or an active fragment thereof, such as a fragment from Ala67 to Leu206. Preferably, FGF4 is human FGF4. Human FGF4 is a 176 AA long protein derived by cleavage of the N-terminal 30 AA of the precursor protein. FGF-4 contains a single N-linked glycosylation site. Unglycosylated FGF-4 is a polypeptide truncated with two NH2-terminal residues, which has better heparin affinity and biological activity than the wild-type protein.

[0137] Recombinant FGF4 protein is commercially available (eg, from Sigma Aldrich, where the recombinant FGF4 protein is produced in baculovirus, or from Invitrogen, where the recombinant FGF4 protein is produced in E. coli).

[0138] As used herein, the term "pharmaceutically acceptable carrier" refers to one or more formulation materials suitable for achieving or enhancing the delivery of FGF4.

[0139] The term "therapeutically effective amount" refers to the amount of FGF4 used to support observable levels of one or more biological activities, such as the ability to reduce blood sugar, insulin, triglycerides or cholesterol levels; reduce body weight; or improve glucose tolerance, energy expenditure or insulin sensitivity. In the present invention, "patient" is preferably a human, but can also be an animal, preferably a mammal such as a dog, pig, cow, horse, rat, mouse, guinea pig, etc.

[0140] With respect to human subjects, "obesity" may be defined as a body weight that is more than 20% above the ideal body weight for a given population (RH Williams. Textbook of Endocrinology, 1974, pp. 904-906).

[0141] "Diabetes" is characterized by excessive urination and constant thirst and includes two types. Type II diabetes is characterized by excessive glucose production despite the availability of insulin and excessive body glucose levels due to insufficient glucose clearance. In this embodiment, the diabetes can be type 1 diabetes, and it is believed that the effect of FGF4 on the treatment of type 2 diabetes is much stronger than that of type 1 diabetes. The inventors have found that FGF4 is more beneficial for the treatment of type 2 diabetes.

[0142] The term "insulin resistance" refers to a condition in which normal amounts of insulin produce a subnormal biological response.

[0143] The term "hyperinsulinemia" is defined as higher than normal levels of insulin in the blood.

[0144] The term "glucose intolerance" as used herein is defined as an abnormal sensitivity to glucose.

[0145] The term "hyperglycemia" is defined as high levels of sugar (glucose) in the blood.

[0146] The pharmaceutical composition may contain one or more pharmaceutically acceptable agents for modifying, maintaining or preserving, for example, the pH, osmotic rate, viscosity, clarity, color, isotope, odor, sterility, stability, dissolution or release rate, adsorption or penetration of the composition. Suitable agents include, but are not limited to, amino acids (such as glycine, glutamine, aspartic acid, arginine or lysine), antimicrobial agents, antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite), buffers (such as borates, bicarbonates, Tris-HCl, citric acid, phosphates or other organic acids), auxiliary materials (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinyl pyrrolidone, β-cyclodextrin, or hydroxypropyl β-cyclodextrin), fillers, monosaccharides, disaccharides and other carbohydrates (such as glucose, mannose or dextrin), proteins (such as serum albumin, gelatin or immunoglobulins), colorants, flavorings and diluents, emulsifiers, hydrophilic polymers (such as polyvinyl pyrrolidone), low molecular weight polypeptides, salt-forming ions (such as sodium), preservatives (such as benzyl chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methyl terephthalic acid, propyl terephthalic acid, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerol, propylene glycol or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronic; PEG; sorbitan esters; polysorbates such as polysorbate 20 or polysorbate 80; triton; trimethylamine; lecithin; cholesterol or tyloxapol), stability enhancers (such as sucrose or sorbitol), sensitizers (such as alkali metal halides - preferably sodium chloride or potassium chloride or mannitol sorbitol), delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants (Remington's Pharmaceutical Sciences, A.R. Gennaro, ed., Mack Publishing Company).

[0147] The main vehicle or carrier in the pharmaceutical composition can be aqueous or non-aqueous. For example, a suitable vehicle or carrier for injection can be water or physiological saline solution.

[0148] The pharmaceutical composition of the present invention can be administered by any route known to those skilled in the art, such as oral administration, rectal administration, sublingual administration, intrapulmonary administration, peritoneal administration, transdermal administration, iontophoresis administration, vaginal administration, intracerebral administration, and intranasal administration. However, intraperitoneal administration of a large dose of FGF4 only results in a temporary reduction in blood glucose levels in db / db mice. Preferably, according to the mechanism discovered by the inventors, the pharmaceutical composition of the present invention is administered by intracerebral administration or intranasal administration.

[0149] The pharmaceutical composition for intranasal administration in the present invention may be in the form of liposomes. In an embodiment, FGF4 is loaded into 100 nm liposomes.

[0150] A single intracerebral or intranasal administration of FGF4 may result in a sustained chronic hypoglycemic effect. The inventors have found that a single intracerebral administration of FGF4 may even produce a hypoglycemic effect that lasts for more than 4 months. At least the frequency of intracerebral administration may be less than once every 2 weeks, preferably less than once every 5 weeks, for example, once every 6 weeks to 16 weeks. In addition, the frequency of intranasal administration may be less than once every 2 days, preferably less than once every 5 days, for example, once every 6 days to 9 days.

[0151] The dosage will vary according to the formulation, the time course required, and the patient to be treated, and the physician can determine the feasible dosage in the treatment based on the actual situation (e.g., the patient's condition, body weight, etc.). For a general adult, the dosage of the pharmaceutical composition of the present invention can be 1ng-10mg of FGF4 or its variant for every kg of adult body weight. For the injection route, the dosage is preferably 10ng-1mg per kg of body weight, more preferably 100ng-100ug per kg of body weight, and further more preferably 1ug-10ug per kg of body weight, for example 2ug, 5ug, 7ug or 8ug.

