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Department of Internal Medicine, Karl Franzens University (H.D.), Graz, Austria; and the Department of Orthopedics, The Mayo Foundation (R.T.T.), Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Russell T. Turner, Ph.D., Orthopedic Research, Room 3–69, Medical Science Building, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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The potent anabolic actions of PTH on rat bone has stimulated considerable interest in the possible therapeutic use of the hormone for treating human skeletal disorders and has been recently reviewed by Mosekilde and Reeve (14). An especially important currently unresolved clinical problem that might be amenable to PTH therapy is the restoration of bone to the severely osteopenic skeleton. There are, however, serious questions regarding possible limitations of PTH therapy, including undesirable side-effects and parenteral mode of administration of the hormone.
Recent reviews document that continuous exposure to elevated PTH in humans and laboratory animals results in hypercalcemia and a variety of abnormalities in bone (14, 15, 16). The purpose of the present study was to more precisely determine the duration of exposure to PTH that results in increased bone formation without hypercalcemia. Additionally, we performed these studies to establish whether programmed administration of implanted hormone is a feasible alternative to daily injection.
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Materials and Methods |
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Preliminary study: clearance of PTH
The time course for clearance of PTH from the circulation was
estimated after sc administration of human PTH-(1–34) (hPTH; Bachem,
Torrance, CA; 80 µg/kg BW) to 22 14-month-old virgin female rats.
hPTH was dissolved in a solution containing 150 mM NaCl, 1
mM HCl, and 2% heat-inactivated rat serum. The rats were
anesthetized with ethrane and killed with exsanguination via the
abdominal aortic artery. Serum was frozen at -70 C until assayed for
immunoreactive PTH (iPTH).
Study 1: effects of programmed administration of hPTH for 6
h/day
The purpose was to compare programmed infusion of hPTH for 6
h/day with sc administration of the hormone. Three groups of virgin
6-month-old female rats with six or seven animals in each group were
studied. One group received hPTH by sc injection (80 µg/kg·day)
each day for 6 days. The two other groups received osmotic pumps with a
tubing system external to the pump that was designed to deliver vehicle
alone or hPTH (80 µg/kg·day) for 6 h/day for 6 days.
The reservoir volume of the pumps was filled with saline solution, and the pumps were primed at 37 C for 6 h sitting in a microcentrifuge tube also filled with saline before the delivery portal of the pump was connected to a coiled external tubing system consisting of PE60 polyethylene tubing (Becton Dickinson, Sparks, MD). The tubing itself was prepared in the following way; a regular straight piece of tubing material was wrapped around a pencil with both ends fixed to the pencil, then submerged in boiling water for 3 min and chilled in an ice-cold water bath. The coil was then loaded with sesame oil alone (vehicle pump group) or interspaced with 6 µl hPTH solution to produce a regular pulsatile release of 6 h "on hormone" followed by 18 h "off hormone," respectively. A methylene blue dye mark of 2 µl was positioned at the end of the coil facing the outlet of the pump. This marker migrated toward the opening end of the tubing system during the course of the study and indicated proper emptying of the vehicle or hormone at the time of death.
A calcein (20 mg/kg BW; Sigma Chemical Co., St. Louis, MO) and tetracycline (20 mg/kg BW; Sigma) label was given by perivascular tail injection on days 0 and 5 to all animals.
Study 2: effects of programmed (1 and 2 h/day) and continuous
administration of hPTH
The purpose was to further refine the optimal duration of
infusion of hPTH by programmed administration of the hormone. Five
groups of 4-month-old male rats, with 4–10 animals in each group, were
studied. One group received hPTH daily (80 µg/kg·day) for 6 days by
sc injection. The remaining 4 groups were implanted with Alzet osmotic
pumps as described for study 1 (Alza Corp., Palo Alto, CA), which were
designed to deliver for 6 days vehicle solution only, continuous hPTH
(40 µg/kg·day), and intermittent hPTH at 1 and 2 h/day (80
µg/kg·day) at a nominal pumping rate of 1 µl/h.
Serum chemistry
Total serum calcium, serum phosphorous, and alkaline phosphatase
activity were determined using a Hitachi 717 (Hitachi, Hialeah,
FL).
Serum iPTH was measured using an immunoradiometric assay for rat PTH (Nichols Institute, San Juan Capistrano, CA) that exhibits cross-reactivity for hPTH of approximately 100%. Rat serum samples with expected high values of hPTH were diluted up to 20-fold with the 0 pg/ml rat PTH standard and assayed. In rats that were not injected or infused with hPTH, the measured concentrations reflect endogenously secreted "rat" PTH. For measurements of 1,25-dihydroxyvitamin D (1,25D), serum samples were dilipidated, extracted with a highly specific solid phase monoclonal anti-1,25D, and quantitated by RIA (IDS, Boldon, UK). High 1,25D concentrations were measured after a 10-fold dilution of the samples with the zero calibrator.
