A 7-day continuous infusion of PTH or PTHrP suppresses bone formation and uncouples bone turnover

Authors


Abstract

Human in vivo models of primary hyperparathyroidism (HPT), humoral hypercalcemia of malignancy (HHM), or lactational bone mobilization for more than 48 hours have not been described previously. We therefore developed 7-day continuous-infusion models using human parathyroid hormone(1–34) [hPTH(1–34)] and human parathyroid hormone–related protein(1–36) [hPTHrP(1–36)] in healthy human adult volunteers. Study subjects developed sustained mild increases in serum calcium (10.0 mg/dL), with marked suppression of endogenous PTH(1–84). The maximal tolerated infused doses over a 7-day period (2 and 4 pmol/kg/h for PTH and PTHrP, respectively) were far lower than in prior, briefer human studies (8 to 28 pmol/kg/h). In contrast to prior reports using higher PTH and PTHrP doses, both 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and tubular maximum for phosphorus (TmP/GFR) remained unaltered with these low doses despite achievement of hypercalcemia and hypercalciuria. As expected, bone resorption increased rapidly and reversed promptly with cessation of the infusion. However, in contrast to events in primary HPT, bone formation was suppressed by 30% to 40% for the 7 days of the infusions. With cessation of PTH and PTHrP infusion, bone-formation markers abruptly rebounded upward, confirming that bone formation is suppressed by continuous PTH or PTHrP infusion. These studies demonstrate that continuous exposure of the human skeleton to PTH or PTHrP in vivo recruits and activates the bone-resorption program but causes sustained arrest in the osteoblast maturation program. These events would most closely mimic and model events in HHM. Although not a perfect model for lactation, the increase in resorption and the rebound increase in formation with cessation of the infusions are reminiscent of the maternal skeletal calcium mobilization and reversal that occur following lactation. The findings also highlight similarities and differences between the model and HPT. © 2011 American Society for Bone and Mineral Research

Introduction

Parathyroid hormone (PTH) and parathyroid hormone–related protein (PTHrP) are similar in structure and are associated with similar but not identical pathophysiologic syndromes—primary hyperparathyroidism (HPT) and humoral hypercalcemia of malignancy (HHM), respectively. Human PTH and PTHrP both are secreted as large prohormones that are then metabolized into a number of active forms, of which hPTH(1–34) and hPTHrP(1–36) have been employed in studies in humans and rodents, as well as in vitro.1, 2 hPTH(1–34) and hPTHrP(1–36) bind to and signal via a common PTH-PTHrP G protein–coupled receptor, the PTH-1 receptor or PTH1R.3 They have been shown to signal via the same intracellular signaling pathways and until recently were thought to bind with identical affinity and signal with identical potency to the PTH1R. More recently, Dean and colleagues4 have demonstrated that while both peptides associate with the PTH1R with equivalent affinity and rapidity, hPTH(1–34) remains bound to the PTH1R, is internalized, and continues to signal for more than an hour despite removal of the ligand.4 In contrast, PTHrP(1–36) dissociates from the PTH1R and ceases signaling almost immediately after removal of PTHrP from the perfusion medium.4

In normal physiology, PTH and PTHrP play distinct functions.1, 5 PTH is a classic endocrine peptide hormone produced exclusively by the parathyroid glands. Its principal function is to maintain normal serum calcium homeostasis through its direct actions on the skeleton and kidney and indirect actions on the intestine. In contrast, PTHrP is a ubiquitously expressed, locally acting paracrine, autocrine, and intracrine factor that variously regulates growth, survival, and differentiation in the myriad of cell types in its local environment. Unlike PTH, with only rare exceptions, PTHrP normally does not enter the systemic circulation. One exception is the normal systemic or endocrine secretion of PTHrP by the mammary gland during lactation, whereby PTHrP activates osteoclastic bone resorption and also activates renal calcium retention so that large quantities of skeletally derived calcium are available to be transported into milk.6

Overproduction of the two hormones produces similar but distinct pathophysiologic syndromes, HPT and HHM. In both syndromes, patients display hypercalcemia, hypercalciuria, hypophosphatemia, phosphaturia, and increases in osteoclastic bone resorption.7–10 On the other hand, osteoblastic bone formation is increased in HPT but reduced in HHM.7–10 Also, circulating 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is increased in HPT but reduced in HHM.7, 8 Why continuous secretion in vivo of two similar peptides that share a common receptor should produce opposite effects on two fundamental calcitropic peptide target organs has never been made clear.

