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Parathyroid Hormone-Related Protein-(1–36) Is Biologically Active When Administered Subcutaneously to Humans¹

Divisions of Endocrinology, Veterans Administration Connecticut Healthcare System, West Haven, Connecticut 06516; and Yale University School of Medicine, New Haven, Connecticut 06520

Address all correspondence and requests for reprints to: Andrew F. Stewart, M.D., Research 151/C, Veterans Administration Medical Center, 950 Campbell Avenue, West Haven, Connecticut 06516.

Abstract

PTH-related protein (PTHrP) is responsible for most cases of humoral hypercalcemia of malignancy (HHM). It mimics the actions of PTH as a result of its structural homology with PTH and its ability to bind to and signal via the PTH/PTHrP receptor in bone and kidney. PTHrP-(1–36) appears to be one of several secretory forms of PTHrP. This peptide has been administered iv to normal volunteers previously and has been shown to produce effects that are qualitatively and quantitatively the same as those produced by PTH-(1–34). To determine whether PTHrP-(1–36) could be used sc in humans as a diagnostic reagent for elucidating the differences between HHM and hyperparathyroidism, we performed a 12-h dose-finding study examining whether sc PTHrP-(1–36) could elicit effects on mineral homeostasis.

PTHrP-(1–36) administered sc in three doses (0.82, 1.64, and 3.28 µg/kg) to 21 normal women produced increases in circulating PTHrP-(1–36), reductions in serum phosphorus and the renal phosphorus threshold, increments in fractional calcium excretion and nephrogenous cAMP excretion, and increases in plasma 1,25-dihydroxyvitamin D. These changes were highly significant in statistical terms and were observed at doses that had no effect on serum calcium or endogenous PTH.

These studies demonstrate the feasibility of using PTHrP-(1–36) as a diagnostic probe for future studies aimed at elucidating the differing pathophysiologies of HHM and hyperparathyroidism.

PTH-RELATED protein (PTHrP) was initially discovered because of its role as a causative agent responsible for humoral hypercalcemia of malignancy (HHM). It is now known that the initial PTHrP messenger ribonucleic acid translation products, PTHrP-(1–139), PTHrP-(1–141), and PTHrP-(1–173), serve as preprohormones, which undergo posttranslational endoproteolytic processing by prohormone convertases to yield a spectrum of mature secretory forms of the peptide (1, 2, 3, 4). One of these cleavage sites is in the -1 to +1 position, and another is in the +37 position, cleavages that would yield PTHrP-(1–36) as one of the several secretory forms of PTHrP (1, 2, 3, 4). Multiple other secretory forms of PTHrP exist, but have not been as fully characterized as peptides derived from the amino-terminus of the peptide. The PTHrP-(1–36) region is important, for this region of the peptide is homologous with PTH and contains amino acids that appear to be involved in binding to and transducing signals through the PTH/PTHrP receptor in bone and kidney (1, 2, 3, 4).

Recently, we have synthesized PTHrP-(1–36) and have shown that it is equal in potency to human (h) PTH-(1–34) as well as to other amino-terminal PTHrP peptides in its ability to stimulate adenylyl cyclase in human bone cells in vitro (3). These studies also explored the potency and actions of PTHrP-(1–36) when administered iv in vivo to humans (3). In these studies, a 6-h infusion of PTHrP-(1–36) to healthy volunteers produced all of the effects expected of exogenously administered PTH-(1–34) and endogenous PTH-(1–84); serum calcium rose, serum phosphorus fell, the tubular maximum for phosphorus (TmP/GFR) fell, the fractional excretion of phosphorus (FEPi) rose, nephrogenous cAMP (NcAMP) excretion rose, and endogenous PTH-(1–84) fell. The fractional excretion of calcium (FECa) rose initially and then fell, suggesting both calciuric and anticalciuric effects for PTHrP-(1–36). Plasma concentrations of 1,25-dihydroxyvitamin D [1,25-(OH)₂D] rose. Importantly, PTHrP-(1–36) appeared to be as potent as hPTH-(1–34) in producing these effects.