[0152] Publications cited in this application are used to illustrate the present invention and are incorporated herein by reference as if they were written herein.

[0153] In order to better understand the present invention, the present invention will be described in more detail with reference to specific examples now.It should be pointed out that these examples only illustrate the present invention, and should not be interpreted as limiting the scope of the present invention.According to the description of the application, various modifications and variations of the present invention are obvious to those skilled in the art.

[0154] Example

[0155] The following examples are illustrations of specific embodiments of the present invention and various uses thereof. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way. The materials used in the examples are commercially available.

[0156] Example 1. Materials and methods

[0157] Experimental model and subject details

[0158] Mouse Model

[0159] Adult male db / db(B6.BKS(D)-Lepr db / J, 8-12 weeks old) were purchased from Jicui Pharmaceutical Co., Ltd. (Nanjing, China). C57BL / 6J (wt) mice were from Shanghai Jihui Experimental Animal Breeding Co., Ltd. flox / flox Mice (accession number 007671) were obtained from Jackson Laboratory. flox / flox Mice and Lepr Cre / + Mouse (#008320, Jackson Laboratory) and Gfap Cre / + Mice (gift from JW Zhou) were crossed to generate Fgfr1 conditional knockout mice (Lepr:CKO, Lepr stands for leptin receptor; GFAP:CKO, GFAP stands for glial fibrillary acidic protein). Mice were housed in a 12-h light / 12-h dark cycle (light, 8PM-8AM) under constant temperature and humidity. Mice were randomly assigned to experimental groups and had ad libitum (ad libitum) access to water and standard laboratory chow or high-fat diet (HFD; D12492, High Fat Diet, USA) unless otherwise stated. All animal procedures were performed in strict accordance with the Animal Research: Reporting In Vivo Experiments guide. The care and use of animals were reviewed and approved by the ShanghaiTech University, Shanghai Model Animal Co., Ltd., Zhangjiang Laboratory National Protein Science Facility Animal Facility and government regulations.

[0160] Method details

[0161] Expression constructs and recombinant protein production

[0162] Human FGF1 (Met1-Asp155), FGF4 (Ala67-Leu206), FGF7 (Cys32-Thr194), FGF8a (Gln23-Arg204), FGF20 (Met1-Thr211) and FGF21 (His29-Ser209) were expressed and purified according to published protocols (Beenken et al., 2012; Bellosta et al., 2001; Kalininna et al., 2009; Kharitenkov et al., 2005; Olsen et al., 2006; Sher et al., 2003). Briefly, the cDNA fragment encoding FGF4 (Ala67-Leu206) was PCR amplified and subcloned into pET-15b at the NcoI site and Xhol site, respectively. In an incubation shake flask at 37°C and 200 rpm, E. coli BL-21 (DE3) cells transformed with the FGF4 construct were cultured in LB medium (2% glucose and 30 kg / mL kanamycin). At an optical density of 0.8-1.0 (measured at 600), cells were induced at 37°C for 4 hours with 1.0 mM isopropyl-L-thio-β-D-galactopyranoside (IPTG). Then, cells were collected and lysed in 25 mM Na / K phosphate buffer (pH 7.5) containing 300 mM NaCl using an Emulsiflex-C3 (Oat Protein Company, Datian, Ontario, Canada) high volume homogenizer. The lysate was clarified by centrifugation at 20,000 rpm for 30 minutes at 4°C, and the soluble recombinant protein was purified by heparin affinity column (Hitrap Heparin HP, GE Healthcare, Piscataway, NJ, column volume 5 ml), using NaCl (0M-2.0M) linear gradient elution in HEPES (25mM, pH7.5). The purity of the target protein determined by 12% SDS-PAGE analysis was then combined, concentrated and further purified using gel molecular sieve (SuperdexTM-75 GE Healthcare, Piscataway, NJ) in mobile phase buffer (1.0M NaCl, 25mM Tris-HCl, pH 8.0). The purity of the recombinant protein was estimated to be >98%. Other FGFs were prepared in a similar manner.

[0163] Stereotaxic brain injection

[0164] FGF (~3.0 μg, unless otherwise stated) or AAV virus (~200 nl) was delivered by drawing glass pipettes and pen-type oil-pressure syringes (MO-10, Tritech Research, the United States) with custom manipulators. Injection coordinates were calculated according to Paxinos & Franklin mouse brain coordinates (3rd edition) by using a small animal stereotaxic instrument (David Kopf Instrument, #PF-3983; RWD Life Sciences, #68030; Qianao Xingke Nanjing Biotechnology Co., Ltd., #SH01A) under isoflurane general anesthesia. Feedback heaters were used to keep mice warm during surgery. Before mice were transferred to a cage, mice were recovered in a warm support. Coordinates are as follows: 1) for intracerebroventricular (icv) administration, lateral ventricle (LV): -0.40 mm posterior to bregma; 1.10 mm lateral, 2.50 mm below skull surface; 2) for hypothalamic delivery, ventral hypothalamic nucleus (VMH): -1.65 mm posterior to bregma; 0.40 mm lateral, 5.70 mm below skull surface, anatomical structures are shown directly on the associated figures. For pharmacology studies, mice were cannulated (RWD Life Sciences) in the LV.

