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Original Studies |
Department of Pathology, Wake Forest University School of Medicine (R.B., C.E.H., C.J.L., M.W.S., C.P.J.), Winston-Salem, North Carolina 27157-1040; and Lilly Research Laboratories (J.M.H.), Indianapolis, Indiana 46285
Address all correspondence and requests for reprints to: Robert Brommage, Ph.D., Section on Comparative Medicine, Department of Pathology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157-1040. E-mail: brommage{at}wfubmc.edu
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Abstract |
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Adult female cynomolgus monkeys were divided into sham-vehicle (n = 21), ovariectomized (OVX)-vehicle (n = 20), and OVX groups given daily sc injections of rhPTH-(1–34) at 1 (n = 39) or 5 (n = 41) µg/kg for 12 months. Whole body bone mineral content was measured, as was bone mineral density (BMD) in the spine, proximal tibia, midshaft radius, and distal radius. Serum and urine samples were also analyzed. rhPTH-(1–34) treatment did not influence serum ionized Ca levels or urinary Ca excretion, but depressed endogenous PTH while increasing serum calcitriol levels. Compared to that in the OVX group, the higher dose of rhPTH-(1–34) increased spine BMD by 14.3%, whole body bone mineral content by 8.6%, and proximal tibia BMD by 10.8%. Subregion analyses suggested that the anabolic effect of rhPTH-(1–34) on the proximal tibia was primarily in cancellous bone. Similar, but less dramatic, effects on BMD were observed with the lower dose of rhPTH-(1–34). Daily sc rhPTH-(1–34) treatment for 1 yr increases BMD in ovariectomized monkeys without inducing sustained hypercalcemia or hypercalciuria.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although fluoride therapy can increase bone mass, several problems have prevented its widespread acceptance as a treatment for osteoporosis. These problems include determining its optimal dose and formulation, gastric complications, nonresponders, the production of the more brittle bone mineral fluoroapatite in place of hydroxyapatite, and a presumed failure of some patients to appropriately increase their intestinal absorption of calcium to supply the calcium required for new bone formation (1).
Considerable attention has recently been devoted to the possible use of PTH and its analogs as skeletal-specific anabolic agents (2, 3, 4). PTH administration increases bone mass in dogs (5, 6), osteoporotic men (7), premenopausal women with endometriosis receiving GnRH analog therapy to block ovarian function (8), and postmenopausal women with osteoporosis (9, 10). The effectiveness of PTH administration to increase bone mass in rats under a variety of physiological conditions has been extensively characterized (2, 3, 4). Although the exact biochemical mechanism for this anabolic effect has not been fully elucidated (11), PTH must be given intermittently rather than continuously to increase bone mass (5, 12, 13).
The purpose of this study was to examine the skeletal actions of recombinant human PTH-(1–34) [rhPTH-(1–34)] given by daily sc injections for 12 months to adult ovariectomized (OVX) cynomolgus monkeys, a species that undergoes Haversian remodeling of cortical bone. This paper focuses on bone densitometry and serum and urine parameters related to bone metabolism. Initial data obtained after 6 months have been presented in abstract form (14).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Monkeys were fed a purified diet containing 0.3% calcium (Ca), 0.3% phosphorus (P), 29.5% protein, 55% carbohydrate, 4.9% fat, and 9% fiber. Therefore, fat provided 13.3% of the total calories, and Ca consumption was 1734 mg/2000 cal, a value similar to that of women taking a daily 1-g Ca supplement.
rhPTH-(1–34), LY333334, was produced at Lilly Research Laboratories (Indianapolis, IN) using recombinant DNA technology and stored lyophilized at -20 C. Doses were prepared daily in plastic CZ vials (West Co., Lionville, PA) in a sterile vehicle of saline containing 20 mmol/L NaH2PO4. Subcutaneous injections (0.1 ml/kg) in the dorsal region were given 7 days/week starting the day after OVX/sham surgery. Monkeys were injected between 0900–1300 h, during which time they did not have access to their diet. On 20 separate days during the course of the study, duplicate aliquots of the dosing solutions were diluted with an equal volume of double strength high performance liquid chromatography (HPLC) solvent, frozen, and subsequently analyzed for rhPTH-(1–34) purity and concentration by peak area analysis during chromatography on a C18 reverse phase column. rhPTH-(1–34) was detected by its absorbance at 214 nm, and the mobile phase was 27% acetonitrile, 0.1% 18 mol/L sulfuric acid, and 53.9% 50 mmol/L NaCl dissolved in water. rhPTH-(1–34) was always found to be pure and at the expected concentrations of 10 and 50 µg/mL.