One goal of the studies described in this report was to lay the groundwork for future studies exploring the pathophysiological differences between HHM and primary hyperparathyroidism (HPT) (5, 6, 7). The clinical, biochemical, and skeletal histomorphometric features of these two common syndromes resemble one another in many ways, but differ in at least three important and unexplained ways. In contrast to patients with HPT, patients with HHM display 1) uncoupling of osteoclastic resorption from osteoblastic formation (8, 9), 2) reductions instead of elevations in circulating concentrations of 1,25-(OH)₂D (5, 7), and 3) relatively greater rates of renal calcium excretion (5, 10), although this point is controversial (11, 12). The availability of a parenteral form of PTHrP that could be administered to healthy subjects and for which normal response profiles had been defined could prove useful as an experimental tool or probe for exploration of the pathophysiology of HHM.

At present, a number of amino-terminal PTH peptides are under investigation as anabolic skeletal agents for the treatment of osteoporosis (13, 14, 15, 16, 17, 18). A second overall goal of these studies was to begin to explore the potential of PTHrP-(1–36) as a therapeutic anabolic skeletal agent, because of its structural and functional similarity to PTH. The studies described herein, therefore, address four initial questions. First, is PTHrP-(1–36) active in humans when administered sc? Second, if so, what doses are required to elicit effects on mineral metabolism? Third, as clinical and diagnostic use of PTHrP-(1–36) would be limited if administration were accompanied by the development of hypercalcemia, could doses of PTHrP-(1–36) be identified that would have such effects without causing hypercalcemia? Finally, as pharmacokinetic studies have not been performed with PTHrP-(1–36) in humans, what are the MCR and circulating half-life (t_1/2) of PTHrP-(1–36)?

Subjects and Methods

Study subjects

For the MCR and t_1/2 studies, 3 healthy subjects, 2 men and 1 woman, aged 40–60 yr, were studied. For the sc PTHrP protocol, 21 healthy women, ranging in age from 24–65 yr, were recruited from the greater New Haven area. Exclusion criteria for both studies included renal, skeletal, hepatic, gastrointestinal, cardiovascular, hematological, neurological, or endocrine disease, specifically disorders of calcium metabolism or nephrolithiasis. Seventeen subjects were taking no prescribed or over the counter medications, except estrogen, as indicated below. Four were taking medications (cimetidine, a ß-adrenergic blocker, levothyroxine, and lovastatin, 1 subject each). Eighteen subjects were postmenopausal. The number of years since menopause ranged from 1–18. Seven subjects were receiving estrogen replacement therapy, and 14 were not. All had normal physical examinations. All provided written consent to the studies described herein, and the studies were approved by the Yale University School of Medicine human investigation committee.

Protocols

For the MCR studies, subjects were admitted to the out-patient General Clinical Research Center after an overnight fast. Three baseline plasma samples were obtained for the measurement of PTHrP-(1–36), as shown in Fig. 1, and a 3-h continuous iv infusion of PTHrP-(1–36) at 80 pmol/kg·h was begun. Plasma samples were collected, as described in Fig. 1, at 60, 90, 120, 150, and 180 min, and the infusion was abruptly terminated at 180 min. Samples were then collected at 3-min intervals for PTHrP-(1–36) assay until 218 min, as shown in Fig. 1. MCR was calculated as follows: MCR (mL/min) = [infusion rate (pmol/kg·min)]/[steady state plasma concentration (pmol/mL)].

View larger version (17K): [in this window] [in a new window]   Figure 1. Upper panel, Results of 3-h steady state PTHrP-(1–36) iv infusion in three normal healthy subjects. The plateau phase during the continuous infusion was used to calculate the MCR. The infusion was abruptly terminated at 180 min, and the degradation rates were used to calculate the t1/2. Lower panel, This figure shows the same data as in the upper panel, but the x-axis is expanded to allow ready visualization of the disappearance rate of PTHrP-(1–36). Note that the y-axis is a log scale, and that approximately 90% of the decay displays linear kinetics from the plateau concentration (mean, 54 pmol/L) until approximately 5 pmol/L. The dotted lines indicate the first two 50% decay positions of a representative subject.

View larger version (18K): [in this window] [in a new window]   Figure 2. Mean plasma immunoreactive PTHrP-(1–36) determined using a two-site IRMA for PTHrP-(1–36) in the three study subject groups after sc injection of PTHrP-(1–36). In this and subsequent figures, the following symbols are used: closed squares, the 3.28 µg/kg dose; closed triangles, the 1.64 µg/kg dose; open squares, the 0.82 µg/kg dose. Bars in this and subsequent figures represent the SE. For simplicity, the SE bars are shown only for the 15 min point in this figure. The changes from baseline are highly significant in each of the three groups with P values of 0.0006, 0.0037, and 0.0006 at 15 min in the high, medium, and low dose groups. The differences between the two lower doses are not significant.