[0165] Physiological measurements

[0166] Glucose levels were measured by handheld blood glucose meter (Ako-Qiak Excellent Gold Glucose Meter, Roche, Switzerland). Unless otherwise stated, blood samples were collected from the tail vein of mice fed ad libitum. In food intake determination, mice were implanted with cannulas (RWD Life Sciences, 62002, 62102) in the lateral ventricle, and the cannulas were fixed to the skull with dental cement (C&B metabolic fast adhesive cement system). All intubated mice were caged separately for 7-10 days after surgery and recovered with ad libitum food and water. FGF4 (3.0 μg) or saline was delivered to mice by cannula, and food was weighed every day. Body composition was measured using quantitative magnetic resonance spectroscopy (minisec body composition analyzer LF50, Bruker, Karlsruhe, German). Heat production, physical activity and RER (respiratory exchange rate) were monitored with a comprehensive laboratory animal monitoring system (CLAMS, Columbus Instruments). For plasma analysis, samples were collected from mice fed ad libitum. Plasma insulin, corticosterone, ACTH, epinephrine, and nonesterified free fatty acids (NEFA) concentrations were determined by Elisa kits according to the manufacturer’s instructions (#E0448m, #E0540Ge, #E0836m, #E0858Ge from EIAab, Wuhan, China; #A042-2-1 from Jiancheng Bioengineering Institute, Nanjing, China).

[0167] Intraperitoneal glucose tolerance test (ipGTT) and intraperitoneal insulin tolerance test (ipITT)

[0168] ipGTTs were performed in mice after an intraperitoneal injection of 1.0 g / kg body weight of glucose (20% glucose) followed by an overnight fast of 16 hours. ipITTs were performed in mice fasted for 6 hours by injecting 0.75 units / kg body weight (for wt mice) or 1.0 units / kg (for db / db and HFD mice) of insulin (#40112ES25, Yeasen 2016, China). Blood samples were collected from the tail vein at the indicated times.

[0169] Micro-PET-CT imaging and analysis

[0170] MicroPET-CT analysis was performed in db / db mice 3 days after icv injection of FGF (~3.0 μg). 18 Overnight fasting mice of F-FDG (259.1 ± 17.76 μCi, iv) were briefly anesthetized with 1.5% isoflurane in oxygen at 2L / min, and scanned for 1 hour in Inveon preclinical multimodal PET-CT (Siemens, Munich, Germany). Use the three-dimensional ordered subset expectation maximum (3DOSEM) algorithm and then use the maximum a posteriori (MAP) to reconstruct the image. Under the guidance of CT images, 3D regions of interest (ROI) were drawn on brain, liver and muscle. Use Inveon research workplace (Siemens, Munich, Germany) to measure the radioactivity of corresponding tissues.

[0171] AAV vectors

[0172] AAV vectors carrying specific genes (titer > 10 12 ) were purchased from Taitool Bioscience (Shanghai, China), including AAV2 / 8-CMV_bGI-Cre-EGFP-pA, AAV2 / 8-CMV_bGI-EGFP-WPRE-pA, AAV2 / 8-hSyn-Cre-EGFP-WPRE-pA, and AAV2 / 8-hSyn-EGFP-WPRE-pA.

[0173] Reverse transcription and quantitative real-time PCR

[0174] Total RNA was extracted from the hypothalamus or liver using Trizol reagent (Invitrogen, USA). Reverse transcription PCR was performed using PrimeScript RT kit (RR047A, Takara, Japan), and SYBR PCR kit (RR820A, Takara, Japan) was used with ViiATM Real-time PCR was measured using the ELISA® 7 Real-Time PCR System (Applied Biosystems, USA) according to the manufacturer's instructions. -Actin was used as an internal control for real-time PCR amplification.

[0175] Immunohistochemistry and RNA in situ hybridization

[0176] Mice were anesthetized and perfused with PBS followed by 10% formalin. Brains were excised, fixed overnight and cut into ~50 μm sections. After collection and washing three times with PBST (PBS containing 0.3% Triton X-100), cryostat sections were incubated with blocking solution (PBS containing 2.5% goat serum and 0.3% Triton X-100), then treated with primary antibody (chicken anti-GFP, ab13970, Abcam, 1:1000) at 4°C overnight, and then samples were incubated with secondary antibody (goat anti-rabbit Alexa 488 secondary antibody, Invitrogen, A11039) for 2 hours at room temperature. Images were captured on a Zeiss LSM800 confocal microscope or an Olympus VS120 virtual microscope slide scanning system. The slides were scanned using the ELISA kit according to the manufacturer's instructions. Fgfr1 RNA in situ detection was performed using the 2.5HD kit (#322350, Advanced Cell Diagnostics). The Fgfr1 probe was designed by Advanced Cell Diagnostics, and the DapB probe was used as a negative control.

[0177] Electrophysiological recording slices

[0178] In brief, hypothalamic slices containing VMH region were prepared from adult male wt, db / db and db / db with icv FGF4. Before perfusion through the heart with ice-cold oxygenated (95% O2 and 5% CO2) high sucrose solution, mice were anesthetized with sodium pentobarbital (100mg / kg i.p.), and the high sucrose solution was composed of the following (in mM): 213 sucrose, 26 NaHCO3, 1.25 NaH2PO4, 2.0 Na2HPO4, 2.5 KCl and 2 MgSO4 (osmotic pressure concentration was adjusted to 300mOsm / kg, pH 7.4). The brain was removed rapidly and placed in ice-cold oxygenated high sucrose solution. Coronal hypothalamic slices (250 μm) were cut with a vibrating microtome (VT1200S, Leica Microsystems, Germany). Before being transferred to the recording chamber, the slices were incubated at 31°C in oxygenated artificial cerebrospinal fluid (ACSF in mM: 124NaCl, 5KCl, 1.24KH2PO4, 1.3MgSO4, 26NaHCO3, 2.4CaCl2 and 2.5glucose, pH 7.2-7.4, 300-310mOsm / kg) for >1 hour. ACSF was perfused at 1.2ml / min. Hypothalamic slices were observed with infrared differential interference contrast (ID-DIC) video microscopy using an upright microscope (AxioDetector, Zeiss, Germany) and an infrared camera (IR-1000, DAGE-MTI, USA).