Histomorphometry
Histomorphometric procedures were carried out using an imaging
system that has been described in detail previously (11).
Cancellous bone measurements made on stained sections
The metaphysis was dehydrated, embedded without
demineralization, and sectioned as previously described (11).
Bone architecture measurements and calculations
Cancellous bone area was determined in unstained 5-µm thick
sections as the area of total cancellous bone per mm2
metaphyseal tissue within the sampling site and expressed as a
percentage (tissue referent). Cancellous bone perimeter was expressed
as the perimeter of cancellous bone per mm2 metaphyseal
sampling area (tissue referent). Calculations relating to cancellous
bone perimeter and area (17) were the following: 1) trabecular
thickness, calculated as the cancellous bone perimeter (millimeters)
divided by the cancellous bone area (square millimeters per
mm2), then dividing that number by 2 and multiplying by
1000 (millimeters); 2) trabecular number, defined as the cancellous
bone area (square millimeters per mm2) divided by the
trabecular thickness (microns); and 3) trabecular separation,
calculated as the trabecular thickness (millimeters) divided by the
cancellous bone area (square millimeters per mm2).
Bone cell measurements
The cancellous bone perimeters lined by osteoblasts and
osteoclasts were measured and expressed as percentages (perimeter
referent). Briefly, undemineralized 5-µm thick sections were stained
with toluidine blue. Osteoclast perimeter was determined as the
cancellous perimeter lined by multinucleated cells. These cells usually
had other characteristics of osteoclasts, including a foamy cytoplasm
and location in a pronounced lacuna. Osteoblasts were identified as a
palisade of large basophilic cuboidal cells directly lining a bone
perimeter. The peritrabecular fibrotic marrow perimeter was identified
as the bone perimeter lined by multiple layers of fibroblasts (see Fig. 3
).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The maximum concentration of iPTH
(>15,000 pg/ml) was observed 15 min after hormone administration. The
serum levels of the hormone declined rapidly; they were less than 10%
of the peak value at 1 h and did not differ from the pretreatment
value at 6 h. The calculated t1/2 for the
disappearance of serum iPTH was 11 min. Subsequent studies designed to
determine the optimal duration of programmed infusion of hPTH focused
on pulses of 6 h in duration or less.
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(A and B). Infusion of hPTH at
concentrations of 2 h/day and higher led to a significant decrease in
body weight, with a mean reduction of up to 11% in the group with
continuous hPTH infusion. Unexpectedly, programmed infusion of hPTH for
6 h/day led to a mortality rate of 43% (three of seven rats died). In
the subsequent study, the dose rate in the group continuously infused
with hPTH was reduced to decrease the risk of life-threatening
hypercalcemia; none of the animals in the other groups died during the
course of the study.
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(A and B) and Fig. 2. Daily sc hPTH administration for 7
days to female rats (Table
2A) had no effect on serum chemistry, which
consisted of measurements of calcium, phosphorous, alkaline phosphatase
activity, and iPTH. In contrast, programmed infusion of hPTH for 6
h/day resulted in severe hypercalcemia, no change in phosphorous,
depressed alkaline phosphatase activity, and greatly elevated iPTH. In
male rats (Table 2B), sc PTH had no effect on calcium or alkaline
phosphatase, and increased phosphorous and 1,25D3 levels
(Fig. 2). Programmed infusion of hPTH for either 1 or 2 h/day had no
effect on serum chemistry. In contrast, continuous infusion of hPTH
resulted in hypercalcemia, no change in phosphorous, depressed alkaline
phosphatase activity, increased iPTH, and decreased
1,25D3.
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, continuous
(male rats) and programmed infusion of hPTH for 6 h/day (female rats)
resulted in similar marked histological changes in the proximal tibia,
consisting of multiple layers of fibroblastic cells encircling
trabecular profiles. Additionally, focal increases in resorption
lacunae with osteoclasts were observed. However, equal or larger doses
of hPTH administered sc or by programmed infusion for up to 2 h/day did
not result in abnormal bone histology.
The effects of hPTH on cancellous bone histomorphometry were evaluated
in male rats and are summarized in Tables 3–5. Short term sc treatment, continuous
infusion, and programmed infusion for 2 h/day with hPTH had no effect
on indexes of cancellous bone architecture, consisting of bone area,
trabecular number, trabecular thickness, and trabecular separation
(Table 3). In contrast, programmed infusion of hPTH for 1 h/day
resulted in increases in bone area and trabecular number, no change in
trabecular thickness, and a decrease in trabecular separation.
