Cancer Therapy: Preclinical
Increased Renal Calcium Reabsorption by Parathyroid Hormone ^
Related Protein Is a Causative Factor in the Development of
Humoral Hypercalcemia of Malignancy Refractory to
Osteoclastic Bone Resorption Inhibitors
Etsuro Onuma,1 Yumiko Azuma,1 Hidemi Saito,1 Toshiaki Tsunenari,1 Toshihiko Watanabe,2
Manabu Hirabayashi,2 Koh Sato,1 Hisafumi Yamada-Okabe,1 and Etsuro Ogata3
Abstract
Purpose: Bisphosphonate and calcitonin lower blood calcium in humoral hypercalcemia of
malignancy (HHM) by suppressing osteoclastic bone resorption, but repeated administration
of these drugs often leads to relapse. In this study, we examined the roles of parathyroid hormone ^
related protein (PTHrP) in the development of bisphosphonate- and calcitonin-refractory HHM.
Experimental Design: Nude rats bearing the LC-6 JCK tumor xenograft (LC-6 rats) exhibited
high bone turnover and HHM. Repeated administration of alendronate induced a sustained suppression of the bone resorption, but it caused only early and transient reduction of the blood calcium levels, leading to unresponsiveness to the drug. Because high blood levels of PTHrP were
detected in the LC-6 rats, those that developed alendronate-refractory HHM were treated with an
anti-PTHrP antibody.
Results: Administration of anti-PTHrP antibody to animals that received repeated administration
of alendronate, thereby developing alendronate-refractory HHM, resulted in an increase in fractional excretion of calcium and a marked decrease of blood calcium level. Drug-refractory HHM
was also observed in animals that received another osteoclast inhibitor, an eel calcitonin analogue
elcatonin. The blood calcium level decreased after the initial administration of elcatonin, but it
eventually became elevated during repeated administration. Administration of the anti-PTHrP
antibody, but not of alendronate, effectively reduced the blood calcium of the animals that
developed elcatonin-refractory HHM.
Conclusion: High levels of circulating PTHrP and the resulting augmentation of renal calcium
reabsorption is one of the major causes of the emergence of osteoclast inhibitor-refractory HHM.
Thus, blockage of PTHrP functions by a neutralizing antibody against PTHrP would benefit
patients who develop bisphosphonate- or calcitonin-refractory HHM.
Humoral hypercalcemia of malignancy (HHM) is one of the
most common paraneoplastic syndromes that considerably
deteriorate the quality of life of cancer patients (1). Increased
bone resorption has been thought to play important roles in
the development of HHM and current therapies for HHM focus
rather on inhibiting the osteoclastic bone resorption. In fact,
bisphosphonate drugs that interfere with the osteoclasts have
become the gold standard for the treatment of HHM (2).
However, there are patients who do not respond to these drugs
Authors’Affiliations: 1Pharmaceutical Department IV, Chugai Research Laboratories, Chugai Pharmaceutical, Co., Ltd., Kanagawa, Japan; 2Pharmacology and
Pathology Research Center, Chugai Research Institute for Medical Science, Inc.,
Shizuoka, Japan; and 3Cancer Institute Hospital, Tokyo, Japan
Received 12/8/04; revised 2/19/05; accepted 3/7/05.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Hisafumi Yamada-Okabe, Pharmaceutical Research
Department IV, Kamakura Research Laboratories, Chugai Pharmaceutical, Co.,
Ltd., 200 Kajiwara, Kamakura, 247-8530 Kanagawa, Japan. Phone: 81-467-454382; Fax: 81-467-45-6782; E-mail: okabehsf@ chugai-pharm.co.jp.
F 2005 American Association for Cancer Research.