[0179] The recording pipette was pulled out from the borosilicate glass capillary using a P-97 pipette puller (Sutter Instruments, USA). In order to record spontaneous discharges extracellularly in slices with current clamps, the pipette was filled with a solution (in mM: 130 potassium gluconate, 1 CaCl2, 10 EGTA, 10 HEPES, 2.0 Mg-ATP, 2.0 Na3-GTP) (pH 7.2-7.4, 295 mOsm / kg). Voltage signals were recorded with an EPC-10 amplifier and PatchMaster10 acquisition software (HEKA, Germany). These signals were digitized at 10 kHz and filtered with a low-pass filter at 2 kHz. In order to determine the glucose sensitivity of each recorded VMH neuron, the 2.5 mM glucose in the ACSF was replaced with 0.1 mM glucose, as shown in the figure. The spontaneous discharge activity of neurons was continuously recorded, and a holding current was not applied to neurons during changes in extracellular glucose. After identification, neurons were recorded during perfusion of ACSF containing 2.5mM glucose for 20 minutes. Then control ACSF was replaced with the experimental ACSF containing 9μg / ml FGF4 according to the experiment, and the experimental ACSF had or did not have 10μM FGFR1 selective inhibitor (PD166866) for about 5 minutes. Then experimental ACSF was replaced with control ACSF for 20 minutes to wash out the drug.

[0180] For GE / GI neurons, the peak of the firing rate change was defined as the minimum / maximum value of the firing rate in 0.1 mM glucose perfusion minus 100% (baseline, t0). The response curves of GE / GI neurons to 0.1 mM glucose were fitted by 2nd order smoothing. The time to peak of GE / GI neurons was defined as the duration from t0 (0.1 mM glucose perfusion) to the time when FR reached the minimum / maximum value. The criterion for excitation or inhibition was set as a change in firing rate of ∼30% (Toda et al., 2016).

[0181] Preparation, characterization and treatment of FGF4-loaded liposomes

[0182] FGF4 is dissolved in 20% poloxamer 188 covalently bound heparin copolymer solution, and further diluted to a final concentration of 1.68mg / ml with 2.0% gelatin solution. Then the mixture is ultrasonically (110w, 15°C, 15s) and lyophilized to obtain a powder containing FGF4. Afterwards, the powder is dispersed in the tert-butyl alcohol solution containing hydrogenated soybean phospholipids (HSPC) and cholesterol. The mixed solution is ultrasonically processed (90w, 25°C, 20s) and lyophilized again to obtain a powder containing FGF4-liposomes. Finally, FGF4-liposomes are reconstructed to form a liposome suspension in double distilled water. In these processes, the gelatin solution without FGF4 is used as a blank control.

[0183] The morphology of blank and FGF4-loaded liposomes was observed by transmission electron microscopy (TEM) (1230, Jeol Jem Co., Tokyo, Japan) using negative staining with 1% phosphotungstic acid. The zeta potential of blank and FGF4-liposomes was determined by dynamic light scattering using the Zeta Potential / Particle Sizer Nicomp as described previously. TM The 380 ZLS (PSS. Nicomp, Santa Barbara, CA, USA) (Zhao et al., 2016) was used for determination.

[0184] Then, db / db mice were intranasally administered with equal volumes of blank or FGF4-liposomes (30 μg FGF4) to achieve nose-to-brain delivery, respectively. Blood glucose was measured at the indicated times and ipGTTs / ipITT experiments were performed as described above.

[0185] Methods-only references

[0186] Beenken, A., Eliseenkova, A. V., Ibrahimi, O. A., Olsen, S. K., and Mohammadi, M. (2012). Plasticity of the interaction between the N-terminus of fibroblast growth factor 1 (FGF1) and FGF receptors underlies the multifunctionality of FGF1. J Biol Chem 287, 3067-3078.

[0187] Bellosta, P., Iwahori, A., Plotnikov, AN, Eliseenkova, AV, Basilico, C., and Mohammadi, M. (2001). Identification of heparin binding sites in receptors and fibroblast growth factor 4 based on a structural mutagenesis approach. Mol Cell Biol 21, 5946-5957.

[0188] Kalinina, J., Byron, S. A., Makarenkova, H. P., Olsen, S. K., Eliseenkova, A. V., Larochelle, W. J., Dhanabal, M., Blais, S., Ornitz, D. M., Day, L. A., et al. (2009). Homodimerization controls receptor binding and heparan sulfate-dependent diffusion in the extracellular matrix of fibroblast growth factor 9 subfamily members. Mol Cell Biol 29, 4663-4678.

[0189] Kharitonenkov, A., Shiyanova, T.L., Koester, A., Ford, A.M., Micanovic, R., Galbreath, E.J., Sandusky, G.E., Hammond, L.J., Moyers, J.S., Owens, R.A., et al. (2005). A novel metabolic regulator, FGF-21. J Clin Invest 115, 1627-1635.

