rickets 1.pdf

Pharmacological Inhibition of Fibroblast GrowthFactor (FGF) Receptor Signaling AmelioratesFGF23-Mediated Hypophosphatemic Rickets

Simon Wohrle,1 Christine Henninger,1 Olivier Bonny,2 Anne Thuery,1 Noemie Beluch,1 Nancy E Hynes,3

Vito Guagnano,1 William R Sellers,4 Francesco Hofmann,1 Michaela Kneissel,1 and Diana Graus Porta1

1Novartis Institutes for BioMedical Research, Basel, Switzerland2Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland3Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland4Novartis Institutes for BioMedical Research, Cambridge, MA, USA

ABSTRACTFibroblast growth factor 23 (FGF23) is a circulating factor secreted by osteocytes that is essential for phosphate homeostasis. In kidney

proximal tubular cells FGF23 inhibits phosphate reabsorption and leads to decreased synthesis and enhanced catabolism of 1,25-

dihydroxyvitamin D3 (1,25[OH]2D3). Excess levels of FGF23 cause renal phosphate wasting and suppression of circulating 1,25(OH)2D3

levels and are associated with several hereditary hypophosphatemic disorders with skeletal abnormalities, including X-linked hypopho-

sphatemic rickets (XLH) and autosomal recessive hypophosphatemic rickets (ARHR). Currently, therapeutic approaches to these diseases

are limited to treatment with activated vitamin D analogues and phosphate supplementation, often merely resulting in partial correction

of the skeletal aberrations. In this study, we evaluate the use of FGFR inhibitors for the treatment of FGF23-mediated hypophosphatemic

disorders using NVP-BGJ398, a novel selective, pan-specific FGFR inhibitor currently in Phase I clinical trials for cancer therapy. In two

different hypophosphatemic mouse models, Hyp and Dmp1-null mice, resembling the human diseases XLH and ARHR, we find that

pharmacological inhibition of FGFRs efficiently abrogates aberrant FGF23 signaling and normalizes the hypophosphatemic and

hypocalcemic conditions of these mice. Correspondingly, long-term FGFR inhibition in Hyp mice leads to enhanced bone growth,

increased mineralization, and reorganization of the disturbed growth plate structure. We therefore propose NVP-BGJ398 treatment as a

novel approach for the therapy of FGF23-mediated hypophosphatemic diseases. � 2013 American Society for Bone and Mineral

Research.

KEY WORDS: FGF23; PHOSPHATE HOMEOSTASIS; HYPOPHOSPHATEMIC RICKETS; FIBROBLAST GROWTH FACTOR RECEPTOR; TARGETED THERAPY

Introduction

Fibroblast growth factor 23 (FGF23) is a critical, bone-

derived mediator of phosphate homeostasis.(1) In kidney

proximal tubule epithelial cells, FGF23 signaling controls

expression of the vitamin D metabolizing enzymes CYP27B1

and CYP24A1, resulting in decreased synthesis and elevated

turnover of the active vitamin D metabolite 1,25(OH)2D3.(2,3)

In addition, FGF23 impairs expression of the sodium-phosphate

co-transporters NaPi-2a (SLC34A1) and NaPi-2c (SLC34A3) in

the brush border membrane (BBM) of proximal tubular cells,

which mediate the reabsorption of urinary phosphate.(4,5)

FGF23 signaling is transduced bymembers of the FGF receptor

(FGFR) family in conjunction with the essential co-receptor

Klotho, which confers tissue-specificity for endocrine FGF23

signals owing to its predominant expression in kidney.(6,7) Fgf23-

and Klotho-deficient mice show largely overlapping phenotypes,

resembling familial tumoral calcinosis (FTC), which is associated

with hyperphosphatemia, increased or inappropriately normal

levels of 1,25(OH)2D3, and ectopic calcifications.(8–11)

In contrast, excess levels of FGF23 result in hypophosphatemia

and are associated with several hereditary hypophosphatemic

disorders with skeletal abnormalities as a consequence of

impaired bone mineralization and growth, including X-linked

hypophosphatemic rickets (XLH), autosomal dominant hypopho-

sphatemic rickets (ADHR), and autosomal recessive hypopho-

sphatemic rickets (ARHR).(12–16) In addition, in rare cases

secretion of FGF23 by tumor cells has been identified to cause

ORIGINAL ARTICLE JJBMR

Received in original form May 15, 2012; revised form October 18, 2012; accepted October 23, 2012. Accepted manuscript online November 5, 2012.

Address correspondence to: Diana Graus Porta, Novartis Pharma AG, Werk Klybeck, Klybeckstrasse 141, CH-4057 Basel, Switzerland

E-mail: [email protected]

Additional Supporting Information may be found in the online version of this article.

Journal of Bone and Mineral Research, Vol. 28, No. 4, April 2013, pp 899–911

DOI: 10.1002/jbmr.1810

� 2013 American Society for Bone and Mineral Research

899

hypophosphatemia, resulting in tumor-induced osteomalacia

(TIO).(3) Whereas ADHR is characterized by gain-of-function

mutations in FGF23 itself,(17) XLH and ARHR are caused by

inactivating mutations in the PHEX and DMP1 genes, respective-

ly, leading to elevated expression of FGF23 in bone.(13,14,18)

Another form of ARHR is caused by loss-of-function mutations in

the ENPP1 gene, which also cause increased expression of

FGF23.(15,16) The disease-relevant function of FGF23 in XLH and

ARHR has been elucidated in Phex- and Dmp1-deficient mice, in

which targeted deletion of FGF23 not only rescues the

hypophosphatemic conditions of these animal models, but

resembles the FTC phenotype of single Fgf23-null mice.(11,19,20)

Likewise, disruption of Klotho overcorrects hypophosphatemia in

the Hyp mouse model,(21) further highlighting the functional role

of the FGF23/Klotho pathway in XLH and related hypopho-

sphatemic diseases.