Spine (lumbar vertebrae 2–4) BMD and whole body bone mineral content (BMC; including the head) were measured at baseline and 6 and 12 months with a QDR/1500 densitometer (Hologic, Inc., Waltham, MA). BMD at the midshaft radius, distal radius, and proximal tibia was determined at baseline and 12 months by peripheral quantitative computed tomography (pQCT) using a Stratec 960A XCT (Stratec Medizintechnik, Pforzheim, Germany) densitometer. For the proximal tibia and distal radius, four concentric subregions of equal area (designated I, II, III, and IV; defined as the outermost to the innermost zones, respectively) were generated for analysis. These zones do not strictly correspond to specific anatomical locations (such as cortical and cancellous bone), as the thickness of the cortex varies among and within bones. This analysis was chosen to facilitate interpretation of the data, as it was anticipated that the transitional zone (the interface between cortical and cancellous bone) might drift during the study. During these procedures, monkeys were pretreated with 0.07 mg/kg atropine, im, then anesthetized 10 min later with 20 mg/kg ketamine, im, and maintained with isoflurane by inhalation.
Blood was collected at 3-month intervals after an overnight fast under ketamine anesthesia from the femoral artery/vein for serum analyses. In all cases, blood was obtained before the normal daily doses were given, typically about 22 h after the previous dose. A 0.6-mL aliquot of blood was kept in a separate capped plastic tube for measurement of serum ionized Ca levels (corrected to pH 7.4) using an AVL 988/4 Electrolyte Analyzer (AVL Scientific, Roswell, GA). Serum levels of total Ca, inorganic phosphate, creatinine, urea nitrogen, and total alkaline phosphatase activity were determined with automated procedures using a COBAS FARA II system (Roche Diagnostic Systems, Montclair, NJ). Baseline serum levels of 25-hydroxyvitamin D were measured by RIA (INCSTAR Corp., Stillwater, MN). Serum levels of estradiol (RIA from Diagnostic Products, Los Angeles, CA), osteocalcin (RIA from Diagnostic Systems Laboratories, Inc., Webster, TX), calcitriol (RIA from INCSTAR Corp.), and intact PTH (immunoradiometric assay from Diagnostic Systems Laboratories, Inc.) were measured at the Yerkes Regional Primate Research Center (Lawrenceville, GA).
Urine was collected over 23–25 h by placing the monkeys in individual metabolic cages. Aliquots were analyzed for Ca and creatinine by automated procedures as described above, for cAMP by RIA (from INCSTAR Corp.) at Yerkes, and for the C-terminal cross-linked peptide of type I collagen (CrossLaps, Diagnostic Systems Laboratories, Inc.) by enzyme-linked immunosorbent assay.
Data are presented as the mean ± SEM. All statistical analyses were performed by analysis of covariance comparing the sham monkeys and two treatment groups to the OVX monkeys receiving vehicle, with corrections for multiple comparisons using the Bonferroni procedure. Serum and urine data obtained for each individual monkey at 3, 6, 9, and 12 months were averaged with individual baseline values used as covariates. Data for serum creatinine and urea nitrogen were treated in a similar fashion, except that these parameters were only measured at 9 and 12 months. For the DXA data, an additional analysis was performed to examine whether the changes in each group were different between the first 6 months and the second 6 months of treatment.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Serum values presented in Table 1
show that ovariectomy lead to elevated levels of total Ca but no change
in ionized Ca levels. Treatment with rhPTH-(1–34) did not influence
either total or ionized Ca values. Serum P levels were increased by
ovariectomy, but decreased to levels observed in sham monkeys with
rhPTH-(1–34) treatment. There were no effects of ovariectomy or
treatment on serum levels of creatinine and urea nitrogen. Ovariectomy
did not influence serum levels of intact PTH, but decreased serum
calcitriol levels (Fig. 1). Endogenous
levels of intact PTH were reduced with rhPTH-(1–34) treatment, whereas
this treatment increased serum calcitriol to levels similar to those
observed in sham monkeys.
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) of bone
formation markers (osteocalcin and alkaline phosphatase) together with
increased urinary excretion (Fig. 3) of
the C-terminal type I collagen cross-linked peptide (CrossLaps), a
marker of bone resorption. Monkeys treated with rhPTH-(1–34) tended to
have even higher levels of osteocalcin, alkaline phosphatase, and
CrossLaps, but this effect was not statistically significant, except
for alkaline phosphatase in the high dose treatment.
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. There was no effect of treatment on
urinary Ca excretion. Although ovariectomy did not influence urinary
cAMP excretion, this parameter was increased slightly with the low dose
and 2-fold with the high dose of rhPTH-(1–34).