View larger version (13K): [in this window] [in a new window]   Figure 3. Upper panel, Mean total serum calcium concentrations in the three groups before and after sc PTHrP-(1–36) injection. The symbols and error bars are the same as in Fig. 2. The SE bars in this figure are too small to be apparent. Middle panel, Mean ionized serum calcium values in the three groups. The symbols and error bars are the same as in Fig. 2. The SE bars in this figure are too small to be apparent. Lower panel, Mean circulating concentrations for endogenous PTH-(1–84), as determined using a two-site PTH-(1–84) IRMA. The symbols and error bars are the same as in Fig. 2.

View larger version (18K): [in this window] [in a new window]   Figure 4. Mean values for FECa in the three groups. The symbols and error bars are the same as in Fig. 2. P values at each time point in the three groups, from high, middle, and low doses, respectively, are as follows: 2 h, 0.014, 0.019, and 0.022; 4–10 h, NS.

View larger version (17K): [in this window] [in a new window]   Figure 5. The symbols and error bars are the same as in Fig. 2. Upper panel, Mean serum phosphorus concentrations in the three groups. P values at each time point in the three groups, from high, middle, and low doses, respectively, are as follows: 1 h, 0.015, NS, and 0.02; 3 h, 0.004, 0.039, and NS; 5 h, NS, NS, and NS; 7 h, NS, NS, and 0.01; 9 h, NS, 0.018, and NS. Middle panel, Mean FEPi in the three groups. P values at each time point in the three groups, from high, middle, and low doses, respectively, are as follows: 2 h, 0.005, 0.002, and NS; 4 h, 0.006, 0.002, and 0.001; 6 h, 0.006, 0.007, and 0.004; 8 h, 0.004, NS, and 0.008; 10 h, 0.024, NS, and 0.008. Bottom panel, TmP/GFR in the three groups. P values at each time point in the three groups, from high, middle, and low doses, respectively, are as follows: 2 h, 0.002, 0.002, and 0.007; 4 h, 0.0005, less than 0.0001, and 0.0009; 6 h, 0.0026, 0.003, and 0.0006; 8 h, NS, NS, and 0.0001; 10 h, 0.025, NS, and 0.0005.

View larger version (18K): [in this window] [in a new window]   Figure 6. NcAMP excretion in the three groups. The symbols and error bars are the same as in Fig. 2. P values at each time point in the three groups, from high, middle, and low doses, respectively, are as follows: 2 h, 0.0006, 0.004, and 0.001; 4 h, 0.0009, 0.004, and 0.002; 6 h, 0.0032, 0.0015, and 0.003; 8 h, NS, 0.02, and NS; 10 h, NS, NS, and NS.

View larger version (18K): [in this window] [in a new window]   Figure 7. Mean plasma 1,25-(OH)2D concentrations in the three groups. The symbols and error bars are the same as in Fig. 2. P values at each time point in the three groups, from high, middle, and low doses, respectively, are as follows: 1 h, NS, NS, and NS; 3 h, NS, 0.017, and NS; 5 h, 0.006, 0.022, and 0.031; 7 h, 0.006, NS, and NS; 9 h, 0.01, NS, and NS.

PTHrP-(1–36) was synthesized at the William Keck Peptide Synthesis Facility at Yale University School of Medicine. Its structure, purity, and biological and immunologic activities were documented using mass spectroscopy, amino acid analysis, analytical reverse phase high performance liquid chromatography, SaOS-2 adenylyl cyclase assay, and PTHrP-(1–36) RIA, as previously described (3), and purity and sterility were confirmed using the limulus amebocyte gel clot assay and standard aerobic and anaerobic bacterial culture. Approval for use in human subjects was provided both by the Yale University School of Medicine Human Investigation Committee and an investigational new drug application from the U.S. FDA. The doses of peptide employed were based on peptide content, as determined by amino acid analysis. The lot used in the current study is the same as that used in our prior report on the iv administration of PTHrP-(1–36) (3). The peptide, lyophilized in 300-µg aliquots, was suspended immediately before use in 0.5–1.0 mL sterile saline.