In vitro, FGF23/Klotho signaling has been shown to be

mediated by FGFR4 or the IIIc isoforms of FGFR1 and FGFR3.(6,7)

In vivo, genetic depletion of FGFR family members only partially

resembles the loss of Fgf23 or Klotho, owing to embryonic lethal

events as well as potential redundancy or compensatory

mechanisms within the FGFR family.(22–25) However, pharmaco-

logical inhibition of FGFRs using a pan-specific FGFR kinase

inhibitor effectively blocks FGF23 function in wild-type mice,

emphasizing the essential role of FGFR signal transduction in

phosphate and 1,25(OH)2D3 homeostasis.(26) In Hyp mice,

simultaneous deletion of FGFR3 and FGFR4 partially corrects

the hypophosphatemic condition.(22) Therefore, FGFRs consti-

tute promising therapeutic targets for hypophosphatemic

disorders caused by aberrant FGF23 expression.

Hereditary hypophosphatemic diseases typically present in

early childhood with short stature and skeletal abnormalities,

such as bending deformities of the legs and rickets.(27) Current

therapeutic approaches to these diseases are mainly limited

to treatment with activated vitamin D analogues, such as

1,25(OH)2D3 (Rocaltrol) and 1a-hydroxyvitamin D (Alfacalcidol),

and phosphate supplementation, resulting in partial correction

of skeletal abnormalities, the extent of which depends on

disease severity. Despite therapy, patients typically exhibit

reduced adult height and require careful monitoring on

treatment to avoid complications such as abdominal pain,

diarrhea, secondary hyperparathyroidism, and ectopic calcifica-

tions.(28)

In this study, we preclinically assessed the potential use of

FGFR inhibitors for the therapy of FGF23-mediated hypopho-

sphatemic disorders using NVP-BGJ398, a novel selective, pan-

specific FGFR inhibitor.(29) Using two different mouse models of

FGF23-mediated hypophosphatemic rickets, Hyp and Dmp1-null

mice,(30,31) resembling the human diseases XLH and ARHR,

we find that pharmacological inhibition of FGFRs efficiently

suppresses aberrant FGF23 signaling and alleviates the hypo-

phosphatemic and hypocalcemic conditions of these mice.

Correspondingly, long-term FGFR inhibition in Hyp mice leads

to the normalization of bone mineralization and a striking

reorganization of the disturbed growth plate structure.

We therefore propose FGFR inhibitor therapy as a potential

approach for the treatment of FGF23-mediated hypopho-

sphatemic diseases.

Methods

Mice

Wild-type C57BL/6 and Hyp (B6.Cg-PhexHyp/J) mice were

obtained from The Jackson Laboratory (Bar Harbor, ME, USA).

Dmp1-null mice were generated by Feng and colleagues(30) and

were licensed from the University of Missouri–Kansas City

(Kansas City, MO, USA). All mice were kept in cages under

standard laboratory conditions with constant temperature of 20

to 248C and a 12-hour–12-hour light-dark cycle. Mice were fed on

a standard rodent diet (3302; Provimi Kliba SA, Penthalaz,

Switzerland) containing 1.15% calcium, 0.85% phosphate, and

1000 UI/kg vitamin D with water ad libitum. Protocols, handling,

and care of the mice conformed to the Swiss federal law for

animal protection under the control of the Cantonal Veterinary

Office Basel-Stadt, Switzerland.

FGFR inhibitor treatment

The FGFR inhibitor NVP-BGJ398 is a small molecular weight

compound featuring an N-aryl-N0-pyrimidin-4-yl urea motif.(29)

NVP-BGJ398 (50mg/kg body weight; Novartis, Basel, Switzerland)

or vehicle only (PEG-300 [Applichem]/Glucose 5% [B. Braun,

Melsungen, Germany], 2:1 mix) was administered by oral gavage.

Mice were used at 5 to 7 weeks of age in the case of single-dose

administrations. For long-term treatment over 8 weeks, dosing

was initiated at 5 weeks of agewith a schedule of three treatments

per week (3qw). Mice were anesthetized by isoflurane inhalation

and blood was collected from the caval vein. Mice were

euthanized by exsanguination and kidney and tibial and femoral

bones were obtained. Concentrations of NVP-BGJ398 in kidney at

7 hours and 24 hours posttreatment were determined by liquid

chromatography/tandem mass spectroscopy (LC/MS-MS).

Serum parameters

Serum was separated from whole blood using clot activator

centrifugation tubes (Sarstedt, Numbrecht, Germany). Serum

(100mL) was used for determination of phosphate and calcium

levels using the VetScan diagnostic profiling system (Abaxis,

Darmstadt, Germany). Serum concentrations of 1,25(OH)2D3

were determined using a radio receptor assay kit (Immundiag-

nostic, Bensheim, Germany). FGF23 serum levels were analyzed

by an ELISA detecting intact FGF23 (Kainos, Tokyo, Japan).

Determination of PTH levels was performed using a mouse PTH

ELISA (Immutopics, San Clemente, CA, USA).

RNA purification and quantitative real-time PCR

For isolation of kidney RNA, approximately 60mg of tissue was

homogenized in 1.5mL RTL buffer (Qiagen, Hilden, Germany)

with a rotor-stator homogenizer (Digitana, Yverdon-les-Bains,

Switzerland) and RNA was purified with the RNeasy Mini kit.

Random hexamer primed cDNA was synthesized with 0.5 to 2mg

RNA and MultiScribe MuLV reverse transcriptase (Applied

Biosystems, Carlsbad, CA, USA). Quantitative real-time PCR was

performed in an iQ5 Real-Time PCR Detection System (BioRad,

Hercules, CA, USA) using a quantitative PCR (qPCR) core kit for

probe assay (Eurogentec, Seraing, Belgium) and an equivalent of

900 WOHRLE ET AL. Journal of Bone and Mineral Research

40 or 80 ng RNA of each sample. The data were normalized to

Gapdh expression. TaqMan assays and primer sequences used

are indicated in the Supplemental Material.

Radiography and micro–computed tomography analyses

Radiographs of femur and tibia were taken ex vivo using a high-

resolution radiography system (Faxitron MX-20; Faxitron, Buffalo

Grove, IL, USA). Micro–computed tomography (mCT) measure-

ments were performed ex vivo using a Scanco vivaCT 40 system

(voxel size 6mm; high resolution; Scanco Medical, Bruttisellen,

Switzerland). For cancellous and cortical bone analyses a fixed

threshold of 200 was used to determine the mineralized bone

fraction from 50 slices. A Gaussian filter was applied to remove

noise (s¼ 0.7; support¼ 1). Cancellous bone mineral density

and bone volume per tissue volume and trabecular number,

thickness, and separation were determined in the distal femur

metaphysis. In addition cortical thickness and cortical bone

mineral density and bone volume per tissue volume were

determined.