[0190] Olsen, SK, Li, JY, Bromleigh, C., Eliseenkova, AV, Ibrahimi, OA, Lao, Z., Zhang, F., Linhardt, RJ, Joyner, AL, and Mohammadi, M. (2006). Structural basis for alternative splicing regulation of FGF8 organizing activity in the brain. Genes Dev 20, 185-198.

[0191] Sher, I., Yeh, BK, Mohammadi, M., Adir, N., and Ron, D. (2003). Structure-based mutational analysis in FGF7 identifies novel residues involved in specific interactions with FGFR2IIb. FEBS Lett 552, 150-154.

[0192] Toda, C., Kim, J. D., Impellizzeri, D., Cuzzocrea, S., Liu, Z. W., and Diano, S. (2016). UCP2 regulates mitochondrial fission and glucose responsiveness in the ventromedial nucleus. Cell 164, 872-883.

[0193] Zhao, Y. Z., Zhang, M., Tian, ​​X. Q., Zheng, L., and Lu, C. T. (2016). Early intervention of diabetic cardiomyopathy using basic fibroblast growth factor nanoliposomes combined with ultrasound. Int J Nanomedicine 11, 675-686.

[0194] Quantitative and statistical analysis

[0195] Statistics

[0196] Mice were randomly assigned to each group, and the age and weight of the mice were matched. Unless otherwise stated, animals were not excluded from the study. Unpaired Student's t test was used for two-group comparisons. RM one-way ANOVA was used for multiple group comparisons, and two-way ANOVA followed by Sidak's or Tukey's multiple comparisons was used for groups mixed by time factor design. Data were analyzed using Prism8.0 and expressed as mean ± sem.

[0197] Example 2. Results

[0198] A single injection of FGF4 into the CNS alleviates hyperglycemia

[0199] First, four human recombinant FGFs (rFGF4, rFGF7, rFGF8a, and rFGF20) were purified as representative members of their paracrine subfamily ( Figure 1 A). These rFGFs were then administered into the CNS of db / db mice and tested for their antihyperglycemic activity. Human recombinant FGF1 and FGF21 (rFGF1 and rFGF21) were used as controls. Notably, a single dose of rFGF4 (FGF4, 3.0 μg) administered into the lateral ventricle of db / db mice significantly reduced blood glucose for >7 weeks without affecting body weight, similar to the effect of FGF1 ( Figure 1 B and Figure 1 C). In contrast, rFGF7, rFGF8a, rFGF20, and rFGF21 lack this anti-hyperglycemic activity ( Figure 1 B. Figure 1 C and Figure 5 Interestingly, intracerebroventricular (icv) administration of FGF4 (hereafter referred to as icv FGF4) had a more pronounced effect than FGF1 on glucose tolerance (measured using an intraperitoneal glucose tolerance test, ipGTT) 7 days after injection ( Figure 1 D). 50 days after injection, this difference was no longer significant ( Figure 1 E). As expected, higher doses of FGF4 were more effective in lowering blood sugar and improving glucose tolerance, again without affecting body weight ( Figure 5 B to Figure 5 D). As little as 0.3 μg of FGF4 is sufficient to lower blood glucose for 4 days ( Figure 5 B). Similar to FGF1 (Scarlett et al., 2016), intraperitoneal (ip) administration of FGF4 (3.0 μg) did not affect blood glucose in db / db mice ( Figure 5 E), while the 30 μg dose induced a transient decrease ( Figure 5 F). Notably, central administration of FGF1 or FGF4 to wild-type (wt) mice did not affect blood glucose, body weight, or glucose tolerance ( Figure 5G to Figure 5 L and (Scarlett et al., 2016)), suggesting that the risk of these two FGFs inducing hypoglycemia is negligible.

[0200] Next, we documented the peripheral responses to central administration of FGF. We measured plasma hormones from db / db mice 7 days after icv FGF4 and found no significant changes in insulin or corticosterone concentrations, whereas adrenocorticotropic hormone (ACTH) levels increased significantly ( Figure 5 J). These results suggest that the glucose-lowering effects of FGF4 are unlikely to be due to increases in insulin or decreases in stress hormones associated with the hypothalamic-pituitary-adrenal (HPA) axis. In addition, food intake was transiently reduced after icv FGF4 ( Figure 5 K), which is similar to the effect after icv FGF1 (Scarlett et al., 2016). Meanwhile, body fat / lean mass, physical activity, and respiratory exchange ratio (RER) did not change 7 days after icv FGF4 ( Figure 5 to Figure 5 N). However, heat production increases ( Figure 5 O). In addition, insulin resistance (as measured by the intraperitoneal insulin tolerance test (IPITT)) was significantly improved 10 days after icv FGF4 ( Figure 5 P). To identify the target organs responsible for glucose disposal, micro PET / CT studies were performed on db / db mice 3 days after icv FGF4 or FGF1. Deep enrichment of radiolabeled fluorodeoxyglucose (18F-FDG) was observed in the liver, skeletal muscle, and brain of mice treated with FGF4 and FGF1 ( Figure 1 F and Figure 5 Q). Consistent with the results, hepatic glycolytic genes Pklr, Gck, and Gys2 were upregulated, while the gluconeogenic gene Pck1 was downregulated ( Figure 1 G). Liver and skeletal muscle glycogen also increased significantly ( Figure 1 H) Taken together, these data suggest that transient anorexia, elevated heat production, and increased glucose uptake and use are important peripheral responses to centrally administered FGF to lower blood glucose.