In this regard, lactation and HHM are particularly intriguing because both conditions are associated with strikingly rapid bone loss. Normal lactating women lose up to 10% of their skeletal mass during 6 months of lactation.6, 11 Patients with HHM may lose 50% of their skeletal mass in a matter of a few months.7, 9 In contrast, many or most patients with HPT do not lose bone mass rapidly but gradually over a period of years.12–14 These differences presumably reflect the tightly coupled increases in bone resorption and formation that occur in HPT, in contrast to the markedly uncoupled bone turnover in patients with HHM.7, 9 Some degree of uncoupling also must occur in lactating women because marked bone loss occurs, but confirmatory quantitative bone histomorphometry has never been performed during human lactation. Thus the explanation for the complete uncoupling of resorption and formation by PTHrP in HHM, as compared with the tight coupling that occurs in HPT, remains enigmatic despite some 30 years of study.7–9

In an effort to understand how or why turnover can be coupled in response to one hormone but uncoupled in response to a similarly acting one, we have developed subacute 6-, 12-, and 48-hour infusion models using PTH and PTHrP in healthy human volunteers.15–18 We found that infusion of both peptides causes hypercalcemia, phosphaturia, and increases in osteoclastic bone resorption. Surprisingly, we found that PTHrP, despite being reported to bind to the receptor with equivalent potency, failed to stimulate 1,25(OH)2D3 production with the efficiency of PTH, appearing some twofold less potent than PTH.15–18 We also found that both peptides, when infused continuously for 12 to 48 hours, suppress markers of bone formation,17, 18 as is seen in HHM7, 9, 10 but in contrast to events in HPT.9, 12, 13 While this had been reported for short-term PTH infusions,19–21 it has never been reported previously for PTHrP.

Reasoning that increases in 1,25(OH)2D3 and in bone formation by both PTH and PTHrP likely would occur eventually but might require more prolonged infusions, we have developed, for the first time, parallel 7-day continuous-infusion models for both PTH and PTHrP. Here we report a number of surprising and novel observations. These help to further define the normal physiologic and pathophysiologic actions of PTH and PTHrP. Finally, they provide important tools for future human studies.

Methods

Study subjects

Twenty-two healthy normal young adults between the ages of 24 and 35 years, recruited from the local community, participated in this study (Table 1). Exclusion criteria included subjects of African-American descent, smokers, and anyone with a chronic disease such as cardiac, vascular, pulmonary, endocrine, musculoskeletal, or hematologic disease or malignancy; body mass index (BMI) > 30 kg/m2; anemia; a history of alcohol or drug abuse; or baseline hypertension. Subjects were required to have normal screening laboratory values [ie, serum calcium, albumin, phosphorous, creatinine, 25(OH)D, intact PTH(1–84), and hemoglobin/hematocrit], a negative urine drug screen, and a normal physical examination. All procedures were approved in advance by the University of Pittsburgh Institutional Review Board, and all subjects provided written informed consent.

Table 1. Baseline Demographics (±SD)
 PTHrP 2 (n = 3)PTHrP 4* (n = 6)PTHrP 5 (n  = 3)PTH 2* (n = 5)PTH 4 (n = 5)
  • *

    Maximum tolerated dose; no statistically significant differences between PTHrP 4 and PTH 2 pmol/kg per hour groups (p ≥ .05).

Age (years)31.0 ± 6.128.1 ± 3.127.3 ± 2.926.4 ± 1.729.8 ± 3.2
Height (cm)170.9 ± 7.5169.0 ± 10.0170.9 ± 5.9172.8 ± 5.8171.5 ± 8.9
Weight (kg)69.0 ± 12.966.4 ± 10.971.6 ± 16.374.1 ± 15.572.4 ± 9.6
BMI (kg/m2)23.5 ± 3.023.4 ± 4.224.4 ± 5.024.7 ± 4.324.5 ± 1.16
Male/Female2/13/32/12/34/1
Creatinine (mg/dL)1.13 ± 0.060.83 ± 0.050.77 ± 0.061.04 ± 0.241.12 ± 0.25
Calcium (mg/dL)9.50 ± 0.209.78 ± 0.409.63 ± 0.329.70 ± 0.179.56 ± 0.48
Phosphorus (mg/dL)3.73 ± 0.253.38 ± 0.443.47 ± 0.514.12 ± 0.703.46 ± 0.26
Albumin (g/dL)4.37 ± 0.324.30 ± 0.464.37 ± 0.554.50 ± 0.214.36 ± 0.35
PTH(1–84) (pg/mL)33.7 ± 20.421.5 ± 10.047.3 ± 4.223.6 ± 9.630.8 ± 9.3
1,25(OH)2D3 (ng/mL)30.0 ± 9.648.8 ± 47.732.0 ± 8.534.2 ± 16.025.0 ± 6.7

Study design

This study was designed as a standard dose-escalation, dose-finding pilot study; the primary outcome measures were safety outcomes (ie, hypercalcemia, hypophosphatemia, hemodynamic measurements, and symptoms) during a progressive dose escalation. Secondary outcome measures were serum intact PTH(1–84), bone turnover markers, tubular maximum for phosphorus (TmP/GFR), plasma 1,25(OH)2D3, and urinary calcium excretion measures.

Subjects initially were assigned to receive either PTH or PTHrP at 2 pmol/kg per hour, one-quarter the minimal dose used in prior studies of shorter duration.15–21 A modified Fibronacci dose-escalation scheme was employed such that three subjects were scheduled to receive the initial 2 pmol/kg per hour dose. If no dose-limiting toxicity (DLT; see below) occurred, three more subjects were infused at the next higher dose, 4 pmol/kg per hour, for 7 days. If DLT again did not occur, the dose was increased further until DLT occurred. If DLT occurred in one of the three subjects, three more were studied at that dose. When DLT occurred in more than two subjects at a given dose, the prior dose was defined as the maximal tolerated dose (MTD) or the dose was deesclated in 1 pmol/kg per hour increments.