Bone histomorphometric analyses

The left femur was fixed for 24 hours in 4% phosphate-buffered

paraformaldehyde, dehydrated, defatted at 48C, and embedded

in methylmethacrylate resin. A set of 5mm nonconsecutive

longitudinal sections was cut in the frontal mid-body plane

(Leica RM2155 microtome; Leica Microsystems, Heerbrugg,

Switzerland). Osteoblast number and osteoid surface per bone

surface and osteoid width were determined on Goldner-stained

sections in the secondary spongiosa of the distal metaphysis

and epiphysis using a Leica DM microscope fitted with a SONY

DXC-950P camera and adapted Quantimet 600 software (Leica).

Osteoid width was determined in addition at the endocortical

surface of the distal metaphysis. Microscopic images were

digitized and evaluated semiautomatically on screen (200-fold

magnification). Sections were stained for tartrate-resistant acid

phosphatase 5b (TRAP5b) activity for determination of osteoclast

number per bone surface in the secondary spongiosa of the

distal metaphysis and epiphysis using a Merz grid (200-fold

magnification). Bone histomorphometric nomenclature was

applied as recommended by Parfitt and colleagues.(32)

Statistical analysis

All data shown represent mean� standard error of the mean

(SEM). Statistical analyses were performed using Student’s t test

(two-tailed). The significance level is indicated by asterisks:�p< 0.05; ��p< 0.01; ��p< 0.001.

Results

FGF23 is the disease-causing factor in several hypophosphatemic

conditions including X-linked hypophosphatemic rickets (XLH).(1)

We have recently provided evidence for a functional role of

FGFRs in renal FGF23/Klotho signaling in vivo by means of

pharmacological inhibition of FGFRs using the preclinical tool

compound PD173074.(26) Here, we aimed to determine whether

FGFR inhibition also counteracts pathological FGF23 signaling

and to provide preclinical proof of efficacy in hypophosphatemic

rickets for NVP-BGJ398, a novel inhibitor in Phase I clinical trials

for cancer patients with FGFR genetically altered tumors.(33) To

this end, we used the Hyp and Dmp1-null mouse models,(18,31,34)

and analyzed the effect of treatment with NVP-BGJ398, which

inhibits the kinase activity of all four FGFR family members at

nanomolar one-half maximal inhibitory concentration (IC50)

values and displays high specificity for FGFRs in cellular kinase

profiling assays.(29)

FGFR inhibition using NVP-BGJ398 suppresses renalFGF23 signaling

FGF23 exerts its hypophosphatemic functions in part by

transcriptional regulation of the 1,25(OH)2D3-metabolizing

enzymes CYP27B1 and CYP24A1 in the kidney.(2,3) Despite the

elevated FGF23 levels present in Hypmice, Cyp27b1 and Cyp24a1

expression and 1,25(OH)2D3 serum levels in Hyp mice were not

significantly different compared to wild-type mice (Fig. 1A–C), in

line with previous reports.(35,36) To initially demonstrate the

inhibitory effect of NVP-BGJ398 on FGF23 signaling in wild-type

and Hyp mice we performed a single-dose, short-term treatment

study. Based on the pharmacokinetic profile of the compound

(Supplemental Fig. S1) we analyzed renal FGF23 target gene

expression at 7 hours postdosing to illustrate the immediate

effects of FGFR inhibition before the onset of feedback regulations

upon release of pathway inhibition.(26) We found that in both

wild-type and Hyp mice, NVP-BGJ398 treatment led to increased

Cyp27b1 levels and an almost complete loss of Cyp24a1

expression (Fig. 1A, B). Accordingly, this resulted in a strong

increase in 1,25(OH)2D3 serum levels in both wild-type and Hyp

mice at 7 hours postdosing of NVP-BGJ398 (Fig. 1C). These results

illustrate that pharmacological inhibition of FGFRs with NVP-

BGJ398 counteracts FGF23 signaling in wild-type and Hyp mice.

In addition, we also observed effects of FGFR inhibitor

treatment on PTH serum levels (Supplemental Fig. S2). In wild-

type mice PTH levels were significantly increased after 7 hours of

NVP-BGJ398 treatment, whereas reduced levels were observed

at 24 hours postdosing, consistent with the effect observed with

the FGFR inhibitor PD173074.(26) In contrast, PTH levels were

higher in Hyp mice but not affected by NVP-BGJ398 treatment.

Furthermore, we noted a transient repression of FGF23 bone

mRNA and serum levels upon NVP-BGJ398 treatment (Supple-

mental Fig. S3A, B), in line with the previously reported

regulatory function of FGFR signaling on FGF23 expression.(26,37)

Short-term FGFR inhibition did not affect NaPi-2a and NaPi-2c

mRNA levels in the kidney (Supplemental Fig. S4A, B) and

NaPi-2a expression in the brush border membrane (Supplemen-

tal Fig. S4C). Consequently, no significant changes in urinary

phosphate levels were observed over a 24-hour time-course

following FGFR inhibition (Supplemental Fig. S4D) and NVP-

BGJ398 treatment did not impinge on fractional excretion of

phosphate in Hyp mice, whereas it only mildly affected the

phosphate filtration rate in wild-typemice (Supplemental Fig. S4E).

NVP-BGJ398 treatment ameliorates the hypophosphatemicconditions of Hyp and Dmp1-null mice

A single dose of NVP-BGJ398 induced elevated serum calcium

and phosphate levels in both wild-type and Hypmice at 24 hours

postdosing, thus alleviating the severe hypocalcemia and

Journal of Bone and Mineral Research TARGETED THERAPY OF FGF23-MEDIATED HYPOPHOSPHATEMIC DISEASES 901

hypophosphatemia observed in Hyp mice. Serum calcium levels

of NVP-BGJ398-treated Hyp mice were indistinguishable from

vehicle-treated wild-type mice (Fig. 1D), whereas serum

phosphate concentrations in the inhibitor treated group were

still significantly lower compared to wild-type mice (Fig. 1E).