[0201] To test whether these FGFs could induce sustained diabetic remission in other diabetic models, a hyperglycemic mouse model of diet-induced obesity (DIO) was generated by feeding a high-fat diet (HFD). Significantly, central administration of FGF4 significantly reduced blood glucose, and the effect was maintained for >2 weeks ( Figure 1 I). In contrast, FGF1 did not alleviate hyperglycemia or affect body weight in DIO mice ( Figure 6 A and Figure 6B), which is consistent with previous reports (Scarlett et al., 2016). As expected, FGF4 significantly improved glucose tolerance and insulin sensitivity in DIO mice ( Figure 1 J and Figure 6 C). In addition, the glucose lowering effect in DIO mice was independent of body weight, fat / lean tissue content, food intake, physical activity, and RER ( Figure 1 K. Figure 1 L and Figure 6 D to Figure 6 F). For example, in db / db mice, heat production increases ( Figure 1 M), ip administration of 30 μg FGF4 only transiently reduced blood glucose in DIO mice ( Figure 6 G) Thus, unlike FGF1, central injection of FGF4 induces sustained remission of diabetic DIO mice.

[0202] Fgfr1 in hypothalamic neurons is required for the anti-hyperglycemic function of FGF4

[0203] To determine whether Fgfr1 in the CNS is a target of FGF, we first characterized the Fgfr1 expression pattern by mRNA in situ hybridization. + Relative to Dapi + Cellular quantification revealed that Fgfr1 is expressed in most cells throughout the hypothalamus, including the VMH, dorsomedial hypothalamus (DMH), and ARC ( Figure 2 A to Figure 2 C and Figure 7 A to Figure 7 C). The highest level of expression was observed in the VMH ( Figure 2 C and Figure 7 C) Then, by adding Fgfr1 flox / flox Fgfr1 was non-selectively knocked out in the hypothalamus of mice by injecting an adeno-associated virus expressing Cre under the constitutive CMV promoter (AAV8-CMV-CreGFP). Figure 2 D) The CMV promoter drives CreGFP expression in ∼33% of cells in the VMH, ∼24% of cells in the ARC, and ∼53% of cells in the DMH ( Figure 7 E and Figure 7 F, resulting in an overall decrease of Fgfr1 expression in the hypothalamus by ∼40% ( Figure 7 G). Notably, mice lacking Fgfr1 in the hypothalamus (Fgfr1 CMV-Cre ) showed hyperglycemia and impaired glucose tolerance 6 weeks after virus injection ( Figure 2 F and Figure 7 I). Interestingly, these mice began to gain weight 3 weeks after injection ( Figure 7 H), which means that Fgfr1 also has an important regulatory effect on body weight. In order to directly test the function of Fgfr1 in FGF4-mediated anti-hyperglycemia, FGF4 was injected into the Fgfr1 CMV-Cre mice. It was found that icv FGF4 did not alleviate hyperglycemia, which proves that the ability of FGF4 to lower glucose is blocked ( Figure 2 G), weight is not affected ( Figure 7 J) Thus, Fgfr1 is a key receptor in the hypothalamus for central administration of FGF4 to lower blood glucose.

[0204] To determine which hypothalamic cell type mediates the effects of FGF4 on blood glucose, the Lepr-Cre line was used to knock out Fgfr1 in cells expressing the leptin receptor (Lepr), as Lepr is known to regulate metabolism (Xu et al., 2018; Zhang et al., 1994). Surprisingly, conditional knockout of Fgfr1 specifically in these cells did not affect blood glucose or body weight in mice fed a standard chow or HFD ( Figure 8 A to Figure 8 F). This knockout also did not affect the ability of icv FGF4 to lower blood glucose in mice fed a HFD ( Figure 8 G and Figure 8 H). We then used the Gfap-Cre line to knock out Fgfr1 in astrocytes and found that it did not block the glucose-lowering effect of FGF4 ( Figure 8 I arrive Figure 8 M). Next, AAV8-hSyn-CreGFP was injected into Fgfr1 flox / flox In the mouse hypothalamus ( Fig. 9 A), used to selectively delete Fgfr1 in hypothalamic neurons (Fgfr1 hSyn-Cre Interestingly, Fgfr1 hSyn-Cre The mice showed a significant increase in body weight, but blood sugar levels were not affected ( Fig. 9 B and Fig. 9 C), indicating that the effects of Fgfr1 deficiency on weight gain and hyperglycemia are separable. The mice were then switched to a HFD and Fgfr1 hSyn-Cre Body weight and blood glucose in mice increased dramatically compared with GFP controls ( Figure 2 H to Figure 2 J) More importantly, icv FGF4 did not alleviate the expression of Fgfr1 hSyn-Cre Hyperglycemia in mice ( Figure 2 K), which is in sharp contrast to the GFP control ( Figure 2L). Body weight was not affected after icv FGF4 or GFP control in Fgfr1hSyn-cre mice ( Figure 2 M and Figure 2 N). Taken together, these results suggest that the glucose-lowering effects of FGF4 require Fgfr1 in hypothalamic neurons.

[0205] Fgfr1 showed the highest expression level in VMH ( Figure 2 C), which led to the hypothesis that the VMH is the primary target of FGF4. Therefore, FGF4 was injected directly into the VMH of DIO mice to test its effect. Indeed, half the icv dose (1.5 μg) was sufficient to alleviate hyperglycemia and improve glucose tolerance without affecting body weight ( Figure 2 O to Figure 2 Q), which suggests that VMH may be the primary target area for FGF4 to exert its glucose-lowering effect.