DLT was defined as achieving one major criterion or two minor criteria. The major criteria were defined as symptomatic orthostatic hypotension (systolic blood pressure fall > 30 mm Hg), tachycardia (pulse > 120 beats/min), hypertension (systolic blood pressure >160 mm Hg on two occasions), hypercalcemia (serum calcium ≥ 12 mg/dL), and hypophosphatemia (serum phosphorous < 1.5 mg/dL). Minor criteria included symptoms such as flushing, nausea, abdominal or muscle cramps, dizziness, light-headedness, and palpitations. Subjects were asked if they were experiencing any of these or additional symptoms prior to each blood draw. If any symptoms were present, the subject was asked to grade them on a scale of 0 to 3 (0 = none, 1 = mild, 2 = moderate, and 3 = severe). Any symptom rated 2 or greater was considered a significant minor criterion.

PTHrP and PTH peptides

Human PTH(1–34) (referred to hereafter as PTH) and human PTHrP(1–36) (referred to hereafter as PTHrP) were prepared using solid-phase synthesis at the WM Keck Peptide Synthesis Facility at Yale University (New Haven, CT, USA) and purified using preparative-scale reversed-phase high-performance liquid chromatography (RP-HPLC), as described previously.15–18, 23–25 Purity was defined using analytic-scale RP-HPLC and mass spectrometry. Peptide was sterile filtered and aliquotted into individual sterile vials, lyophilized, and stored at −80°C until the day of use. Vials were tested for sterility, peptide content, and bioactivity as described in detail previously.15–18, 23–25 PTH and PTHrP were approved for use by the Food and Drug Administration under Investigational New Drugs (INDs) numbers 60,979 and 49,175, respectively.

Human infusion protocol

On the day of study, subjects were admitted to the University of Pittsburgh Clinical Translational Research Center (CTRC), and an indwelling central catheter was inserted and attached to a continuous infusion pump. For the inpatient overnight portions of the study, a Baxter Auto Syringe A550 pump (Baxter Healthcare, Round Lake, MN, USA) was used. The PTH or PTHrP infusions were commenced at 11:00 am on day 1 of the study. Every 48 hours, fresh aliquots of PTH or PTHrP were suspended in 100 to 200 mL of 0.9% NaCl containing 1.0 mL of the study subject's own blood and then loaded into sterile plastic syringes and refrigerated until use. Syringes were replaced at least every 24 hours, as described previously,16–18 and the infusion was continued for 7 days. On days 3 to 6 of the infusion, after 7:00 am laboratory determinations, a second morning void was obtained, and if hemodynamically stable and not significantly hypercalcemic (7:00 am serum calcium < 11.0 mg/dL), subjects were allowed to leave the hospital between 9:00 am and 3:00 pm for up to 6 hours. To facilitate this, subjects were switched to a miniaturized continuous infusion pump (CADD-Micro Ambulatory Infusion Pump Model 5900, SIMS Deltec, Saint Paul, MN, USA) and provided with a cellular telephone to contact the research staff should the need arise. Meals contained 15% protein, 55% carbohydrate, 30% fat, 400 mg of calcium and 800 mg of phosphorus per day and were prepared at the CTRC, including bag lunches in the event that subjects elected to leave the CTRC during the daytime.

Measurements

Vital signs were obtained every 8 hours, including supine and standing blood pressure and pulse. Blood was obtained at multiple times each day, as depicted in the figures and as discussed below. A second morning void also was obtained each day for calcium, phosphorus, and creatinine determinations. On the last day of the study, a 24-hour urine also was obtained for calcium and creatinine determinations. Finally, 10 days after completion of the study, subjects returned for a follow-up visit to undergo a final blood test, as depicted in the figures.

Analyses

Serum and urine calcium, serum ionized calcium, phosphorus, creatinine, plasma 25(OH)D, and serum intact PTH(1–84) were measured at the University of Pittsburgh Medical Center Clinical Laboratories. Ionized calcium samples were collected aerobically in a heparin sodium tube and transported on ice for immediate analysis of whole blood on an ABL700 Series instrument that automatically corrected the sample for pH (Radiometer ABL700 Series Reference Manual, Radiometer, Copenhagen, Denmark). Fractional excretion of calcium and TmP/GFR were calculated as described previously.15–18, 24 1,25(OH)2D3 was assayed using an RIA as described previously.26 Amino-terminal telopeptides of procollagen 1 (P1NP), serum amino-terminal telopeptide of collagen 1 (sNTX), and carboxy-terminal telopeptide of collagen 1 (CTX) were measured using commercial kits from Orion Diagnostics RIA [Espoo, Finland; intraassay coefficient of variation (CV) 4.3%, interassay CV 7.7%], Osteomark ELISA (Ostex International, Seattle, WA, USA; intraassay CV 4.2%, interassay CV 9.6%), and Crosslaps ELISA (Nordic Bioscience Diagnostics, Inc., Herlev, Denmark; intraassay CV 3.8%, interassay CV 8.4), respectively. Bone-specific alkaline phosphatase (BSAP) was measured using commercial kits from Ostase EIA (Hybritech Incorporated, Fullerton, CA, USA; intraassay CV 3.6%, interassay CV 7.2%). The safety laboratory determinations (ie, calcium, ionized calcium, phosphorous, and creatinine) all were run immediately after collection, whereas aliquots were frozen and batched together at completion of the study for all other measurements.