Clin Cancer Res 2005;11(11) June 1, 2005
and others often suffer relapse after repeated administration
of bisphosphonate drugs. In a recent study by Body et al. (3),
the percentage of patients who responded to a first dosing
of pamidronate was 90%, but decreased to 80% and 70% at
second and third dosings, respectively. Furthermore, the
response rate to pamidronate was higher in patients with lower
blood parathyroid hormone – related protein (PTHrP) levels
(between 2 and 12 pg/mL) than those with higher PTHrP levels
(higher than 12 pg/mL), suggesting the involvement of PTHrP
in bisphosphonate-refractory HHM (4).
Bisphosphonate-refractory HHM has also been observed in
animal models. Human pancreatic cancer xenografts (FA6)
caused HHM when transplanted into nude mice (5). Administration of a bisphosphonate drug (pamidronate) lowered the
blood calcium levels, but those levels eventually increased even
during the treatment. Although the efficacy of 22-oxa-1,25dihydroxyvitamin D3 (OCT), a vitamin D analogue, was only
moderate, it reduced the blood calcium levels in the
pamindronate-resistant FA6 xenograft model (5). Because
OCT diminishes the expression of parathyroid hormone
(PTH) and PTHrP at a transcriptional level (6), it seems likely
that not only insufficient suppression of bone resorption by
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Role of PTHrP on Osteoclast Inhibitor ^ Resistant HHM
bisphosphonate but other mechanisms are attributable to
emerging bisphosphonate-refractory HHM.
PTHrP, often secreted from tumor cells, is considered to play
a major role in HHM (7, 8). PTHrP and PTH share the parathyroid hormone receptor 1, and the PTHrP fragment consisting
of NH2-terminal 34 amino acids is fully capable of binding and
activating parathyroid hormone receptor 1 (9). The secretion of
PTHrP from tumor cells not only activates osteoclasts but also
enhances renal reabsorption of calcium and consequently
causes hypercalcemia (10, 11). Previously, it was shown that
administration of either monoclonal or polyclonal antibodies
against human PTHrP into nude mice bearing tumor xenografts
that caused hypercalcemia lowered the serum calcium levels
and prolonged survival (12, 13). Furthermore, a humanized
antibody against human PTHrP(1-34) was generated for the
clinical application (14).
In this study, we explored the role of PTHrP in the
bisphosphonate- and calcitonin-refractory HHM and show that
the HHM that is refractory to osteoclastic bone resorption
inhibitors is largely attributable to a high level of circulating
PTHrP and the resulting augmentation of renal reabsorption of
calcium. Thus, suppression of both osteoclastic bone resorption
and renal calcium reabsorption are important for the treatment
of HHM.
the drugs were first administered to the rats that had received five
dosings of alendronate or 12 dosings of elcatonin and thereby
developed alendronate- or elcatonin-refractory HHM. Whole blood
and urine were collected on the indicated days before and after the
administration of drugs.
Determination of the concentrations of parathyroid hormone, parathyroid hormone – related protein, calcium, creatinine, rat osteocalcin, and
rat Laps (rat serum C – telopeptide of type I collagen) in blood and/or
urine. Concentrations of calcium and creatinine in blood and urine
were measured with an automatic analyzer (Hitachi 7170; Hitachi, Ltd.,
Tokyo). Plasma concentrations of the intact rat PTH and human PTHrP
were examined with immunoradiometric assay kits (rat PTH IRMA,
Nihon Mediphysics, Tokyo, Japan and PTHrP IRMA, Mitsubishi
Chemical, Tokyo, Japan). Serum concentrations of rat osteocalcin and
rat Laps [rat serum C – telopeptide of type I collagen (CTX)] were
determined with BIOTRAK rat osteocalcin ELISA kit (Amersham
Pharmacia Biotech, Piscataway, NJ) and Rat Laps ELISA kit (Nordic
Bioscience Diagnostics, Herlev, Denmark), respectively. Fractional
excretion of calcium, which precisely portrays tubular calcium excretion
function in kidney, was determined as the ratio of calcium clearance to
creatinine clearance (glomerular filtration rate).