A mouse model for ARHR, the genetically engineered Dmp1-

null strain, was also examined following FGFR inhibition. As seen

with the Phex-deficient Hyp model, renal expression of Cyp27b1

was increased whereas Cyp24a1 levels were repressed upon

treatment with NVP-BGJ398 (Supplemental Fig. S5A, B).

Moreover, as observed for Hyp mice, FGFR inhibition led to

increased serum phosphate and calcium levels in Dmp1-null

mice (Supplemental Fig. S5C, D).

Long-term FGFR inhibition enhances body weight andtail length development in Hyp mice

Because single-dose treatments with NVP-BGJ398 alleviated the

hypocalcemic and hypophosphatemic phenotypes of Hyp and

Dmp1-null mice, we aimed to monitor a potential amelioration of

the rickets-like bone phenotypes upon long-term FGFR inhibi-

tion. Here, we focused on the Hyp model, given the substantial

improvements of mineral ion homeostasis upon single-dose

FGFR inhibitor treatment. Treatments were performed over a

course of 8 weeks. Owing to the persistence of elevated calcium

and phosphate levels for at least 48 hours post-NVP-BGJ398

administration (Supplemental Fig. S6A, B)—extending beyond

the clearance of the compound from the kidney (Supplemental

Fig. S1)—mice were treated only 3qw with NVP-BGJ398 (50mg/

kg body weight) or vehicle.

Before the onset of therapy treatment, at 5 weeks of age, Hyp

mice displayed a reduced body weight compared to wild-type

littermates. Although body weight of both vehicle and NVP-

BGJ398-treated Hyp mice remained significantly lower com-

pared to wild-type littermates during the course of treatment,

pharmacological FGFR inhibition in Hyp mice led to a significant

increase in body weight compared to the vehicle control group

from day 31 of treatment on (Fig. 2A). Overall, the total body

Fig. 1. FGFR inhibitor treatment induces 1,25(OH)2D3 biosynthesis and alleviates hypocalcemia and hypophosphatemia in Hyp mice. Regulation of the

renal FGF23 target genes Cyp27b1 (A) and Cyp24a1 (B) upon FGFR inhibition in vivo. Wild-type or Hypmice received a single oral dose of the FGFR inhibitor

NVP-BGJ398 (50mg/kg) or vehicle and were studied 7 hours after administration of the compound. Kidneys were sampled, total RNA was isolated, and

gene expression was analyzed by quantitative real-time PCR. Expression values were normalized to GapdhmRNA copies. Data are shown as relative levels

to the wild-type control group (relative expression of 100) and are given as means with SEM (n� 6). (C) Serum 1,25(OH)2D3 levels of wild-type and Hyp

mice treated with NVP-BGJ398 for 7 hours as described in A and B were determined by radio receptor assay. Calcium (D) and phosphate (E) levels at 24

hours postadministration in wild-type and Hyp mice treated with a single oral dose of NVP-BGJ398 (50mg/kg) or vehicle. Phosphate and calcium levels

were determined from serum. Data are given as means with SEM (n� 6). Data were compared by unpaired Student’s t test; �p< 0.05; ��p< 0.01;��p< 0.001; n.s.¼ not significant.


weight gain in NVP-BGJ398-treated Hyp mice was similar to

vehicle-treated wild-type mice (Fig. 2B). A shorter tail is a

pronounced feature of the hypophosphatemic rickets pheno-

type of Hyp mice, reflecting the impaired bone formation.(36)

Again, the tail length of both Hyp groups was significantly

shorter compared to wild-type littermates throughout the

treatment period. However, during the 8 weeks of treatment

NVP-BGJ398-treated Hyp mice displayed a much stronger

increase in tail length compared to control Hyp mice (Fig. 2C).

Moreover, the tail length gain in Hyp mice treated with the FGFR

inhibitor was also significantly higher compared to vehicle-

treated wild-type littermates (Fig. 2D).

Long-term therapy with NVP-BGJ398 restores mineral ionhomeostasis in Hyp mice

To examine the effect of continuous FGFR inhibition on

phosphate and calcium homeostasis in Hyp mice, we analyzed

serum calcium and phosphate concentrations at the end of the

8-week study. To distinguish immediate short-term responses to

NVP-BGJ398 treatment from steady-state effects of continuous

FGFR inhibition, serum was prepared at 24 hours after terminal

dosing of the 8-week study, a time point when pharmacological

inhibition was relieved based on the pharmacokinetic profile of

NVP-BGJ398 in wild-type and Hyp mice (Supplemental Fig. S1).

We found that in contrast to single-dose FGFR inhibitor

administration (Fig. 1D, E), long-term therapy with NVP-BGJ398

led to a complete normalization of both calcium and phosphate

levels in Hyp mice (Fig. 3A, B). Despite the transient repressive

effect of FGFR inhibition on FGF23 expression (see Supplemental

Fig. S3), long-term treatment with NVP-BGJ398 led to a further

increase of FGF23 serum concentrations in Hyp mice (Fig. 3C),

which was accompanied by a normalization of PTH levels

(Fig. 3D), whereas 1,25(OH)2D3 was not significantly different

among the treatment groups (Fig. 3E). Also, renal Klotho

expression was not affected by FGFR inhibitor treatment

(Supplemental Fig. S7). Taken together, these results illustrate

the beneficial effect of pharmacological FGFR inhibition in the

context of aberrant FGF23 signaling and point toward an

alleviation of the bone formation deficiency of Hyp mice.