Statistical methods

Frequencies and percentages were used to describe the gender distribution by peptide dose group, whereas measures of central tendency and dispersion were used to describe continuous-type descriptors and the baseline values of endpoints by group. To assess for possible group imbalances, groups were compared using the F test from an analysis of variance (or Kruskal-Wallis test if non–normally distributed) for continuous-type descriptors and the baseline values of endpoints (eg, BMI, serum calcium, and P1NP). The differences in the change in the five groups over time on serum calcium, fractional calcium excretion, plasma 1,25(OH)2D3, serum phosphorous, TmP/GFR, endogenous PTH(1–84), and the percentage change in biomarkers (ie, CTX, NTX, BSAP, serum osteocalcin, and P1NP) relative to baseline values were analyzed using repeated-measures analysis with a linear mixed modeling approach that included fixed between-subject effects for the peptide dose group, fixed within-subject effects for time, and their two-way interactions. Subject was treated as a random effect. The PROC MIXED procedure in SAS (Version 9.2, SAS Institute, Cary, NC, USA) was employed for fitting these linear mixed models. The level of statistical significance was set at .05 (two-tailed). Repeated-measures analyses via linear mixed modeling also were performed, focusing only the two MTDs, PTHrP 4 pmol/kg per hour and PTH 2 pmol/kg per hour.

Results

Baseline demographics

The demographics of the study subjects are summarized in Table 1. As can be seen, there were 13 male and 9 female participants with a mean age of 28 years. There were no significant differences in age, BMI, or other parameters between the groups at the two MTDs (defined below).

Serum total and ionized calcium

The mean total serum calcium in each group is shown in Fig. 1. The mean baseline calcium was 9.65 ± 0.07 mg/dL. Among subjects receiving PTH at a dose of 2 pmol/kg per hour, five completed the study, with one dropping out on day 2 because of infiltration of the intravenous line. The 2 pmol/kg per hour dose yielded a statistically significant increase in serum calcium (mean serum calcium during the infusion = 10.0 mg/dL; p < .0001). DLT did not occur, and per protocol, the dose therefore was escalated to 4 pmol/kg per hour. Subjects receiving this dose displayed a similar calcemic response (10.0 mg/dL), but in this group, DLT occurred in two subjects: One developed a serum calcium level greater than 12.0 mg/dL on day 3 (PTH was terminated), and another developed tachycardia (pulse = 148 beats/min) but was able to complete the study. Based on these considerations, a dose of 2 pmol/kg per hour of hPTH(1–34) was identified as the MTD.

Figure 1.

Changes in serum calcium and phosphorus. (Upper left) Total serum calcium. The legend describes the line colors and symbols for the five patient groups. The light-gray bar in the middle of the panel indicates the baseline calcium concentration (9.65 ± 0.07 mg/dL; mean ± SD). The dark-gray bar in the top of the panel indicates the duration of the PTH and PTHrP infusions. Vertical bars indicate SEM. There was no statistical difference among the groups at baseline (p = .3), but there was a statistically significant increase over time in the 2 pmol/kg per hour of PTH and 4 pmol/kg per hour of PTHrP groups (p < .0001). There was no statistically significant difference in calcemic response between these two groups. (Upper right) Ionized serum calcium. There was no difference among the groups at baseline, but there was a statistically significant increase in time for both the 2 pmol/kg per hour of PTH and 4 pmol/kg per hour of PTHrP groups (p < .0001). (Lower panel) Serum phosphorus. There were no significant differences in serum phosphorus at baseline nor important changes during the infusions.

Among subjects receiving PTHrP, the 2 pmol/kg per hour dose had little effect on total serum calcium (Fig. 1) and produced no DLT. Per protocol, the dose was escalated to 4 pmol/kg per hour. This group developed a statistically significant (p < .0001) calcemic response (mean serum calcium level during the infusion = 10.0 mg/dL), comparable with the 2 pmol/kg per hour dose of PTH. A total of six subjects at 4 pmol/kg per hour (three initially and three after the 5-pmol group was tested) completed the study without DLT. Accordingly, per protocol, after the initial three subjects, the PTHrP dose was escalated to 6 pmol/kg per hour in two additional subjects. Among these two, one had a sustained serum calcium level approaching 12.0 mg/dL, and one developed a DLT by minor criteria. Since the required three subjects were not achieved at this dose, these subjects were excluded from further analysis. Accordingly, an additional group was recruited to receive PTHrP at a dose of 5 pmol/kg per hour. Among these three subjects, one reached DLT based on minor criteria, and a second was terminated on day 4 for sustained hypertension. An additional three subjects then were studied at 4 pmol/kg per hour, which was defined as the MTD for PTHrP.

Figure 1 also displays the ionized serum calcium and phosphorus levels. As can be seen, the ionized calcium results are similar to those observed for total serum calcium and support selection of 2 pmol/kg per hour of PTH and 4 pmol/kg per hour of PTHrP, respectively, as the MTDs (p < .0001). Infusion of PTHrP and PTH at the doses administered had no significant effect on serum phosphorus (Fig. 1) or creatinine (data not shown).

