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.
902 WOHRLE ET AL. Journal of Bone and Mineral Research
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.
Journal of Bone and Mineral Research TARGETED THERAPY OF FGF23-MEDIATED HYPOPHOSPHATEMIC DISEASES 903
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.
904 WOHRLE ET AL. Journal of Bone and Mineral Research
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.
Journal of Bone and Mineral Research TARGETED THERAPY OF FGF23-MEDIATED HYPOPHOSPHATEMIC DISEASES 905
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.
906 WOHRLE ET AL. Journal of Bone and Mineral Research
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.
Journal of Bone and Mineral Research TARGETED THERAPY OF FGF23-MEDIATED HYPOPHOSPHATEMIC DISEASES 907
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.
908 WOHRLE ET AL. Journal of Bone and Mineral Research
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.
Journal of Bone and Mineral Research TARGETED THERAPY OF FGF23-MEDIATED HYPOPHOSPHATEMIC DISEASES 909
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Journal of Bone and Mineral Research TARGETED THERAPY OF FGF23-MEDIATED HYPOPHOSPHATEMIC DISEASES 911