FGFR inhibition enhances longitudinal bone growth inHyp mice

We therefore analyzed the effect of long-term FGFR inhibition

on longitudinal growth of femur and tibia by radiography and

Fig. 2. Long-term FGFR inhibition enhances body weight and tail length development in Hyp mice. Wild-type or Hyp mice were treated with the FGFR

inhibitor NVP-BGJ398 (50mg/kg) or vehicle 3qw for 56 days, and body weight (A) and tail length (C) development was monitored. Total body weight (B)

and tail length gain (D) over the course of the treatment. Data are shown as means with SEM (n� 6). Data were compared by unpaired Student’s t test;�p< 0.05; ��p< 0.01; ��p< 0.001; #p< 0.05 versus vehicle-treated Hyp mice; n.s.¼ not significant.


found that NVP-BGJ398-treated Hyp mice displayed significant

elongation of both femur (Fig. 4A, C) and tibia (Fig. 4B, D)

compared to the vehicle-treated control group. Still, the

enhanced bone growth did not result in femur or tibia

sizes comparable to wild-type mice, but FGFR inhibition did

partially alleviate the widening of both femoral and tibial

growth plate areas, which is typically observed in rickets

(Fig. 4A, B).

Long-term NVP-BGJ398 treatment amelioratesosteoid abundance and impaired matrix mineralizationin Hyp mice

To determine the effect of FGFR inhibitor treatment on bone

structure in more detail we performed mCT analyses of the distal

femoral metaphysis. This analysis revealed impaired mineraliza-

tion of the cortical bone area in Hyp mice, apparent given the

gaps and holes within the Hyp femoral cortex structure (Fig. 5A,

indicated by arrowheads). Consistent with this observation the

relative bone volume within the cortical compartment, which

approaches 100% in healthy rodents, was reduced in vehicle-

treated Hyp mice compared to wild-type controls (Fig. 5B).

Accordingly, cortical bone mineral density was decreased

(Table 1). Moreover, animals presented with decreased

average cortical thickness (Fig. 5C). In contrast, cortex of

NVP-BGJ398-treated Hyp mice appeared intact (Fig. 5A) and

relative cortical bone volume was indistinguishable from wild-

type mice (Fig. 5B). Also, cortical bone mineral density was

partially rescued (Table 1) and cortex thickness was significantly

increased compared to vehicle-treated Hyp mice (Fig. 5C).

In line with those observations, the histomorphometric

analysis revealed a significant amelioration of the abnormal

endocortical osteoid width in Hyp mice (Table 1). Metaphyseal

cancellous bone volume and bone mineral density were

markedly reduced in Hyp mice owing to a decrease in trabecular

number and a concomitant increase in trabecular separation

compared to wild-type controls, whereas trabecular thickness

was unaltered. FGFR inhibition in Hyp mice did not correct these

abnormalities as determined by mCT (Table 1). However,

histomorphometric analysis demonstrated that NVP-BGJ398

treatment also significantly normalized matrix mineralization

in the cancellous bone compartment as reflected in the

reduction in osteoid width and surface present in Hyp

mice (Table 1). Because in Hyp mice, irrespective of treatment,

the amount of metaphyseal trabecular bone surfaces available

for quantitative evaluation was low, we aimed to confirm our

histomorphometric findings at a site with higher cancellous

bone volume. Visual inspection suggested that this was the case

in the distal femur epiphysis, where indeed bone volume, when

evaluated on the sections as the sum of the mineralized and

Fig. 3. Long-term FGFR inhibition restores mineral ion homeostasis in Hyp mice. Wild-type or Hyp mice were treated with the FGFR inhibitor NVP-BGJ398

(50mg/kg) or vehicle 3qw for 56 days, and calcium (A), phosphate (B), FGF23 (C), PTH (D) and 1,25(OH)2D3 (E) levels were determined from serum 24 hours

after the last administration at the end of the 8-week treatment. Data are shown as means with SEM (n� 4). Data were compared by unpaired Student’s

t test; �p< 0.05; ��p< 0.01; ��p< 0.001; n.s.¼not significant.


unmineralized bonematrix, was higher in all groups compared to

the metaphyseal site and not different between groups (Fig. 6A,

and data not shown). At this site, NVP-BGJ398 treatment also led

to a substantial improvement of matrix mineralization as

reflected by a significant decrease in the osteoid surface and

width in Hyp mice (Table 1).

Osteoblast number was nonsignificantly elevated in the

metaphysis and significantly increased in the epiphysis in Hyp

mice. However, at both sites osteoblast number of NVP-BGJ398-

treated Hyp mice was indistinguishable from wild-type litter-

mates. Osteoclast count was comparable between all groups in

the metaphysis. In the epiphysis, we observed an increased

osteoclast number in vehicle-treated Hyp mice, whereas

osteoclast count in NVP-BGJ398-treated Hyp mice was compa-

rable to wild-type controls (Table 1). These data indicate that

FGFR inhibitor treatment significantly reduced themineralization

defects present in Hyp mice at all skeletal envelopes, as well as

any concomitant abnormalities in histomorphometric indices.

Taken together, the radiography (Fig. 4), microtomography

(Fig. 5, Table 1) and histomorphometric (Fig. 6, Table 1) analyses

revealed a favorable effect of FGFR inhibition on longitudinal

growth, structural integrity, and mineralization of bone in Hyp

mice.

Treatment with NVP-BGJ398 corrects growth plateorganization in Hyp mice

In addition, we found an ameliorative effect of NVP-BGJ398

treatment on growth plate organization in tibial histological

sections of NVP-BGJ398-treated Hyp mice (Fig. 6A). In vehicle-

treated Hyp mice the columnar organization and directional

growth of chondrocytes was disturbed in contrast to the highly

ordered structure in wild-type mice. In NVP-BGJ398-treated Hyp

mice, however, we observed a striking reorganization of the

growth plate area (Fig. 6A, left panels), and a reformation of the

columnar stacks of chondrocytes along with an increased height

of the proliferative zone (Fig. 6A, right panels).

In summary, our data indicate that pharmacological inhibition

of FGFRs might be sufficient to inhibit aberrant FGF23 signaling

and to alleviate the hypophosphatemic rickets phenotype of

XLH and potentially other FGF23-related hypophosphatemic

diseases, such as ARHR.