Calcitropic hormones

Endogenous serum PTH(1–84) declined significantly in all groups (Fig. 2), even the one with the subhypercalcemic (2 pmol/kg per hour) dose of PTHrP. Although prior studies have demonstrated that infusion at higher doses of PTH (8 to 16 pmol/kg per hour) and PTHrP (8 to 28 pmol/kg per hour) induce marked increases in plasma 1,25(OH)2D3,15–18, 21, 23 the current, lower doses produced no change in plasma 1,25(OH)2D3 for PTH or an actual decline by day 8 for PTHrP (p = .01; Fig. 2) despite clear changes in serum calcium and other measures (see below).

Figure 2.

Changes in endogenous PTH(1–84) and plasma 1,25(OH)2D3. See Fig. 1 for explanations of bar, line, and symbol colors. Endogenous serum PTH declined rapidly and robustly during the PTH and PTHrP infusions, even in the PTHrP 2 pmol/kg per hour group, in which serum calcium did not change appreciably. Plasma 1,25(OH)2D3 did not increase in response to the infusions and actually decreased by day 8 for the PTHrP 4 pmol/kg per hour group (p = .01).

Renal mineral handling

Fractional calcium excretion increased significantly in the 2 pmol/kg per hour of PTH and 4 pmol/kg per hour of PTHrP groups (p = .002; Fig. 3), reflecting increases in total and ionized serum calcium and therefore the filtered load of serum calcium. Twenty-four-hour urinary calcium excretion (Fig. 3) was normal in the subjects receiving 2 pmol/kg per hour of PTHrP. In contrast, it was at the upper limits of normal and comparable in the other groups, as expected by their increases in serum calcium and reductions in PTH(1–84). As with the serum phosphorus values but in contrast with higher PTH and PTHrP doses in prior studies, the TmP/GFR did not change with these low doses of PTH and PTHrP (p = .61; Fig. 3).

Figure 3.

Changes in renal calcium and phosphorus excretion. See Fig. 1 for explanations of bar, line, and symbol colors. (Upper left) Fractional calcium excretion. The upper light bar represents the fractional calcium excretion (mean ± SD = 6.5) in response to a calcium infusion in which the serum calcium was “clamped” at 10.3 mg/dL and endogenous PTH suppressed by infusion of calcium chloride.16 Fractional calcium excretion increased significantly (p = .002) during the infusion, reflecting the increase in filtered calcium load, but was far below the 6.5% range observed with similar degrees of hypercalcemia and filtered load observed with the calcium clamp, revealing comparable anticalciuric effects of PTH and PTHrP even at these low doses. (Upper right) Twenty-four-hour calcium excretion expressed as calcium per gram of creatinine and obtained at the conclusion of the study approached the hypercalciuric range. The mean 24-hour urine creatinine values in the two MTD groups were 1.06 ± 0.3 mg/dL and 1.49 ± 0.1 mg/dL for the PTH and PTHrP groups, respectively. (Botton panel) TmP/GFR. There were no changes in TmP/GFR, indicating that these low doses of PTH and PTHrP did not induce phosphaturia.

Bone turnover

With respect to bone resorption, both serum CTX and NTX increased significantly in the face of both PTH and PTHrP infusion, even with the lowest, subcalcemic 2 pmol/kg per hour dose of PTHrP (Fig. 4A). Also, bone resorption reversed rapidly, returning to normal when PTH and PTHrP infusions were discontinued.

Figure 4.

(A) Changes in bone-resorption markers. See Fig. 1 for explanations of bar, line, and symbol colors. Both serum NTX and CTX increased rapidly and significantly in response to low-dose PTH and PTHrP infusion (p < .008 and p < .0001, respectively) and declined abruptly with cessation of the infusions. (B) Changes in bone-formation markers. Serum P1NP was suppressed and remained so throughout the 7-day infusions (p < .0001) and promptly rebounded with cessation of the infusions (p = .01). BSAP also declined significantly during the course of the study (p = .0001), but less robustly than for P1NP. The significant decline is mostly attributable to the PTHrP 4 pmol/kg per hour group (p = .001).

In contrast, bone formation, as assessed using serum P1NP (Fig. 4B), declined and remained suppressed for the entire infusion (p = .0001). At the MTDs for the two peptides, BSAP also declined significantly during the course of the study (p = .0001; Fig. 4B) but less robustly than for P1NP, likely reflecting its lower sensitivity as a measure of bone formation. The decline in BSAP was significant in the 4 pmol/kg per hour of PTHrP group (p = .001) but not in the 2 pmol/kg per hour of PTH group when looking at the groups individually. When the PTH or PTHrP infusions were discontinued, P1NP rapidly returned to normal and even overshot the baseline (p = .01).

Discussion

Important differences between the clinical HPT and HHM syndromes were defined some 30 years ago, but the reasons for these differences remain incompletely understood. The mechanisms that lead to bone loss and restitution associated with lactation are also incompletely understood. One obstacle to fully understanding these syndromes is that rodent models do not faithfully mimic the human syndromes in all regards. Additionally, human infusion studies with PTH and PTHrP have been relatively brief, lasting only 6 to 48 hours.15–22 This contrasts with primary HPT, with HHM, and with normal lactation, which typically last for months or years. Since PTH1R modulation, recycling, signaling, and expression, as well as physiologic and pathophysiologic regulation of renal and skeletal responses to PTH and PTHrP, likely take far longer than 6 to 48 hours to achieve steady state, we sought to develop longer models of PTH and PTHrP infusion into healthy human volunteers that might effectively and faithfully mimic human HPT, HHM, and possibly lactation and also do so in a way that was safe and well tolerated. To the best of our knowledge, prior infusion studies of this duration have never been performed in humans.