Measurements

Serum and urinary total and ionized calcium, phosphorus, and creatinine were measured by standard methods in the Yale-New Haven Hospital Clinical Chemistry Laboratory. Serum PTH-(1–84) was measured using the Nichols Institute (San Juan Capistrano, CA) Allegro Intact PTH two-site immunoradiometric assay (IRMA) as described previously (3). Plasma and urinary cAMP and plasma 1,25-(OH)₂D were measured, and NcAMP, FECa, FEPi, and TmP/GFR were calculated, as we have reported previously (3, 6, 19, 20). Plasma PTHrP-(1–36) was measured using a two-site IRMA developed in our laboratory and described in detail previously (21). Plasma samples were collected for PTHrP-(1–36) IRMA in a cocktail of protease inhibitors (5) and 10% glycerol and stored at -70 C until the day of assay.

Statistics

Data were analyzed using Statview (Abacus Concepts, Berkeley, CA) software operated on a Macintosh computer using Student’s paired t test as shown in the figures, where each posttreatment time point was compared to the baseline values for the same patient groups. P < 0.05 was considered significant. P values are described in the figure legends to simplify the figures.

Results

MCR and half-life studies

Three normal subjects were infused with PTHrP-(1–36) under steady state conditions at an infusion rate of 80 pmol/kg·h. This dose was chosen based on pilot studies that demonstrated that the infusion of this dose yielded plasma levels of PTHrP-(1–36) that were sufficiently high that reliable measurements could be made during the decay portion of the curve at the completion of the infusion. The results of these studies are shown in Fig. 1. As shown in the upper panel, circulating PTHrP-(1–36) concentrations were remarkably constant during the infusion. A steady state was achieved by the initial sampling time (60 min) and continued throughout the duration of the infusion. The mean ± SD MCR was calculated to be 24.35 ± 0.067 mL/min·kg based on an infusion rate of 80 pmol/kg·h; mean plasma concentrations of 54.8, 54.4, and 54.3 pmol/L; and individual body weights of 62, 101, and 108 kg in the three subjects. As shown in the lower panel of Fig. 1, in which the x-axis of the left panel is expanded, the decay in plasma PTHrP-(1–36) was linear when plotted on a log scale until plasma concentrations had fallen by 90%. From the figure, the t_1/2 can be seen to be approximately 6 min in two subjects and 5 min in the third.

Subcutaneous administration of PTHrP-(1–36)

Figure 2 shows the plasma concentrations of PTHrP achieved after sc administration of PTHrP-(1–36). Plasma levels rose promptly, reaching a peak 15–30 min after injection, and then declined to values similar to baseline within 2–3 h. Subjects receiving the highest dose achieved the highest plasma levels, and those receiving the lower two doses had similar circulating concentrations. The lack of a clear dose response with the two lower doses would appear to reflect individual variation in the subjects in these two groups, as a clear-cut dose response was observed in other parameters (see below).

The effects of sc administration of PTHrP-(1–36) on serum total and ionized serum calcium are shown in Fig. 3. Despite the changes in circulating PTHrP-(1–36) concentrations in Fig. 2, there was no change in the serum total or ionized calcium during the 10 h after injection. No important change in parathyroid gland function, as measured using plasma concentrations of endogenous PTH-(1–84), was observed during the study.

The effects of sc PTHrP-(1–36) on FECa are shown in Fig. 4. The FECa rose at 2 h in each of the 3 groups, and these changes were highly significant. The FECa increased at 2 h in each of the 21 subjects studied. By 6 h, the FECa had returned to baseline in all 3 groups. FECa rose in some subjects at 8 and 10 h, but these changes did not achieve statistical significance.

Serum phosphorus, FEPi, and TmP/GFR are shown in Fig. 5. There was a gradual decline in the serum phosphorus in each of the three groups during the course of the study from a baseline of approximately 3.6–3.8 mg/dL to approximately 3.2–3.3 mg/dL at the conclusion. The changes were significant in each groups. The middle panel of Fig. 5 shows the FEPi values for the three groups and demonstrates that increases in FEPi occurred in each that were highly significant. These increases in FEPi together with the mild declines in serum phosphorus were reflected in decrements in the TmP/GFR (Fig. 5, bottom panel), changes that again were highly significant.

Subcutaneous PTHrP-(1–36) administration resulted in changes in NcAMP excretion (Fig. 6). These changes were observed during the first 2-h urine collection, as would be expected from the changes in circulating PTHrP-(1–36) shown in Fig. 2. The changes in NcAMP excretion were highly significant in statistical terms and demonstrated a clear dose response. NcAMP excretion had returned to baseline by 4–8 h, depending on the dose of PTHrP-(1–36) administered.