Statistical analyses. Statistical analyses of the experimental results
were carried out with an SAS statistical package (version 6.12).
Differences in mean values between day 0 and indicated days and
those among study groups were examined by the Dunnett’s multiple
comparison test. Unless otherwise specified, all values indicated are
mean F SD.
Results
Materials and Methods
Cells and animals. The human lung cancer cell line LC-6-JCK
derived from the primary tumors of human lung large cell cancer was
purchased from the Central Institute for Experimental Animals
(Kawasaki, Japan). The cells were maintained in vivo in nude mice
(BALB/cAJcl-nu) and small pieces of tumor tissues (f10 mm3) were
s.c. transplanted into 5-week-old male F344/N Jcl-rnu nude rats. Rats
displaying hypercalcemia and weight loss between 42 and 60 days
after tumor transplantation and those with blood ionized calcium
levels higher than 1.8 mmol/L and at least 0.5 mmol/L higher than
the nontumor bearing rats were used as HHM rats. Nude mice and
nude rats were purchased from Clea Japan, Inc. (Tokyo, Japan), kept
in sterilized cages, and given water and CE2 (Clea Japan). The animals
used in this experiment were treated in accordance with the ethical
guidelines of animal care, handling, and termination of Chugai
Pharmaceutical.
Drugs. Alendronate (10 mg/4 mL ampoule) was purchased from
Teijin, Inc. (Osaka, Japan). Elcatonin (40 units/1 mL ampoule) was
purchased from Asahi Chemical Industry, Co., Ltd. (Osaka, Japan). A
humanized anti-PTHrP antibody version q, which was raised against
the human PTHrP(1-34), was used for the experiments (14).
Treatment. Nude rats showing hypercalcemia were divided into
three groups, each of which consisted of five to seven rats and were
given i.v. saline (1 mL/kg, Otsuka, Japan) as a vehicle control, 3.0 mg/
kg humanized anti-PTHrP antibody (version q; ref. 14), or 5.0 mg/kg
alendronate. The first administration was done f42 days after tumor
transplantation and was defined as day 0. Whole blood and urine were
collected just before the first administration (day 0) and on the
indicated days before and after administration of drugs.
An alendronate-refractory HHM and an elcatonin-refractory HHM
were established by the administration of 2.5 mg/kg alendronate five
times (twice a week, i.v.) and 10 units/kg elcatonin for 6 consecutive
days (twice a day, i.v.), respectively, to the nude rats that developed
HHM. Nude rats used as the alendronate-refractory and the elcatoninrefractory HHM models were divided into three groups, each of which
consisted of five to seven rats, and were given i.v. saline (1 mL/kg),
3.0 mg/kg humanized anti-PTHrP antibody (version q; ref. 14), or
2.5 mg/kg alendronate starting on day 0. Day 0 represents the day when
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Transient decrease of blood ionized calcium and sustained
suppression of bone resorption by alendronate. Nude rats
carrying the human LC-6-JCK tumor xenograft (designated
LC-6 HHM rats in this study) displayed HHM. The blood
ionized calcium of the LC-6 HHM rats was 2.06 mmol/L on
day 49, which was f1.2 to 1.4 mmol/L higher than the
nontumor-bearing rats. In addition, the LC-6 HHM rats
displayed significantly higher levels of serum CTX (70 ng/mL)
and serum osteocalcin (50 ng/mL) compared with those of the
Fig. 1. Changes in blood calcium after a single administration of alendronate. Nude
rats bearing the LC-6-JCK tumor xenograft were given i.v. 5 mg/kg alendronate.
Blood ionized calcium (iCa, o), serum rat CTX (5), and serum rat osteocalcin (4)
after the administration of alendronate are shown. Day 0 represents the day when
alendronate was administered. *, significant difference (P < 0.05) against the values
on day 0. Bars, SD.
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Cancer Therapy: Preclinical
Fig. 2. Changes in blood calcium during repeated administration of alendronate.