The first necessary steps in developing such models would be to define the infused dose ranges of PTH and PTHrP that would be safe but effective in modeling HPT and HHM and to develop a delivery system that is compatible with subacute PTH and/or PTHrP infusions. In this report we describe such systems and safe infused dose ranges. We also define, for this infusion model, the lower dosing thresholds of PTH and PTHrP for driving increases in circulating 1,25(OH)2D3 in humans with intact parathyroids. Importantly, we demonstrate that 7-day infusions of PTH and PTHrP continue to suppress P1NP, arguably the best marker for bone formation, despite markedly activating bone resorption. Finally, we demonstrate that cessation of PTH or PTH infusion leads to a rapid increase in bone formation, as demonstrated by a remarkable rebound and even overshoot in circulating P1NP.

The optimal peptide doses

The lowest dose of PTH or PTHrP employed in earlier studies was 8 pmol/kg per hour over 6 to 48 hours15–22 (approximately 25 µg/d) of PTH(1–34) or PTHrP(1–36). Since earlier studies suggested that serum calcium was on the ascent during 6- to 48-hour infusions of PTH or PTHrP at the 8 pmol/kg per hour dose,15–18 we were concerned that week-long infusions at this dose might result in dangerous hypercalcemia. Accordingly, we selected a lower dose, 2 pmol/kg per hour, as a starting dose for this pilot study. Surprisingly, this proved to be the MTD for PTH.

PTHrP at 2 pmol/kg per hour produced measurable effects on endogenous PTH(1–84) and bone resorption but marginal effects on serum calcium. The decline in PTH(1–84) may reflect a small increase in serum calcium or potential inhibitory effects on PTH secretion.27 An escalated dose of 4 pmol/kg per hour for PTHrP resulted in increases in serum calcium that were both safe and comparable with those observed with 2 pmol/kg per hour of PTH. Higher doses were associated with significant hypercalcemia and/or adverse effects. Thus 4 pmol/kg per hour was selected as the MTD for PTHrP.

Therefore, as noted in earlier studies,15–18, 23 PTHrP appeared to be slightly weaker than PTH(1–34) in inducing hypercalcemia. This is not likely owing to differences in bioactivity or stability between the two peptides because SaOS2 adenylyl cyclase bioassays and peptide content confirmed their equivalence and because measurement of circulating hPTH(1–34) and hPTHrP(1–36) concentrations with the best available assays suggested that circulating levels of PTHrP, if anything, exceed those of PTH in this model.18 Thus this calcemic dose difference presumably reflects intrinsic differences in how the two peptides interact with the PTH1R in their various target organs, as suggested by Dean and colleagues.4 Finally, with regard to the pattern of hypercalcemia, it is interesting to note that the serum calcium level consistently appeared to decline during the middle portion of the study. This did not correlate with inpatient versus outpatient status or other variables and remains unexplained.

Differential activation thresholds for hypercalcemia, phosphaturia, 1,25(OH)2D3, and bone resorption in humans

Prior studies in humans using supraphysiologic doses of PTH and PTHrP demonstrate that PTH and PTHrP are both capable of activating bone resorption, phosphaturia, renal calcium retention, and 1,25(OH)2D3 production.15–23 However, none of these studies has carefully explored the minimal dose thresholds required to activate these responses. Here we show that in this model, the minimal calcemic dose of PTHrP is 4 pmol/kg per hour and the minimal calcemic dose of PTH is less than 2 pmol/kg per hour. Interestingly, all these doses activated osteoclastic bone resorption, as assessed by sCTX and sNTX, but none induced phosphaturia or measurable increases in circulating 1,25(OH)2D3. (It is important to note that some of the second morning urine samples were not fasting. This increases the variability in the TmP/GFR calculations and may have diminished the ability to measure significant changes in this small study.) With these qualifications, these studies therefore suggest that the thresholds for induction of phosphaturia and 1,25(OH)2D3 production are different from those for activation of bone resorption and induction of hypercalcemia using this model.

Suppression of bone formation

One of the principal goals of the study was to determine whether bone formation, which had declined in earlier, briefer studies, eventually would reverse and increase, as occurs in HPT, or would continue to decline and remain suppressed, as occurs in HHM. We observed that even when administered for as long as 7 days, bone formation remained suppressed and showed no signs of becoming activated, as would be expected in HPT.