Plasma 1,25-(OH)₂D rose significantly, but transiently, in subjects receiving the lower two doses, peaking at 3–5 h, then returning to baseline (Fig. 7). In contrast, administration of the highest dose resulted in a sustained and statistically important increase in plasma 1,25-(OH)₂D, which persisted until the conclusion of the study.

With regard to safety, each subject was encouraged to report symptoms of any kind of distress or discomfort during or after the study. No allergic, cardiovascular, or other adverse events or symptoms occurred. Blood pressure and pulse rate were monitored throughout the study and remained stable in each subject.

Discussion

One goal of the current study was to determine the MCR and t_1/2 of PTHrP-(1–36). From the information shown in Fig. 1, the t_1/2 can be seen to be 5–6 min. These values are similar to those reported by Fraher et al. (22), who used the bolus injection technique and curve-stripping algorithms to determine a t_1/2 for PTHrP-(1–34) of 8.28 ± 1.6 min. These values are similar to those reported for PTH-(1–84) and PTH-(1–34) by a number of other researchers and are expected for a small peptide hormone (22, 23). The mean MCR for PTHrP-(1–36) in the current study in three healthy subjects was 24.35 ± 0.067 mL/min·kg. The MCR from the Fraher study was 3950 mL/min, which, when corrected for weight to a 70-kg person, would be 56.43 mL/min·kg, a value approximately twice that observed in the current study. We attribute these differences to differences in the pharmacokinetics of PTHrP-(1–34) vs. PTHrP-(1–36) and to differences in the pharmacokinetic techniques used in the two studies. The site of clearance or degradation of PTHrP-(1–36) has not been identified.

A second goal of the study was to determine whether PTHrP-(1–36) would be effective when given sc, and to determine what doses would be required to elicit biological effects. As noted in Materials and Methods, the doses used were based on our prior iv studies. The lower dose (0.82 µg/kg) is equivalent in a 55-kg female to 45 µg, a dose similar to the those of PTH-(1–34) used in studies in humans with osteoporosis, which generally have been in the 25–40 µg/day range (13, 14, 15, 16, 24). From the responses of FECa, FEPi, and TmP/GFR, which appeared to be maximal, and from the robust NcAMP responses at each dose, it would appear that even smaller doses would have elicited effects.

As shown in Fig. 2, sc PTHrP caused a prompt increase in iPTHrP-(1–36). The rise was more rapid than we had anti-cipated and was more rapid than the rise in PTH-(1–34) observed by Lindsay et al. (24), suggesting that PTHrP absorption from sc sites was more efficient than that described for PTH-(1–34) (24). It is interesting to speculate as to the ultimate consequences of this rapid absorption and disappearance on the potential skeletal anabolic effects, or lack thereof, of PTHrP-(1–36), but clarification of this issue will have to await future studies.

Previously, we have shown that PTHrP-(1–36) when given iv causes hypercalcemia (3). In current study, sc PTHrP-(1–36) did not cause hypercalcemia. Nonhypercalcemic doses of PTHrP-(1–36) were selected for these studies for two reasons. First, in the future we hope to perform longer term studies in patients with osteoporosis and would not want to use doses of PTHrP-(1–36) that would cause hypercalcemia over the long term. Second, as we plan to perform longer term studies on the regulation of plasma 1,25-(OH)₂D and the regulation of renal calcium handling by PTHrP-(1–36), we wanted to use doses that did not cause hypercalcemia, as hypercalcemia could inhibit renal 1,25-(OH)₂D production and would increase the filtered load of calcium. The goal of maintaining a normal serum calcium level during the study was achieved and was further reflected in the maintenance of normal values for iPTH-(1–84).

Interestingly, sc PTHrP-(1–36) resulted in a rise in FECa at 2 h. This was highly significant and was observed in every subject at every dose. This early calciuric effect was also seen in our earlier study in volunteers receiving PTHrP-(1–36) or PTH-(1–34) iv (3). This early calciuric response was not due to nonspecific factors, for it was not seen in our previous study in sham-infused subjects studied under identical conditions in an identical setting (3). The early calciuric effect was not dose related in the three groups, despite clear differences in PTHrP levels achieved with the three doses and in NcAMP excretion in response to these doses. Presumably, as noted above, each of the three doses used in the current study was maximally stimulatory for this effect.