Discussion

In this study we show that pharmacological inhibition of FGFRs

using the novel, pan-specific FGFR inhibitor NVP-BGJ398

Fig. 4. Long-term FGFR inhibition enhances growth of long bones in Hyp mice. Radiographs of femur (A) and tibia (B) from wild-type or Hyp mice treated

with the FGFR inhibitor NVP-BGJ398 (50mg/kg) or vehicle 3qw for 56 days. Quantification of femoral (C) and tibial (D) length. Data are shown as means

with SEM (n� 6). Data were compared by unpaired Student’s t test; �p< 0.05; ��p< 0.01; ��p< 0.001.


counteracts pathological FGF23 signaling, thereby depicting a

potential novel therapeutic approach for the treatment of

FGF23-mediated hypophosphatemic disorders. In particular,

FGFR inhibition corrects hypophosphatemia and hypocalcemia

in the Hyp mouse model of XLH. Consequently, long-term

treatment with NVP-BGJ398 alleviates the rickets-like bone

phenotype in this model and leads to enhanced bone

mineralization, normalization of bone turnover and a striking

restoration of the growth plate organization.

Pharmacological FGFR inhibition as a potential noveltherapeutic approach for FGF23-mediatedhypophosphatemic diseases

XLH and other FGF23-mediated hypophosphatemic diseases

such as ADHR and ARHR commonly manifest clinically in early

childhood with short stature and bowing deformities of the

legs.(28) Current medical therapy consists of phosphate supple-

mentation and treatment with activated vitamin analogues from

time of diagnosis until completion of growth. Although therapy

improves growth and rickets in patients, correction is often

limited and results in impaired postpubertal height.(28) Owing to

the persistence of FGF23 signaling—constituting a continuous

counteractive force—the administration of high doses of

phosphate and vitamin D analogues is required for medical

therapy of XLH and other FGF23-mediated hypophosphatemic

diseases, necessitating close monitoring and dose adjustments

to avoid toxicity risks such as ectopic calcifications or secondary

hyperparathyroidism.(28,38) Therefore, directly targeting patho-

logical FGF23 signaling by blocking FGFR signal transduction

might provide an advantageous therapeutic approach over the

current standard of treatment.

Effects of single-dose short-term FGFR inhibition

To establish an efficacious dose regimen for long-term FGFR

inhibitor therapy and to monitor short-term events of blocking

renal FGF23 signaling we initially performed single-dose

treatments with NVP-BGJ398 in wild-type and Hyp mice. Similar

to our previous observation in wild-type mice using an FGFR

inhibitor tool compound,(26) we noticed immediate effects of

FGFR inhibition by NVP-BGJ398 on renal FGF23 signaling. In line

with the potent suppressive function of FGF23 on 1,25(OH)2D3

synthesis,(2,3) we observed increased 1,25(OH)2D3 serum levels

after treatment with the FGFR inhibitor NVP-BGJ398 in Hyp mice.

Pharmacological inhibition of FGFRs also resulted in increased

serum calcium and phosphate levels in both Hyp and Dmp1-

deficient mice. While FGF23 was reported to inhibit renal

reabsorption of phosphate by decreasing the expression of the

sodium-phosphate co-transporters NaPi-2a and NaPi-2c in the

brush border membrane (BBM) of proximal tubule epithelial

cells,(4,5) renal expression of NaPi-2a and fractional excretion of

phosphate was not significantly affected by FGFR inhibition in

Hyp mice, indicating that the correction of hypophosphatemia

might be mediated via intestinal absorption of dietary phos-

phate in consequence of the increased 1,25(OH)2D3 synthesis.(39)

The pronounced effect of FGFR inhibition on 1,25(OH)2D3 levels

is in line with a more rapid effect of recombinant FGF23 injection

Fig. 5. Long-term FGFR inhibition improves cortex integrity in femoral bone of Hyp mice. (A) mCT scans of femoral cortex (sub–growth-plate metaphyseal

area) from wild-type or Hyp mice treated with the FGFR inhibitor NVP-BGJ398 (50mg/kg) or vehicle 3qw for 56 days. Porosity of cortex is indicated by

arrowheads. Quantification of relative cortical bone volume (B) and average cortex thickness (C). Data are shown as means with SEM (n � 6). Data were

compared by unpaired Student’s t test; �p< 0.05; ��p< 0.01; ��p< 0.001; n.s.¼ not significant.


in mice on vitamin D metabolism compared to changes in NaPi-

2a expression.(2) Therefore, short-term pharmacological FGFR

pathway inhibition might not be sufficient to induce changes in

urinary phosphate excretion in contrast to more sustained FGF23

loss of function approaches, such as genomic depletion or

treatment with FGF23-neutralizing antibodies.(9,36)

FGF23 signaling directly inhibits PTH expression in the

parathyroid gland.(40) Consequently, single-dose FGFR inhibitor

treatment transiently induced PTH serum levels in wild-type

mice after 7 hours of treatment. However, upon clearance of the

compound at 24 hours postdosing, PTH levels were reduced

in wild-type mice, potentially promoting the hypercalcemic

conditions observed in wild-type mice at this time point.

Hyp mice showed higher PTH levels compared to wild-type

littermates in line with previous observations.(19) Interestingly,

NVP-BGJ398 treatment did not affect PTH levels in Hyp mice,

indicating that PTH does not directly contribute to the effects

on mineral ion metabolism observed upon short-term FGFR

inhibition in Hyp mice.

Long-term NVP-BGJ398 treatment alleviates thepathological effects of FGF23 in Hyp mice

Noteworthy, the increase in phosphate and calcium levels in Hyp

mice persists for at least 48 hours after FGFR inhibitor treatment

and thus exceeds the pharmacological inhibition of FGFRs by

NVP-BGJ398, which is cleared from the kidney within 24 hours of

administration. This illustrates that persistent inhibition of FGFRs

is not required for the therapy of FGF23-mediated hypopho-

sphatemia, allowing for intermittent dose regimens. Corre-

spondingly, a 3qw dosing schedule was used in this study.

During the treatment period of 8 weeks, we observed increased

body weight gain in NVP-BGJ398-treated Hyp mice compared to

the vehicle control group, indicating that intermittent FGFR

inhibitor treatment was well tolerated. Moreover, long-term

treatment with NVP-BGJ398 led to a complete normalization of

hypophosphatemia and hypocalcemia and significantly en-

hanced longitudinal growth of the long bones in Hyp mice.