These observations support several possible interpretations. It is possible to argue that if the PTH or PTHrP infusions had continued for a longer period of time, bone formation certainly would have rebounded. In support of this hypothesis, primary HPT, with its characteristic increase in bone formation, has been present for many months or years before it is diagnosed,12, 13 and even intermittent administration of PTH as a treatment of osteoporosis requires 2 weeks to observe an increase in bone-formation markers.28, 29

On the other hand, many studies in animals and in vitro demonstrate that continuous exposure of bone-derived mesenchymal stem cells to PTH or PTHrP causes an arrest in differentiation at a preosteoblast stage30–34 that lasts at least 3 weeks or longer33 and suggests that the characteristic “marrow fibrosis” observed in HPT may represent a partial arrest of osteoblast differentiation.30, 34 More specifically, Turner and colleagues have suggested that continuous exposure of the skeleton to PTH recruits bone mesenchymal stem cells into the osteoblast lineage but arrests them at the preosteoblast stage, where they are morphologically indistinguishable from marrow fibroblasts.30, 34 Thus continuous exposure of the skeleton to PTH creates the appearance of marrow fibrosis with cells that may in fact be preosteoblasts.

Rebound of bone formation on withdrawal of PTH or PTHrP

The largest surprise in this study was the rapid and profound rebound, indeed overshoot, of bone formation (P1NP) following cessation of the PTH and PTHrP infusions. This observation is entirely reminiscent of the rebound osteoblast differentiation, function, and mineralization following withdrawal of continuously administered PTH or PTHrP observed in vitro by others,31–33 suggesting that continuous PTH or PTHrP exposure recruits osteoblast precursors and induces their partial differentiation but prevents them from completing their entire differentiation program. It also may suggest that the rapid increase in bone density that follows parathyroidectomy in HPT12–14 may reflect a combination of not only mineralization of increased preexisting osteoid but also a component of release of preosteoblasts such that they complete their differentiation program and thereby increase new bone-formation rates. Finally, Fig. 4 is very reminiscent of events at the end of lactation, when bone resorption ceases abruptly and bone mass accumulates rapidly in a matter of a few months to prelactation levels.6, 11 We hypothesize that in lactation, continuous PTHrP production by the mammary gland drives resorption and recruits osteoblast precursors but prevents them from differentiating completely. This allows uncoupled bone resorption during lactation as a source of calcium for lactational milk production. In this scenario, when lactation ceases, systemic PTHrP production ceases, and resorption declines abruptly, suddenly freeing preexisting osteoblast precursors from arrest so that they can robustly remineralize the maternal skeleton, as has been so well demonstrated.6, 11, 30–33

Fidelity as a model of HHM

The PTHrP infusion studies provide an almost perfect model of HHM because it reproduces all the features of HHM. It causes bone resorption, suppression of bone formation, inhibition of calcium excretion, phosphaturia, and suppression of endogenous PTH. Interestingly, while 1,25(OH)2D3 concentrations are markedly reduced in HHM, they are also declining here in response to PTHrP, although they remain in the normal range. The reasons for this quantitative difference may be that additional factors [eg, more severe hypercalcemia, secretion of tumor-derived cytokines, greater chronicity, or PTHrP secretory species other than PTHrP(1–36)] may suppress renal 1-α-hydroxylase in HHM or that it may require longer than 1 week for 1,25(OH)2D3 concentrations to decline. Clarifying these issues will be of interest for future studies.

Fidelity as a model of lactation

The PTHrP infusion studies also provide a tool with which to partially model human lactation. Some features of this model are identical to those described in rodent and human lactation6: continuous production and secretion of low levels of PTHrP into the maternal circulation, activation of bone resorption, prevention of renal calcium losses through PTHrP-mediated anticalciuric effects, and mobilization of large amounts of calcium from the skeleton to serve as a source for milk production and neonatal skeletal mineralization. On the other hand, there are a number of differences between this model and lactation. First, the pregnancy-associated rises in estrogen and progesterone and their sudden fluxes that accompany parturition are absent in this model. Second, these subjects lack milk production (and thus a sink for the excess skeletal calcium removal). This potentially may explain why hypercalcemia did develop here but does not occur in lactation. This hypercalcemia and high filtered load, despite an only limited change in fractional excretion of calcium (FECa), likely account for the elevated 24-hour urine calcium excretion in this model compared with the hypocalciuria typical of lactation.6 (Parenthetically, it would have been ideal to have collected a baseline 24-hour urine for calcium determination in addition to the final 24-hour collection.) In addition, P1NP is decreased in this model compared with the variable increases or decreases in formation markers reported by others during human lactation.35–38 Third, serum phosphorus increases in lactation6 but did not in our system, perhaps reflecting the fact that prolactin is increased in lactation but not in this model. And fourth and most important, precisely what happens to bone formation in humans during and at the cessation of lactation remains enigmatic at present. Bone biopsies to elucidate these issues likely will never be performed in human lactation. However, the kind of modeling described here may permit the development of human experimental paradigms that will clarify whether bone formation is reduced and/or arrested in lactation, as suggested here, or increased, as suggested by other studies.35–38

Fidelity as a model of HPT

In some ways, this approach models primary HPT: mild hypercalcemia, bone resorption, increases in renal calcium retention contributing to hypercalcemia, but with net hypercalciuria, reflecting the increased renal calcium filtered load. These features mimic mild primary HPT and over time would be predicted to lead to skeletal mineral loss, also characteristic of some patients with HPT. At higher doses of PTH, 1,25(OH)2D3 is increased and the TmP/GFR decreases,18 but these did not occur in this lower-dose model. In contrast to these similarities with HPT, there is another striking difference: Bone formation is regularly observed to be increased in HPT,12, 13, 39 whereas in the current model, bone formation is reduced with 7 days of continuous PTH infusion (Fig. 5). As noted earlier for PTHrP, it is possible that the 7-day PTH infusion is simply not a sufficient duration to activate bone formation because it is known that increases in bone-formation markers require some 2 weeks of daily injections with PTH(1–34).28, 29 On the other hand, in HPT, PTH is secreted from its neuroendocrine secretory granules in a pulsatile manner,40–42 in contrast to the continuous infusion model here or the continuous secretion of PTHrP in lactation and in HHM, by mammary epithelium and cancers that lack neuroendocrine secretory granules or machinery. Importantly, the tools developed here will permit future, longer, and also pulsatile versus intermittent studies to clarify these issues and develop better models of HPT.