Nude rats bearing the LC-6-JCK tumor xenograft were given i.v. 5.0 mg/kg
alendronate four times on the days indicated by arrows. Blood ionized calcium after
the first administration of alendronate is shown. Day 0 represents the day when
alendronate was administered. *, significant difference (P < 0.05) against the values
on day 0. Bars, SD.
nontumor-bearing rats (26 ng/mL for CTX and 31 ng/mL for
osteocalcin), indicating that bone metabolism occurred at a
high turnover rate in this animal model. When LC-6 HHM rats
were given 5 mg/kg alendronate, the blood ionized calcium
dropped to 1.88 mmol/L on day 1 and reached the nadir
(1.69 mmol/L) on day 3. Thereafter, the blood ionized calcium
level increased after day 5 and continued to increase until day
14. The serum level of rat Laps (rat CTX) that most accurately
represents the degree of bone resorption also decreased after
administration of alendronate. Serum levels started declining
as early as day 1 and reached the nadir (28 ng/mL) on day 5.
On the other hand, decrease of the serum osteocalcin, a bone
formation maker, occurred gradually: It remained at a high
level (f49 ng/mL) until day 1 and declined to 22 ng/mL by
day 14. Although the serum CTX level was slightly elevated
after day 5, it remained low (below 40 ng/mL; Fig. 1). Thus,
after day 5, there was no clear correlation between the blood
ionized calcium and bone resorption: Blood ionized calcium
increased although bone resorption remained suppressed.
Because a single administration of alendronate might not be
sufficient to elicit its efficacy, we repeatedly administered
alendronate and measured the blood ionized calcium. When
the LC-6 HHM rats were given alendronate every 7 days at a
dose of 5.0 mg/kg, the blood ionized calcium decreased from
2.06 to 1.54 mmol/L reaching the nadir on day 3, and then it
gradually increased although the alendronate continued to be
administered (Fig. 2). After the fourth administration of
alendronate, the blood ionized calcium level reached 2.06 F
0.12 mmol/L on day 25, which is almost the same as that on
day 0 and is significantly higher than that of nontumor-bearing
rats without any treatment (1.33 F 0.01 mmol/L; Fig. 2).
To further confirm the recurrence of HHM under suppression
of bone resorption from bisphosphonate, we examined the
levels of bone resorption and formation markers in rats that
developed alendronate-refractory HHM. Alendronate-refractory
HHM was established by administering 2.5 mg/kg alendronate
to the LC-6 HHM rats every 3 or 4 days. The LC-6 HHM rats
that received five dosings of alendronate showed a blood
ionized calcium level (1.96 mmol/L) similar to that of before
the administration of the drug (Fig. 3). In these animals, both
serum CTX and osteocalcin decreased to levels close to those
Clin Cancer Res 2005;11(11) June 1, 2005
of nontumor-bearing rats (Fig. 3). To explore the mechanism
underlying bisphosphonate-refractory HHM, we examined
renal calcium excretion. Fractional excretion of calcium was
higher in the LC-6 HHM rats than in nontumor-bearing rats
(0.11 for the LC-6 HHM rats versus 0.02 for nontumor-bearing
rats), and it was not significantly affected even after five dosings
of alendronate (Fig. 3).
Involvement of parathyroid hormone – related protein in
bisphosphonate-refractory humoral hypercalcemia of malignancy.
Both PTH and PTHrP augment renal absorption of calcium
(15). This prompted us to examine PTH and PTHrP levels
during repeated dosing of alendronate. As shown in Fig. 4, the
LC-6 HHM rats showed a high level of blood PTHrP and it
continued to increase during the course of five dosings of
alendronate. On the other hand, the blood level of PTH was
suppressed in the LC-6 HHM rats, and it remained suppressed
even after five dosings of alendronate (Fig. 4). These results
show that increase in the blood calcium, even under sustained
suppression of the bone resorption by alendronate, is largely
attributable to a high level of PTHrP in the circulation and
resulting augmentation of renal calcium reabsorption even
under increased glomerular load of calcium.