[0206] FGF4 restores the function of VMH GSN in diabetic mice

[0207] It is well known that the GSN in the VMH regulates blood glucose and may be important for the action of FGF4. To test the function of the GSN, the spontaneous firing rate (FR) was recorded using slice preparations from wt mice, db / db mice, and FGF4-treated db / db mice (7-10 days after icv FGF4). As expected, switching from normal glucose (2.5 mM) to low glucose (0.1 mM) inhibited GE neurons and excited GI neurons ( Figure 3 A and Figure 3 B). Then, the number of GE, GI, and glucose-insensitive (INS) neurons was analyzed and it was found that GE neurons were overrepresented in db / db, whereas GI neurons were underrepresented in db / db mice compared to wt mice ( Figure 3 C). Central administration of FGF4 in db / db mice restores the relative number of GI neurons to wt levels ( Figure 3 C). The peak response and time to peak of the GSN in response to low glucose were further analyzed. For GE neurons, the peak response to low glucose appeared to be smaller in db / db mice compared to wt controls, although the difference was not significant. After treatment of db / db with FGF4, the peak response was increased ( Figure 3 D and Figure 3 E). The time to peak was significantly prolonged in db / db mice compared with wt and was completely restored by FGF4 treatment ( Figure 3 F). In GI neurons, peak responses were indistinguishable between wt, db / db, and FGF4-treated db / db mice ( Figure 3 G and Figure 3 H). The time to peak was prolonged in db / db mice compared to wt and was also restored by FGF4 treatment ( Figure 3 I). Taken together, these results suggest that glucose sensitivity and the relative number of GSNs are altered in diabetic conditions and restored by FGF4 treatment.

[0208] Encouraged by these findings, we sought to determine whether GSNs are sensitive to FGF4 if FGF4 is applied directly ( Figure 3 J). Notably, the majority of VMH GI neurons recorded (7 / 9, 78%) were inhibited by FGF4 perfusion, whereas 11% were either excited or insensitive to FGF4 ( Figure 3 K and Figure 3 L). In contrast, FGF4 infusion stimulated or inhibited an equal number of GE neurons ( Fig.10 A to Fig.10 D). To test whether this acute effect is dependent on Fgfr1, PD166866, a selective Fgfr1 inhibitor (Panek et al., 1998), was added to block Fgfr1 during slice recordings ( Figure 3 M). Remarkably, the inhibitor abolished almost all FGF4-induced neural responses (13 / 14, 93%); Figure 3 N and Figure 3 O), indicating that FGF4 inhibition of GI neurons requires intact Fgfr1. In contrast, PD166866 had essentially no effect on FGF4-stimulated GE neurons and partially blocked FGF4-inhibited GE neurons ( Fig.10 E to Fig.10 G).

[0209] Given the ability of FGF4 to inhibit GI neurons, we tested whether central administration of GI inhibitors could mimic the effects of FGF4 in alleviating hyperglycemia. As expected, a single icv injection of N'-nitro-L-arginine methyl ester hydrochloride (L-name), which inhibits GI neurons by inhibiting nNOS (Canabal et al., 2007), significantly alleviated hyperglycemia for 2 days ( Figure 3 P). Anoctamin 4 was recently identified as a chloride channel in the glucose sensing pathway involving VMH GI neurons (He et al., 2020). Its inhibitor, CaCciNH-A01, was found to also reduce blood glucose for 2 days in db / db mice ( Figure 3 P).

[0210] To explore whether FGF4 has similar acute effects on VMH GSNs in DIO mice, the above experiments with db / db mice were repeated in DIO mice. First, it was confirmed that GE neurons can be easily identified by switching to high glucose (5 mM) or low glucose, while GI neurons are more sensitive to low glucose and show little response to high glucose ( Fig.11 A to Fig.11 D). Therefore, for convenience, low glucose was used to distinguish GSN subtypes. As expected, the majority of VMHGI neurons recorded (6 / 11, 55%) were inhibited by FGF4 perfusion, whereas 2 / 11 were excited and 3 / 11 were insensitive ( Figure 3 Q to Figure 3 To test whether this acute effect requires Fgfr1, we used PD166866 to block Fgfr1 ( Figure 3 T), resulting in silencing of 60% (3 / 5) of FGF4-inhibited GI neurons ( Figure 3 U and Figure 3 V). Thus, as in db / db mice, most GI neurons were acutely inhibited by FGF4, and this acute effect was primarily mediated by Fgfr1. In contrast, similar numbers of VMHGE neurons were found to be excited (6 / 18), inhibited (7 / 18), or insensitive (5 / 18) to FGF4 infusion ( Fig.11 E to Fig.11 G). In addition, PD166866 blocked 60% (3 / 5) of FGF4-stimulated GE neurons and 50% (1 / 2) of FGF4-inhibited GE neurons ( Fig.11 H to Fig.11 J). Taken together, these results suggest that FGF4 inhibits most GI neurons in DIO mice and that most inhibition requires Fgfr1, whereas FGF4 has an equal opportunity to acutely excite or inhibit GE neurons and that Fgfr1 mediates only half of this effect.

[0211] Nose-to-brain delivery of FGF4 alleviates hyperglycemia

[0212] Finally, to explore the therapeutic potential of FGF in treating diabetic patients, we used a nose-to-brain delivery system that has been used for brain-targeted drug delivery in human trials (Chapman et al., 2013). FGF4 was loaded into 100-nm liposomes ( Figure 4 A and Figure 4 B), and administered them to db / db mice by the intranasal route. Notably, nose-to-brain delivery significantly reduced blood glucose in db / db mice for nearly a week without affecting body weight ( Figure 4 C and Figure 4 D). Thus, nasally delivered FGF4 improves glucose tolerance and insulin resistance ( Figure 4 E and Figure 4 F). To determine whether repeated central administration of FGF4 would induce resistance, FGF4 liposomes were administered intranasally to db / db mice every 2-3 weeks, and it was found that FGF4 was able to repeatedly and effectively lower blood glucose without diminishing anti-hyperglycemic effects ( Figure 4 G). Therefore, intranasal administration is an effective way to deliver FGF4 to obtain long-term anti-diabetic effects.