Spine BMD values are presented in Fig. 4
as percent changes from baseline to 6 months, 6 months to 12 months,
and cumulative baseline to 12 months. Consistent with our previous
experience, spine BMD increased approximately 6% in the sham group and
declined slightly in OVX monkeys. Both doses of rhPTH-(1–34) increased
spine BMD during both 6-month intervals, with greater increases
occurring during the first 6 months than the second 6 months
(P < 0.05 and P < 0.001 for the low
and high dose treatments, respectively). For the 5 µg/kg dose of
rhPTH-(1–34), there was an inverse correlation (r = -0.59;
P < 0.001) between baseline spine BMD and the 12-month
gain in spine BMD.
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), and again, the sham group gained BMC
(12%), with a smaller gain (5%) observed in the OVX monkeys.
rhPTH-(1–34)-treated monkeys had greater gains in whole body BMC than
the OVX group over 12 months, with this bone gain occurring during the
first 6 months of treatment. For the 5 µg/kg dose of rhPTH-(1–34),
there was an inverse correlation (r = -0.49; P <
0.001) between baseline whole body BMC and the 12-month gain in whole
body BMC.
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. Neither ovariectomy nor rhPTH-(1–34)
administration influenced total BMD in the midshaft or distal radius.
Subregion analysis of distal radius BMD (data not shown) indicated that
rhPTH-(1–34) treatment did not result in a redistribution of bone
within subregions. In the proximal tibia, ovariectomy resulted in a
slight, but nonsignificant, decrease in BMD, whereas both doses of
rhPTH-(1–34) increased BMD.
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) suggested that ovariectomy led to a
consistent decrease in BMD in all four zones, but these declines never
reached statistical significance. As expected, absolute BMD values were
greatest in the two outermost zones, reflecting the cortical bone
present. The increase in total BMD produced by the higher dose of
rhPTH-(1–34) resulted from anabolic actions in the innermost three
zones with no effect in the outermost zone. The response to the lower
rhPTH-(1–34) dose was more variable and of lower magnitude.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although we found no evidence of cortical bone loss, PTH-(1–34) treatment appears to have dramatic effects on cortical bone turnover. A previous 6-month study in our laboratory showed approximately 3-fold elevations in cortical bone osteonal remodeling in the midshafts of both femur and radius of ovariectomized monkeys treated three times per week with 10 µg/kg PTH-(1–34) (15). Similar results were observed in tibia obtained from rabbits treated for 20 weeks with 10 and 40 µg/kg rhPTH-(1–34) (16) and in ribs obtained from dogs treated for 24 weeks with PTH-(1–34) (17).
Recent successful human trials in women have examined PTH-(1–34) doses of 25 µg (10), 40 µg (8), and 50 µg (9) per woman, and our low dose of 1 µg/kg is only slightly higher than these human doses. Compared to the OVX control group, spine BMD increased by 9.4% with this low dose of rhPTH-(1–34). The magnitude of this effect appears to be similar to or slightly greater than the increases in spine BMD observed in the human studies. One advantage of our study is that we also examined a 5 µg/kg dose of rhPTH-(1–34), and although the two doses were not compared statistically, the higher dose appeared to promote greater increases in both spine (14.3% vs. 9.4%) and proximal tibia (68 vs. 33 mg/mm3) BMD as well as whole body BMC (8.6% vs. 3.3%). Thus, our experience in monkeys suggests that higher doses of PTH-(1–34) can be successfully given to women. Interestingly, a single daily injection of about 1.5 µg/kg PTH-(1–34) is effective in treating hypoparathyroid subjects (18), suggesting that the therapeutic dose for osteoporosis is similar to the physiological replacement dose.
We observed increases in spine BMD and whole body BMC after 6 months of treatment with rhPTH-(1–34). This result was not surprising given similar time-course observations in women (8, 9, 10). Because the effects of ovariectomy and rhPTH-(1–34) treatment on serum and urine parameters of bone metabolism appeared to be similar at 3, 6, 9, and 12 months, these values have been averaged for each individual monkey over these quarterly time points. The ability of rhPTH-(1–34) treatment to promote apparently constant changes in urinary cAMP excretion and serum levels of P, calcitriol, and endogenous PTH over 12 months suggests a lack of refractoriness to continued rhPTH-(1–34) administration for these responses. Additional studies are required to examine the time course of the anabolic bone response with continued rhPTH-(1–34) administration.
rhPTH-(1–34) treatment did not promote sustained hypercalcemia or hypercalciuria in the monkeys. These observations are important, because such responses are usually considered early signs of hyperparathyroidism with possible extraskeletal calcification, particularly in the kidneys. Consistent with these normal serum and urinay Ca values was evidence of undisturbed renal function, as indicated by normal values of serum urea nitrogen and creatinine. As serum was obtained approximately 22 h after the previous daily injection of rhPTH-(1–34), we do not have detailed time-course data on potential transient elevations in serum Ca levels during the first 12 h after injection. Such an elevation has been observed in women receiving a single injection of 25 µg PTH-(1–34) (19), but the same investigators have treated woman with this dose of PTH-(1–34) for 3 yr without adverse effects (10).