If PTHrP is a major causative factor of alendronate-refractory
HHM, neutralizing antibody against PTHrP should decrease the
blood ionized calcium in rats that have developed alendronaterefractory HHM. Previously, we reported the generation of a
humanized monoclonal antibody (mAb) against human
PTHrP(1-34), which was fully capable of neutralizing human
PTHrP and reduced blood calcium in the LC-6 HHM rats (14). As
expected, the anti-PTHrP mAb suppressed renal calcium
reabsorption in the LC-6 HHM rats: Single administration of
the humanized anti-PTHrP antibody at the dose of 3 mg/kg
markedly increased fractional excretion of calcium in the LC-6
HHM rats (Fig. 5). Administration of alendronate at 5 mg/kg also
increased fractional excretion of calcium, but the increase in
Fig. 3. Changes of the blood calcium, bone metabolism, and renal calcium excretion
after repeated administration of alendronate. Blood and urine were collected from
nude rats without tumors (nontumor-bearing, NTB) and from those bearing the
LC-6-JCK tumor xenograft that received (+ALN) ordidnot receive (ALN) repeated
dosing of 2.5 mg/kg alendronate. Blood ionized calcium, serum rat CTX (rat CTX),
serum rat osteocalcin (rat OC), and fractional excretion of calcium (FEca) 67 days
after tumor transplantation are shown. Alendronate was administered five times
(twice a week) starting 42 days after tumor transplantation; nontumor-bearing rats
were given nothing. Fractional excretion of calcium was determined from calcium
clearance (Cca) and creatinine clearance (Ccre). *, significant difference (P < 0.05)
between each group. Bars, SD.
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Role of PTHrP on Osteoclast Inhibitor ^ Resistant HHM
blood ionized calcium of the LC-6 HHM rats, but the blood
ionized calcium level eventually increased even during repeated
dosing of elcatonin. The blood ionized calcium started
increasing on day 3 and reached the original level (2.3 mmol/L)
on day 5 (Fig. 7A). Furthermore, alendronate was no longer
effective in reducing the blood ionized calcium of the LC-6
HHM rats that had received repeated dosing of elcatonin (Fig.
7B), indicating that the suppression of bone resorption alone
did not overcome the elcatonin-refractory HHM. This was also
true for alendronate-refractory HHM: Elcatonin failed to
decrease the blood ionized calcium of the LC-6 HHM rats that
had received repeated dosing of alendronate (not shown). On
the other hand, single administration of 3 mg/kg of anti-PTHrP
mAb effectively reduced the blood ionized calcium of the LC-6
HHM rats that developed elcatonin-refractory HHM (Fig. 7B).
Thus, it seems that the HHM, which becomes refractory to
bisphosphonate or calcitonin, is largely attributable to an
increase in renal calcium reabsorption by PTHrP in the
circulation and that a neutralizing anti-PTHrP mAb is highly
effective for bisphosphonate- or calcitonin-refractory HHM.
Discussion
Fig. 4. Blood levels of PTHrP and PTH before and after repeated administration of
alendronate. Blood was collected from nude rats without tumor and from those
bearing the LC-6-JCK tumor xenograft before (before ALN) and after (afterALN)
repeated dosing of 2.5 mg/kg alendronate. Alendronate was administered five times
(twice a week) starting 42 days after tumor transplantation; nontumor-bearing rats
were given nothing. Concentrations of PTHrP in plasma and PTH in blood were
determined by immunoradiometric assays. *, significant difference (P < 0.05) against
nontumor-bearing rats. Bars, SD.
fractional excretion of calcium by alendronate occurred only on
day 1; saline did not affect the fractional excretion of calcium.