Furthermore, the abundance of osteoid tissue observed in

control Hyp mice was markedly reduced in the NVP-BGJ398-

treated group owing to a normalization of bone mineralization,

resulting in significant rescue of bone mass. Also, FGFR inhibitor

led to increased cortex mineralization in Hyp mice and it would

be interesting to address in future studies whether the observed

improvement of bone geometry and mass translates into

increased mechanical bone strength. Body weight and bone

growth in Hyp mice receiving NVP-BGJ398 was, however, still

lower compared to wild-type mice after 8 weeks of treatment.

This is likely owing to the duration of dosing and the age when

FGFR inhibitor therapy was initiated. Because the mineral ion

Table 1. Femoral Bone Structure and Histomorphometric Indices of Wild-Type and HypMice Upon Long-Term TreatmentWith the FGFR

Inhibitor NVP-BGJ398

Wild-type–vehicle Hyp–vehicle Hyp–NVP-BGJ398

Metaphysis (cancellous bone)

BV/TV (%) 9.0� 0.7 2.5� 0.4�� 1.9� 0.3��

BMD (mg/cm3) 154.1� 5.4 84.6� 5.1�� 83.8� 3.7��

Tb.N (1/mm) 3.4� 0.3 0.9� 0.1�� 0.7� 0.1��

Tb.Th (mm) 26.5� 0.5 26.5� 1.4 26.7� 0.7

Tb.Sp (mm) 280� 27 1114� 119�� 1530� 172��

OS/BS (%) 46.7� 8.1 101� 4.4�� 63.2� 0.2yy

O.Wi (mm) 5.6� 0.2 20� 1.3�� 9.1� 0.1��,yy

N.Obl/BS (1/mm) 0.71� 0.13 1.43� 0.34 0.66� 0.07

N.Ocl/BS (1/mm) 2.1� 0.6 2.4� 0.5 2.8� 0.5

Epiphysis (cancellous bone)

OS/BS (%) 31.7� 4.3 91.7� 1.6�� 50.5� 3.9��,yy

O.Wi (mm) 8.4� 0.5 37.5� 2.5�� 16.8� 0.9��,yy

N.Obl/BS (1/mm) 2.5� 0.4 3.9� 0.3� 3.0� 0.4

N.Ocl/BS (1/mm) 0.8� 0.1 4.4� 0.7�� 1.3� 0.2yy

Cortex

BV/TV (%) 92.6� 0.1 83.8� 1.4�� 92.3� 0.4yy

BMD (mg/cm3) 918.9� 3.4 733.1� 14.3�� 828.3� 8.5��,yy

Ct.Th (mm) 108.7� 1.4 79.1� 1.5�� 92.5� 2.0��,yy

O.Wi (mm) 11.0� 0.8 67.6� 11.0�� 33.8� 2.5��,y

Data are shown as mean� SEM and were compared by unpaired Student’s t test.

Hyp¼ hypophosphatemic; FGFR¼ fibroblast growth factor receptor; NVP-BGJ398¼ a novel selective, pan-specific FGFR inhibitor; BV/TV¼ bone

volume/tissue volume; BMD¼bonemineral density; Tb.N¼ trabecular number; Tb.Th¼ trabecular thickness; Tb.Sp¼ trabecular spacing; OS/BS¼ osteoid

osteoid surface/bone surface; O.Wi¼osteoid width; N.Obl/BS¼ number of osteoblasts/bone surface; N.Ocl/BS¼number of osteoclasts/bone surface;Ct.Th¼ cortical thickness at the distal metaphysis.�p< 0.05 versus vehicle-treated wild-type mice.��p< 0.01 versus vehicle-treated wild-type mice.yp< 0.05 versus vehicle-treated Hyp mice.yyp< 0.01 versus vehicle-treated Hyp mice.


defects in Hyp mice were corrected and growth plate

organization was normalized by NVP-BGJ398 treatment, we

hypothesize that earlier initiation and an extended treatment

period could further alleviate or completely reverse the

pathological phenotypes of FGF23-mediated hypophosphate-

mic diseases.

Effects of long-term pharmacological FGFR inhibition ongrowth plate organization in Hyp mice

A likely on-target effect of systemic FGFR inhibition is expected

from the function of FGFR3 in the control of proliferation and

differentiation of chondrocytes. Genetic depletion of FGFR3

causes skeletal overgrowth due to increased chondrocyte

proliferation.(41) Accordingly, we observed an enlargement of

the proliferative zone of the growth plate in NVP-BGJ398-treated

Hyp mice. Enhanced proliferation of chondrocytes in response to

FGFR inhibition may therefore contribute to the increased

longitudinal bone growth in Hyp mice treated with NVP-BGJ398.

Also, normal phosphate levels are essential for terminal

differentiation and subsequent apoptotic clearance of chon-

drocytes, and phosphate deficiency results in the expansion of

hypertrophic chondrocytes in the growth plates of Hyp

mice.(42,43) In addition, 1,25(OH)2D3 exerts a compensatory

function in the maintenance of a normal growth plate

phenotype in NaPi-2a-deficient mice with persistent hypopho-

sphatemia.(44) Therefore, the normalization of phosphate levels,

the transient increase in serum 1,25(OH)2D3 concentrations, and

the potential effect of FGFR inhibition on chondrocyte

proliferation most likely cooperatively contribute to the

Fig. 6. Long-term treatment with NVP-BGJ398 restores growth plate organization Hyp mice. (A) Goldner staining of tibial sections from wild-type or Hyp

mice treated with the FGFR inhibitor NVP-BGJ398 (50mg/kg) or vehicle 3qw for 56 days. Mineralized tissue is shown in green, unmineralized osteoid is

visualized by red staining. (B) Osteoid surface/bone surface and osteoid width (C) determined by histomorphometry in the tibial epiphysis of wild-type or

Hyp mice treated with NVP-BGJ398 (50mg/kg) or vehicle 3qw for 56 days. Data are shown as means with SEM (n� 6). Data were compared by unpaired

Student’s t test; �p< 0.05; ��p< 0.01; ��p< 0.001.


reorganization of growth plate structure in Hyp mice treated

with NVP-BGJ398.