Treatment with rhPTH-(1–34) reduced serum levels of endogenous PTH-(1–84) by approximately 50%; the low dose of rhPTH-(1–34) was as effective as the high dose. A similar suppression of endogenous PTH levels was observed in women during infusions of PTH-(1–34) (20) and with daily PTH-(1–34) administration (8). Pharmacokinetic studies in women have shown that PTH-(1–34) disappears from the circulation within 4 h after sc injections (19). These findings indicate that daily injections of PTH-(1–34) influence Ca homeostasis for a longer time than it remains in the circulation, with the result that less endogenous PTH is required to maintain normocalcemia.
The effect of rhPTH-(1–34) to increase serum levels of calcitriol in OVX monkeys was expected. Although not measured directly, intestinal Ca absorption was presumably elevated during rhPTH-(1–34) treatment, as whole body BMC increased without a decline in urinary Ca excretion. PTH treatment is known to increase serum calcitriol levels and intestinal Ca absorption in rats (21) and dogs (22). This ability of PTH-(1–34) to stimulate intestinal Ca absorption may be critical in promoting increases in cancellous bone mass without inducing cortical bone loss. In contrast to PTH, fluoride treatment does not increase serum calcitriol levels, and patients responding to fluoride therapy can develop Ca deficiency that is correctable by calcitriol administration (1).
The measured concentrations of calcitriol in monkey serum were approximately 200 pg/mL, which are at least 5-fold higher than those observed in humans and most other mammals. Two previous studies found similarly high serum calcitriol levels in female rhesus (23) and cynomolgus (24) macaques. New World monkeys have very high serum levels of and resistance to steroid hormones, including calcitriol. In contrast, the cynomolgus macaques used in the present study are Old World monkeys that generally have serum steroid hormone levels similar to those observed in humans. The binding affinities of calcitriol to the macaque serum vitamin D-binding protein and the intracellular vitamin D receptor are not known. An analog of calcitriol is effective in increasing serum Ca levels and urinary Ca excretion in cynomolgus macaques (24).
Ovariectomy produced the expected increase in bone remodeling, as reflected by increased serum alkaline phosphatase activity (1.6-fold) and osteocalcin (1.8-fold) concentrations, along with a 2.2-fold elevation in the urinary excretion of collagen cross-links. These markers were consistently higher in the OVX monkeys treated with rhPTH-(1–34) than the OVX controls, but the elevations were small and usually not statistically significant.
Reeve et al. (25) measured urinary cAMP excretion in subjects treated with PTH-(1–34) for 1 yr. Compared to baseline values obtained before the initiation of treatment, PTH-(1–34) promoted a 75% increase in cAMP excretion in urine obtained during the 45 min before the normal daily injection. The magnitude of the increase in urinary cAMP excretion measured during the first 3 h after the daily PTH-(1–34) injections was similar upon initiating treatment and after 1 yr of daily PTH-(1–34) administration. rhPTH-(1–34) administration stimulated renal cAMP excretion in monkeys, but as the urine collections were made over about 24 h, no information is available on the daily time course of cAMP excretion.
Although the major focus of this study involved characterizing the skeletal actions of rhPTH-(1–34), two effects of ovariectomy in monkeys deserve comment. First, menopause in women is associated with increases in the serum levels of total Ca and P without changes in the levels of ionized Ca (26, 27, 28), and ovariectomy produced identical changes in monkeys. Secondly, ovariectomized monkeys had reduced serum calcitriol levels. Although such a decline is not usually observed in postmenopausal women, three longitudinal studies (29, 30, 31) have noted similar decreases in women receiving GnRH analogs to suppress ovulation. As serum calcitriol levels are strongly influenced by dietary intakes of Ca and P, our ability to feed monkeys a purified diet with constant levels of these elements undoubtedly minimized serum calcitriol variations among monkeys. Lower serum calcitriol levels after ovariectomy might result in reduced intestinal Ca absorption.
In conclusion, rhPTH-(1–34), given once daily for 12 months to cynomolgus monkeys starting immediately after ovariectomy, increased BMD in the spine and proximal tibia, but did not alter BMD in the midshaft or distal radius. There was no evidence of cortical bone loss, sustained hypercalcemia, or hypercalciuria. As anticipated, rhPTH-(1–34) stimulated urinary cAMP excretion, but did not appear to impair renal function.
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Acknowledgments |
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Footnotes |
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2 Present address: SkeleTech, Inc., Bothell, Washington 98021.
Received April 8, 1999.
Revised June 9, 1999.
Accepted June 18, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxyvitamin D2
to activated dihydroxyvitamin D2 metabolites decreases
endogenous 1,25-dihydroxyvitamin D3 in rats and monkeys. Endocrinology. 136:4749–4753.[Abstract]
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