Next, we examined the effects of the anti-PTHrP mAb on
alendronate-refractory HHM. Additional administration of
alendronate to the LC-6 HHM rats that had already received
five dosing of alendronate was no longer effective in reducing
the blood ionized calcium level despite the fact that the levels
of serum CTX and serum osteocalcin remained low (Fig. 6A, C,
and D). On the other hand, the anti-PTHrP mAb markedly
reduced the blood ionized calcium even after the rats received
five dosings of alendronate and the level of the ionized calcium
after administration of the anti-PTHrP mAb was almost the
same as that of the nontumor-bearing rats by day 5 (Fig. 6A).
Decrease of blood ionized calcium after the administration of
the anti-PTHrP mAb was accompanied by an increase in
fractional excretion of calcium: The anti-PTHrP antibody
significantly increased fractional excretion of calcium until
day 3, whereas additional administration of alendronate did
not affect fractional excretion of calcium (Fig. 6B).
We also asked whether development of alendronate-refractory
HHM was specific to bisphosphonate drugs or a general aspect
of agents that suppress osteoclastic bone resorption. Eel
calcitonin derivative elcatonin, another agent that suppresses
bone resorption, was used to address this question. Administration of 10 units/kg of elcatonin twice a day decreased the
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In this study, we showed that LC-6 HHM rats developed
alendronate-refractory HHM after repeated administration of
alendronate. In the LC-6 HHM rats that received multiple
dosings of alendronate, increase in the blood calcium occurred
even under conditions where bone resorption was fully
repressed. Furthermore, administration of the anti-PTHrP
antibody to these rats indeed markedly reduced the blood
ionized calcium levels. Repeated dosing of elcatonin, another
agent that suppresses osteoclastic bone resorption, also resulted
in elcatonin-refractory HHM. In addition, alendronate was
ineffective after the animals developed elcatonin-refractory
HHM. From these results, we concluded that bisphosphonateand calcitonin-refractory HHM is largely attributable to an
Fig. 5. Effects of anti-PTHrP antibody on fractional excretion of calcium. Nude rats
bearing the LC-6-JCK tumor xenograft were given i.v. vehicle (saline, o), 5.0 mg/kg
alendronate (4), or 3mg/kg anti-PTHrP antibody (5). Blood and urine were
collected from animals without tumor and from those bearing the LC-6 tumor
xenograft. Fractional excretion of calcium determined from calcium clearance and
creatinine clearance after the administration of the indicated drugs is shown. Day 0
represents the day when saline, alendronate, and the anti-PTHrP were administered;
nontumor-bearing rats were given nothing. *, significant difference (P < 0.05)
against the values on day 0. Bars, SD.
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Cancer Therapy: Preclinical
enhanced renal reabsorption of calcium by PTHrP in the
general circulation.
The LC-6 HHM rats showed a high turnover of bone
metabolism. Bisphosphonate, such as alendronate, is rapidly
distributed onto the active surface of the bone and causes
suppression of osteoclasts when it is engulfed by the cells. On
the other hand, suppression of osteoblastic functions is
considered not to be a direct effect of bisphosphonate but
rather a response to suppressed bone resorption (coupling
phenomenon). When the LC-6 HHM rats were given alendronate, suppression of bone resorption had occurred by day 1,
but decrease of bone formation occurred only after day 2. The
resulting dissociation between bone formation and resorption
under the condition of high bone turnover might contribute to
the initial decrease of blood calcium.
Several factors have been reported that control renal calcium
reabsorption (16). These factors include the blood levels of
calcium and phosphate, calcium-regulating hormones,
Fig. 6. Effects of anti-PTHrP antibody on the levels of blood calcium, fractional
excretion of calcium, CTX, and osteocalcin in rats with alendronate-refractory
HHM. Nude rats bearing the LC-6-JCK tumor xenograft were given i.v.
2.5 mg/kg alendronate five times on the days indicated by arrows to induce
alendronate-refractory HHM. Thereafter, they were further given vehicle
(saline, o), 2.5 mg/kg alendronate (4), or 3 mg/kg anti-PTHrP antibody (5).