Subcutaneous administration and programmed infusion of hPTH for 1 h/day
resulted in increases in dynamic bone measurements and calculated bone
values, consisting of double label perimeter, mineral apposition rate,
and bone formation rate (Table 4).
Continuous infusion of hPTH had no effect on dynamic bone
histomorphometry, whereas programmed administration of hPTH for 2 h/day
increased the mineral apposition rate, but did not influence the double
label perimeter or bone formation rate Table 5. The effects of hPTH on cancellous
perimeter lined by osteoblasts, osteoclasts, and intertrabecular
fibrotic marrow in male rats are shown in Table 4. Osteoblast perimeter
was increased in rats treated with hPTH sc and with programmed infusion
of PTH for 1 h/day. Continuous treatment and programmed infusion of the
hormone for 2 h/day had no effect on osteoblast perimeter. The only
change in osteoclast number in males was an increase after continuous
infusion with hPTH. Similarly, peritrabecular fibrotic marrow was only
observed in male rats after continuous infusion with hPTH.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intermittent and programmed administration of hPTH for 1 h/day resulted in large increases in the label perimeter and calculated bone formation rate and small increases in the mineral apposition rate. The dynamic measurements may underestimate the calculated mineral apposition rate and bone formation rate, because the initial fluorochrome label was administered before initiating hormone treatment.
The noted differential effects of chronic and intermittent
exposure to hPTH are well recognized, but the precise mechanisms that
mediate the respective responses have not been identified. Daily sc
hPTH treatment increases bone mass over a wide dose range, without
resulting in hypercalcemia. Additionally, the hormone can be
administered as often as three times per day (3). In contrast,
continuous infusion of hPTH, although leading to much lower peak blood
levels of the hormone (compare Fig. 1 and Table 2) resulted in severe
pathology (Fig. 3). Thus, it is reasonable to assume that the critical
variable is duration of exposure to elevated levels of PTH rather than
peak levels of the hormone.
The time-course study revealed that the vast majority of administered hPTH was cleared from circulation within 1 h after sc administration of the hormone. This finding suggested that programmed infusion of hPTH over a short time frame should result in changes in bone and serum chemistries similar to those after sc administration of the hormone. This was found to be the case. On the other hand, prolonging the infusion of the same total dose of hPTH for 2 and 6 h/day resulted in progressive increases in undesirable changes that more closely emulated continuous hPTH infusion.
Our findings clearly demonstrate that the therapeutic duration of exposure to hPTH is approximately 1 h. Failure to clear the hormone from the circulation in less than 2 h due to excessive dose, retarded absorption, reduced degradation, or decreased clearance is likely to result in undesired side-effects. Although there may be species differences in the clearance of biologically active PTH (bPTH), the t1/2 for the disappearance of serum iPTH in this study of about 11 min in old female rats was very similar to that reported in humans (18). We did not measure bPTH, but studies in humans have demonstrated that bPTH declines more rapidly than iPTH (18).
The rapid clearance of hPTH is the likely explanation for the reported optimal response of the rat skeleton to a three times per day sc dose regimen. Although theoretically possible to mimic using osmotic pumps, this regimen would be technically more difficult to achieve and is unlikely to have pronounced additional beneficial effects.
The increase in serum 1,25D3 after sc administration of
hPTH was expected, and was probably due to up-regulation of
25-hydroxyvitamin D3:1
-hydroxylase in the kidney (19).
The mechanism for the remarkable decrease in 1,25D3 in the
rats made hypercalcemic by continuous infusion of hPTH is less clear,
but may have been due to a direct inhibition of the 1-hydroxylase by
calcium, as reported in previous investigations (19, 20, 21).
Serum alkaline phosphatase was insensitive to the large increase in cancellous bone formation in the proximal tibial metaphysis and was decreased in male and female rats after continuous infusion. This finding indicates that alkaline phosphatase activity is not a good marker for hPTH-induced changes in bone turnover in rats.
Intermittent PTH resulted in a small increase in serum phosphorous in male, but not female, rats. We have no explanation for this difference, but Wronski et al. reported a transient increase in serum phosphorous in PTH-treated female rats, so it is unlikely that the differences observed between male and female rats in the present studies reflect an inherent sex difference (5).
In summary, the results of these studies demonstrate that the therapeutic duration of exposure to hPTH to initiate increases in osteoblast number and bone formation is critical and very brief. Nevertheless, programmed administration of implanted hPTH is feasible as an alternative to sc injection as a route for administration of the hormone.
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Acknowledgments |
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Footnotes |
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Received April 28, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase: measurement and regulation. In: Kumar R (ed)
Vitamin D Metabolism: Basic and Clinical Aspects. Martinus Nihoff,
Boston, pp 175–196
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