This study has a number of limitations. First, although the number of subjects studied was relatively large for this class of study, the numbers of subjects in the individual dose groups were relatively small. Despite this limitation, the long duration of the study with sampling of multiple time points allowed for attaining robust statistical significance. Also, with the definition of appropriate and safe doses of PTH and PTHrP over prolonged time periods described herein, power analyses now can be performed for future studies designed to address some of the questions alluded to earlier.

Second, this study was largely limited to whites and included no African Americans. This was intentional because it is well known that bone turnover, renal calcium handling, PTH concentrations, and vitamin D metabolism all differ in African Americans compared with whites.43, 44 Having developed the continuous-infusion methodology, and having defined the requisite doses, duration, and group sizes, it is now be possible to pursue similar studies in African Americans to explore the biology underlying the ethnic differences noted earlier.

Third, we examined only a single time point for follow-up: 10 days after discontinuation of the PTH and PTHrP infusions. We do not know whether indices of bone formation rose earlier and were declining or would peak at a later time point, nor how long the apparent increase in formation might persist. This will need to be ascertained in future studies.

Fourth, while we have taken all standard precautions to ensure and document stability of the peptides as infused,15–18 in the current absence of sensitive and specific assays for PTHrP(1–36) and hPTH(1–34), we could not measure the circulating concentrations of each peptide at the multiple time points examined. The consistent increases in serum calcium and bone-resorption markers in this and prior studies15–18 support the likelihood that the peptide are stable and active as administered.

And fifth, this study did not include a control saline infusion group, raising the question as to whether the changes in PTH(1–84) and markers of bone turnover and lack of change in 1,25(OH)2D3 or TmP/GFR might reflect adaptive responses to the study diet. This seems unlikely because in our prior study, which did include a saline-infused control group on the same diet,16 there were no changes in PTH(1–84) or TmP/GFR.16 Also, the low-calcium (400 mg) study diet, if anything, might have increased PTH(1–84) and therefore reduced TmP/GFR. It also seems unlikely that a 400-mg calcium/800-mg phosphorus diet would induce a two- to threefold increase in resorption markers and suppress bone formation by 40% in the face of a decrease in endogenous PTH(1–84).

In summary, we have defined the dose responses and safety profiles of PTH and PTHrP infusion in humans with intact parathyroids, with 2 pmol/kg per hour for hPTH(1–34) and 4 pmol/kg per hour for hPTHrP(1–36) being optimal. We have shown that these chronic studies can serve as excellent models for elucidating the physiology and pathophysiology of HHM and at least partially model events in human HPT as well as lactational bone loss and its subsequent recovery. We have made the surprising observation that continuous exposure of the human skeleton to PTH or PTHrP for 1 week leads to sustained but reversible suppression of bone formation. We also have provided the first clear evidence of a rebound increase in bone formation in human skeletons exposed continuously to PTH or PTHrP. These techniques and approaches will be useful for deepening and expanding our understanding of the biology of human lactation and the pathophysiology of HPT and HHM.

Disclosures

AFS is a member of Osteotrophin, LLC. BWH is a consultant for DiaSorin Corp. MJH is a consultant for Merck. All the other authors state that they have no conflicts of interest.

Acknowledgements

We wish to thank the staff of the University of Pittsburgh CTRC and CTSI, who were essential to this study. We thank Dr William Crowley at the Massachusetts General Hospital (Boston, MA, USA) for many helpful discussions in the design phase of these studies. We thank Dr Mohamed Virgi in the University of Pittsburgh Medical Center for analytical advice and support. We also wish to thank the members of our DSMB, including Drs Elizabeth Shane, Susan Greenspan, David Roodman, and Steven Wisniewski. This work was funded by NIH Grants R-01 DK 073039 and DK 51081 and CTSA UL1 RR024153 and M-01 RR000056. Registered with Clinical Trials.gov number NCT 00377312 (registration date 9/14/2006) and number NCT 00580788 (registration date 12/20/2007).

Authors' roles: Study design: MJH and AFS. Study conduct: MBT, LP, RMC. Data collection: MBT, LP, RMC, MJH, BWH, CMG, AB, AG-O. Data analysis: MJH, SMS. Data interpretation: MJH, RMC, AFS. Drafting manuscript: MJH, RMC, SJS, AFS. Revising manuscript content: MJH, SMS, AFS. Approving final version of the manuscript: all authors. MJH, SMS, CMG, BWH, and AFS take responsibility for the integrity of the data analysis.

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