The fall in FECa at 4–6 h was also interesting. This fall may simply reflect the decline in circulating PTHrP-(1–36) shown in Fig. 2, with loss of the early putative PTHrP-(1–36)-mediated calciuric effect. Alternatively, this fall in FECa at 4–6 h could represent activation of distal tubular calcium reabsorbtion. An anticalciuric effect of PTHrP-(1–36) was observed in our earlier iv study (3) and was expected in the current study. The results observed in this study together with those in our earlier study make it clear that studies designed to examine the anticalciuric potency of PTHrP-(1–36) will need to be of steady state design, employing long term continuous infusion of PTHrP-(1–36).

The effects of PTHrP-(1–36) on serum phosphorus, FEPi, and TmP/GFR were precisely those expected. The only surprising feature was the lack of an apparent dose-response curve with the three different doses. Again, this presumably indicates that all doses maximally stimulated FEPi and suggests that even smaller doses of PTHrP-(1–36) would be effective in inhibiting renal phosphorus reabsorbtion. Similarly, as PTHrP was first posited to exist in patients with HHM because of their increase in NcAMP excretion (5, 6), the effects of PTHrP-(1–36) on NcAMP excretion were precisely those expected: a dose-related, early rise with a rapid return to baseline.

Subcutaneously administered PTH-(1–34) produces a rise in plasma 1,25-(OH)₂D concentrations (25), and iv administered PTHrP-(1–34) and PTHrP-(1–36) also produce increases in plasma 1,25-(OH)₂D concentrations (3, 24). On the other hand, patients with HHM display reductions in plasma 1,25-(OH)₂D concentrations despite their elevations in circulating PTHrP concentrations (5, 6, 7). The reason for this discrepancy between short term PTHrP studies in normal subjects and the authentic clinical situation in HHM is unclear. Explanations include the possibilities that patients with HHM become desensitized to the effects of PTHrP, that circulating inhibitors of PTHrP are present in patients with HHM and that PTHrP fragments in addition to PTHrP-(1–36) circulate in HHM and have different effects on renal 1-hydroxylase. With the development of a rapid sc PTHrP-(1–36) plasma 1,25-(OH)₂D stimulation test such as that described here, it will be possible to begin to determine whether patients with HHM are resistant to PTHrP-(1–36) or have inhibition of renal 1-hydroxylase. Along the same lines, with the development of longer term, steady state infusion protocols, it will be possible to determine whether chronic administration of PTHrP-(1–36) to normal subjects desensitizes the renal PTH receptor-1-hydroxylase complex in subjects chronically exposed to PTHrP-(1–36). These are the goals of future studies.

As was the case for iv administered PTHrP-(1–36) (3) and PTHrP-(1–34) in the studies of Fraher et al. (24, 26), no adverse effects of PTHrP-(1–36) were observed in any of the subjects. None had allergic, urticarial, cardiovascular, or other adverse effects. Thus, to date, sc and short term iv administration of PTHrP-(1–36) in the doses described would appear to be safe.

In summary, PTHrP-(1–36) is biologically active when given sc. The responses observed were those predicted by earlier iv studies of PTHrP-(1–36) and PTHrP-(1–34) and earlier studies employing PTH-(1–34) administered sc. With clarification of the doses that can elicit biological effects over the short term without inducing hypercalcemia, it is now possible to design more complex and longer term studies aimed at more precisely defining the pathophysiological discrepancies between HHM and HPT. It should be emphasized that multiple nonamino-terminal secretory forms of PTHrP exist, and as these are more fully characterized in structural terms, they, too, may be employed in similar studies to examine their potential roles in the clinical HHM syndrome. Finally, these studies demonstrate the feasibility of administering PTHrP-(1–36) sc to humans and set the stage for exploring whether this peptide has potential as an anabolic therapeutic agent for osteoporosis.

Acknowledgments

We thank Frances Rife, R.N., and Karynn Gerow, R.N., for their assistance with the performance of these studies. We also thank Terrence Wu for technical support, Peter N. Peduzzi, Ph.D., for biostatistical support, and Valentin Pacqual, R.P.S., for the support of the Yale-New Haven Hospital Pharmacy. Finally, we thank Dr. Anita Shah at Bayer Pharmaceuticals (West Haven, CT) for help in analyzing the MCR data.

Footnotes

¹ This work was supported by the Department of Veterans Affairs, the Adult General Clinical Research Center, Yale University School of Medicine, and NIH Grants RR-125, DK-51081, and T32-DK-07058.

Received June 25, 1996.

Revised September 10, 1996.

Accepted November 20, 1996.

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