[0213] Discussion

[0214] In this study, we systematically evaluated the ability of representative FGFs to lower blood glucose. We provided strong evidence that central or intranasal administration of FGF4 effectively and long-term alleviated hyperglycemia in diabetic db / db mice without inducing hypoglycemia ( Figure 1 and Figure 4 ). Notably, FGF4 has more potent antidiabetic activity than FGF1 in diabetic DIO mice ( Figure 1 FGF4 is a well-defined essential factor at various stages of embryonic development, including during implantation, morphogenesis, and organogenesis (Beenken and Mohammadi, 2009; Itoh and Ornitz, 2008). This study extends the role of FGF4 in treating T2D in a sustained manner.

[0215] attempted to identify the receptors and cell types that underlie the glucose-lowering effects of FGF and discovered that Fgfr1 in the hypothalamus is a key factor ( Figure 2 G). Fgfr1 is widely expressed in different hypothalamic cell types, including neurons and glia. Neuronal Fgfr1 is shown to be essential for FGF4-mediated diabetic remission, as its absence substantially abrogated the glucose-lowering effects of FGF4 in DIO mice ( Figure 2 K). In contrast, Fgfr1 is expressed in lepr + Expression in cells and astrocytes is not essential for FGF4-mediated glucose lowering ( Figure 8 ). Whether Fgfr1 is also required for the sustained effects of FGF1 was not directly tested, as FGF1 failed to lower blood glucose in DIO mice ( Figure 6 A) and (Scarlett et al., 2016). However, based on the strong binding affinity between FGF1 and Fgfr1 (Suh et al., 2014), it is reasonable to speculate that FGF1 will also act through Fgfr1.

[0216] GSNs are important players in glucose homeostasis. Decreases in blood glucose stimulate GI neurons, which leads to sympathetic nerve activation in the liver, adrenal medulla, and pancreas, thereby increasing glucose production (Shimazu and Minokoshi, 2017). In contrast, increases in blood glucose stimulate GE neurons, which then activate sympathetic nerves innervating BAT, heart, and skeletal muscle to increase glucose use (Shimazu and Minokoshi, 2017). However, the role of GSNs in diabetic pathology and prevention has not been fully explored. Here, we show that FGF4 directly inhibits GI neurons via Fgfr1 in the VMH, and that pharmacological inhibition of GI neurons is sufficient to alleviate hyperglycemia ( Figure 3 Consistent with known GI neuronal functions, FGF4 administration decreased hepatic glucose production and increased hepatic glycolysis and glycogen formation ( Figure 1 F to Figure 1 H). Therefore, the data collectively indicate that FGF4 exerts its anti-hyperglycemic function by inhibiting GI neurons. For GE neurons, it was observed that FGF4 had mixed effects on GE neurons, and only part of the effect depended on Fgfr1. However, a subset of GE neurons stimulated by FGF4 may still mediate this glucose-lowering effect, because activation of GE leads to glucose reduction. Whether GE neurons are involved in the anti-hyperglycemic effect of FGF4 remains to be further studied.

[0217] In addition to GSN, Fgfr1 is expressed in other cell types of the hypothalamus, including tanycytes. Heat shock protein 25 and pERK activation have been reported in villous cells after central administration of FGF1 (Brown et al., 2019; Scarlett et al., 2016). In addition, tanycytes are also sensitive to changes in glucose (Benford et al., 2017; Frayling et al., 2011). Whether FGF requires Fgfr1 signaling in stem cells to affect blood glucose is also an important topic for future research.

[0218] Regarding peripheral responses associated with central administration of FGF, insulin and glucagon levels were not altered immediately after central administration of FGF1 or FGF4 ( Figure 5 J and (Scarlett et al., 2019), suggesting that these hormones may not be the primary driver of diabetes remission. Instead, a transient reduction in food intake ( Figure 5 K and (Tennant et al., 2019)), increased glucose uptake ( Figure 1 F to Figure 1 H) and increased energy expenditure ( Figure 1 M and Figure 5 O) may be an important contributing factor.

[0219] In conclusion, we found that FGF4 can effectively and chronically alleviate hyperglycemia in diabetic db / db and DIO mice. FGF4 achieves this function through Fgfr1 and GSNs in the hypothalamus. FGF4 acutely inhibits GI neurons to achieve glucose lowering and resensitizes GSNs and restores their relative number in the long term. Identification of FGF4 as a potent and sustained antidiabetic factor may provide a new avenue for the treatment of T2D and related metabolic diseases.

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Claims

1. Use of a pharmaceutical composition comprising an Fgfr1 ligand in the preparation of a medicament for treating metabolic diseases via intracerebral or intranasal administration, wherein: The metabolic disease is obesity, type 2 diabetes, insulin resistance, hyperinsulinemia, glucose intolerance or hyperglycemia. The pharmaceutical composition further comprises a pharmaceutically acceptable pharmaceutical preparation liposome, and the ligand is FGF4.

2. The use according to claim 1, wherein The intracerebral administration is chronic intracerebral administration.

3. The use according to claim 2, wherein The frequency of intracerebral administration is less than once every 5 weeks.

4. The use according to claim 1, wherein The frequency of intranasal administration is less than once every 5 days.

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