Steady-state effects of persistent FGFR inhibitortreatment

We have previously reported that FGF signaling is essential for

FGF23 expression in bone and activation of the FGFR pathway

was recently linked to the elevated expression of FGF23 in Hyp

and Dmp1-deficient mice.(26,37) Correspondingly, FGFR inhibition

led to a decrease of both FGF23 mRNA expression and serum

protein levels in Hyp mice, but the reduction of FGF23 levels was

transient and closely correlated with the pharmacokinetic of

FGFR inhibition by NVP-BGJ398. In contrast, Hyp mice treated

with NVP-BGJ398 over a course of 8 weeks showed elevated

FGF23 levels. As an indication of the steady-state effects of long-

term FGFR inhibition, serum parameters at the end of the 8-week

treatment period were determined 24 hours after final dosing. At

this time point pharmacological inhibition of FGFRs is likely

relieved owing to the clearance of the compound. Therefore, this

analysis is indicative of the steady-state effect of long-term FGFR

inhibition in contrast to the immediate changes observed upon

single-dose short-term NVP-BGJ398 treatment. Hence, the

increase in FGF23 levels might reflect a feedback regulation

as a consequence of the correction of hypophosphatemia in Hyp

mice by NVP-BGJ398 treatment. In a similar fashion, persistent

inhibition of mitogen-activated protein kinase (MAPK) signaling

downstream of FGF23 leads to elevated FGF23 expression and

serum levels in Hyp mice.(45) Likewise, current therapeutic

approaches involving activated vitamin D analogues and

phosphate supplementation further induce FGF23 serum levels

in XLH patients.(46) Because FGF23 directly impinges on PTH

expression and secretion in the parathyroid gland,(40) elevated

levels of FGF23 presumably mediate the normalization of PTH

serum concentrations observed in Hyp mice upon long-term

NVP-BGJ398 treatment. This might provide a therapeutic benefit

compared to phosphate administration, which is associated with

the induction of hyperparathyroidism.(38,47) Also, Hyp mice

receiving NVP-BGJ398 for 8 weeks showed normal 1,25(OH)2D3

serum concentrations at 24 hours after terminal dosing,

indicating that the enhancing effect of FGFR inhibition on

1,25(OH)2D3 synthesis observed upon single-dose treatment

does not lead to sustained hypervitaminosis D in continuous

therapy, thus depicting another potential advantage compared

to vitamin D analogue–based therapy.

Using a different approach, Aono and colleagues(36) demon-

strated similar therapeutic effects in the Hyp mouse model by

applying an FGF23-neutralizing antibody. Compared to systemic

inhibition of FGFR signaling, specifically blocking FGF23 function

constitutes a more targeted approach for the therapy of XLH, but

the persistent antibody-mediated inhibition of the FGF23

pathway raises the concern of inducing a physiological condition

resembling FGF23 deficiency, resulting in hyperphosphatemia

and associated toxicities.(48,49) In contrast, the transient nature of

pharmacological pathway inhibition potentially facilitates the

adjustment of phosphate/1,25(OH)2D3 homeostasis in FGF23-

mediated hypophosphatemia patients with varying levels of

aberrant FGF23 activity. Noteworthy, while FGF23-neutralizing

antibodies induce transient hyperphosphatemia in Hyp

mice,(36,50) FGFR inhibitor treatment in Hyp mice did not lead

to elevations in serum calcium or phosphate beyond levels

observed in vehicle-treated wild-type littermates.

In summary, our study indicates the use of pharmacological

FGFR inhibition as a potential novel approach for the therapy of

FGF23-mediated hypophosphatemic diseases. In particular, NVP-

BGJ398 is already applied clinically for cancer indications and

thus might hold promise for clinical use in hypophosphatemic

disorders in the future. Whereas FGFR inhibition alone might be

sufficient to alleviate the pathological effects of aberrant FGF23

signaling, a combination therapy including FGFR inhibitor

treatment and phosphate/vitamin D analogue therapy could

provide additional benefit and allow the reduction of drug doses.

Furthermore, when concomitantly blocking FGF23 signaling,

presumably lower doses of phosphate or vitamin D analogues

are required, thereby decreasing the risk of adverse effects of

therapy. Besides XLH, ADHR, ARHR, and TIO, for which

the pathological role of FGF23 is well established, FGFR

inhibitor treatment could be of therapeutic use in several other

hypophosphatemic syndromes such as epidermal nevus

syndrome, osteoglophonic dysplasia, McCune-Albright syn-

drome, and persistent post-renal transplant hypophosphatemia,

which have been associated with increased FGF23 levels.(1,51,52)

In addition, elevated FGF23 levels have recently been reported as

the putative causal factor for the development of left ventricular

hypertrophy (LVH) and cardiovascular disease in patients

with chronic kidney disease (CKD),(53) revealing a novel and

presumably Klotho-independent pathological effect of aberrant

FGF23 signaling. Because concomitant pharmacological inhibi-

tion of FGFRs prevented the development of LVH in a rat model

of CKD,(53) FGFR inhibitor treatment might also be considered as

preventive therapy for LVH in CKD patients in future clinical trials.

Disclosures

SW, CH, AT, NB, VG, WRS, FH, MK, and DGP are employees of

Novartis Institutes for BioMedical Research. NEH and OB state

that they have no conflicts of interest.

Acknowledgments

We thank R. Rebmann, F. Reimann, A. Studer, P. Ingold, M.

Merdes, B. Bohler, D. Sterker, M. Sutterlin, and C. Stoudmann

for excellent technical assistance. We are grateful to J. Feng and

colleagues for kindly sharing the Dmp1-null mice under license

and to I. Kramer for providing technical expertise and for the

interpretation of histological data. We thank H. Schmid and B.

Hanzi for helpful discussions and critical reading of the manu-

script.

Authors’ roles: Study design: SW, WRS, FH, MK, and DGP. Study

conduct and data collection: SW, CH, OB, AT, and NB. Data

analysis and interpretation: SW, CH, OB, VG, NEH, WRS, FH,

MK, and DGP. Drafting manuscript: SW and DGP. Revising

manuscript content: SW, OB, NEH, FH, MK, and DGP. All authors

approved the final version of manuscript. SW and DGP take

responsibility for the integrity of the data analysis.


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