Blood and urine were collected from animals, and blood ionized calcium (A),
fractional excretion of calcium (B), serum rat osteocalcin (C), and serum rat
CTX (D) after the administration of the drugs are shown. Day 0 represents
the day when saline, alendronate, and the anti-PTHrP were administered after
rats received five dosings of alendronate, thereby developing alendronaterefractory HHM. *, significant difference (P < 0.05) against the values of the
control vehicle group. Blood ionized calcium, fractional excretion of calcium,
serum osteocalcin, and blood CTX of nontumor-bearing rats (open columns)
and those of rats bearing the LC-6-JCK tumor xenograft (filled columns).
Bars, SD.
Clin Cancer Res 2005;11(11) June 1, 2005
Fig. 7. Effects of anti-PTHrP antibody on blood calcium levels in rats with
calcitonin-refractory HHM. A, nude rats bearing the LC-6-JCK tumor xenograft
were given i.v. 10 units/kg elcatonin or saline twice a day for 6 consecutive
days as indicated by arrows. Day 0 represents the day when elcatonin was first
administered. Blood ionized calcium of the rats given elcatonin (.) or vehicle
(o) is shown. B, nude rats bearing the LC-6-JCK tumor xenograft were given
i.v. 10 units/kg elcatonin twice a day for 6 consecutive days as indicated by
arrows. Thereafter, they were further given vehicle (saline, o), 2.5 mg/kg
alendronate (4), or 3 mg/kg anti-PTHrP antibody (5). Day 0 represents the
day when the indicated drugs were administered to the rats that received
12 dosings of elcatonin, thereby developing elcatonin-refractory HHM. Blood
ionized calcium of nontumor-bearing rats (open columns) and rats bearing the
LC-6 tumor xenograft (filled columns). Bars, SD. *, significant difference
(P < 0.05) against the control vehicle group. Ab, anti-PTHrP mAb.
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Role of PTHrP on Osteoclast Inhibitor ^ Resistant HHM
electrolytes in the circulatory system and renal blood flow, and
glomerular filtration rate. Among these factors, phosphate
metabolism is found to be dysregulated in patients with HHM
(17, 18). In the LC-6 HHM rats, the blood level of calcium was
higher than that of nontumor-bearing rats, but levels of
phosphate, chloride, albumin, and hemoglobin were lower,
which is in good agreement with HHM patient profiles. In
addition, we observed that the blood level of phosphate was
further decreased after the alendronate treatment in LC-6
HHM rats (not shown). Moreover, plasma level of PTHrP was
high and that of PTH was low in the LC-6 HHM rats
compared with the nontumor-bearing rats; increased PTHrP
and decreased PTH in the circulation has also been observed
in HHM patients (10). Thus, the LC-6 HHM rat model seems
to well represent the human HHM and, therefore, the antiPTHrP antibody will be effective in human patients who develop HHM, which is refractory to osteoclastic bone resorption
inhibitors.
Acknowledgments
We thank F. Ford for proofreading the manuscript.
References
1. Ralston SH, Gallacher SJ, Patel U, Campbell J, Boyle
IT. Cancer-associated hypercalcemia: morbidity and
mortality. Clinical experience in 126 treated patients.
Ann Intern Med 1990;112:499 ^ 504.
2. Fleisch H. Bisphosphonates. Pharmacology and
use in the treatment of tumour-induced hypercalcaemic and metastatic bone disease. Drugs 1991;42:
919 ^ 44.
3. BodyJJ, Louviaus I, DumonJC. Decreased efficacy of
bisphosphonates for recurrences of tumor-inducedhypercalcemia. Support Care Cancer 2000;8:398 ^ 404.
4. Gurney H, Grill V, Martin TJ. Parathyroid hormonerelated protein and response to pamidronate in tumour-induced hypercalcemia. Lancet 1993;341:
1611 ^ 3.
5. Endo K, Katsumata K, Iguchi H, et al. Effect of combination treatment with a vitamin D analog (OCT) and a
bisphosphonate (AHPrBP) in a nude mouse model of
cancer-associated hypercalcemia. J Bone Miner Res
1998;13:1378 ^ 83.
6. Endo K, Ichikawa F, Uchiyama Y, et al. Evidence for
the uptake of a vitamin D analogue (OCT) by a human
carcinoma and its effects of suppressing the transcription of parathyroid hormone-related peptide gene
in vivo. J Biol Chem 1994;269:32693 ^ 9.
www.aacrjournals.org
7. Mundy GR, Martin TJ. The hypercalcemia of malignancy: pathogenesis and management. Metabolism
1982;31:1247 ^ 77.
8. Mundy GR, Guise TA. Role of parathyroid hormonerelated peptide in hypercalcemia of malignancy and
osteolytic bone disease. Endocr Relat Cancer 1998;
5:15 ^ 26.
9. Juppner H, Abou-Samra AB, Freeman M, et al. A G
protein-linked receptor for parathyroid hormone and
parathyroid hormone-related peptide. Science 1991;
254:1024 ^ 6.
10. Mundy GR, GuiseTA. Hypercalcemia of malignancy.
Am J Med 1997;103:134 ^ 45.
11. Syed MA, Horwitz MJ, Tedesco MB, Garcia-Ocana
A, Wisniewski SR, Stewart AF. Parathyroid hormonerelated protein-(1-36) stimulates renal tubular calcium
reabsorption in normal human volunteers: implications
for the pathogenesis of humoral hypercalcemia of malignancy. J Clin Endocrinol Metab 2001;86:1525 ^ 31.
12. Kukreja SC, Shevrin DH, Wimbiscus SA, et al. Antibodies to parathyroid hormone-related protein lower
serum calcium in athymic mouse models of malignancy-associated hypercalcemia due to human tumors.
J Clin Invest 1988;82:1798 ^ 802.
13. Sato K, Yamakawa Y, Shizume, K, et al. Passive
4203
immunization with anti-parathyroid hormone-related
protein monoclonal antibody markedly prolongs survival time of hypercalcemic nude mice bearing
transplanted human PTHrP-producing tumors.
J Bone Miner Res 1993;8:849 ^ 60.
14. Onuma E, Sato K, Saito H, et al. Generation of a humanized monoclonal antibody against human parathyroid hormone-related protein and its efficacy against
humoral hypercalcemia of malignancy. Anticancer
Res 2004;24:2665 ^ 74.
15. Brown EM. Homeostatic mechanisms regulating extracellular and intracellular calcium metabolism. In:
Bilezikian JP, Marcus R, Levine MA, editors. The parathyroids, basic and clinical concepts. NewYork: Raven
Press; 1994. p. 15 ^ 52.
16. GuiseTA, Mundy GR. Cancer and bone. Endocr Rev
1998;19:18 ^ 54.
17. Strewler GJ, Nissenson RA. Skeletal and renal
actions of parathyroid hormone-related protein. In:
Bilezikian JP, Marcus R, Levine MA, editors. The parathyroids, basic and clinical concepts. NewYork: Raven
Press; 1994. p. 311 ^ 20.
18. Sartori L, Insogna KL, Barrett PQ. Renal phosphate
transport in humoral hypercalcemia of malignancy. Am
J Physiol 1988;255:F1078 ^ 84.
Clin Cancer Res 2005;11(11) June 1, 2005
Downloaded from clincancerres.aacrjournals.org on June 16, 2017. © 2005 American Association for Cancer
Research.
Increased Renal Calcium Reabsorption by Parathyroid
Hormone−Related Protein Is a Causative Factor in the
Development of Humoral Hypercalcemia of Malignancy
Refractory to Osteoclastic Bone Resorption Inhibitors
Etsuro Onuma, Yumiko Azuma, Hidemi Saito, et al.
Clin Cancer Res 2005;11:4198-4203.
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