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Original Studies |
Division of Endocrinology, University of Pittsburgh Medical Center (A.F.S.), Pittsburgh, Pennsylvania 15213; the Departments of Orthopedics (C.G.) and Endocrinology (H.P., M.M., A.F.S.), Yale University School of Medicine, New Haven, Connecticut 06520; and the Department of Veterans Affairs (A.F.S.), Pittsburgh, Pennsylvania 15213
Address all correspondence and requests for reprints to: Andrew F. Stewart, M.D., Division of Endocrinology, E-1140 Biomedical Sciences Tower, University of Pittsburgh Medical Center, 3550 Terrace Street, Pittsburgh, Pennsylvania 15213. E-mail: stewart{at}med1.dept-med.pitt.edu
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Abstract |
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Daily sc PTHrP-(1–36) administration caused no significant changes in serum calcium or phosphorus concentrations, fractional calcium excretion, the tubular maximum for phosphorus, fractional calcium excretion, or plasma 1,25-dihydroxyvitamin D concentrations. Nephrogenous cAMP and endogenous PTH-(1–84) declined. Importantly, markers of bone formation trended upward, as reported in subjects treated with PTH. In marked contrast to findings in PTH-treated subjects, in PTHrP-treated subjects, markers of bone resorption declined in a highly significant fashion.
These observations indicate that PTHrP-(1–36) treatment uncouples bone formation from resorption, in favor of formation. This uncoupling, if it were to continue over the longer term, would predict that PTHrP-(1–36) might be a potent anabolic therapeutic agent for osteoporosis.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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With the documentation that intermittently administered PTH peptides can increase bone mass, peptides based on the PTH sequence are currently being employed in human trials for the treatment of osteoporosis (11, 12, 13). In these trials, PTH administration is associated with an increase in both markers of bone formation and those of bone resorption (11, 12, 13), and the increase in bone mass observed would appear ultimately to reflect the marginal or net increase in osteoblastic bone formation over osteoclastic, and perhaps osteocytic, bone resorption. The increase in bone mass has been most striking in the lumbar spine and to a lesser extent the hip (11, 12, 13). These effects may be enhanced when PTH is used in combination with other agents that increase bone mass, such as estrogens (12).
PTH-related protein (PTHrP) was identified as the agent responsible for humoral hypercalcemia of malignancy (HHM) (3, 15, 16). PTH and PTHrP share common structural and functional properties, and PTH and PTHrP-(1–36) function in the kidney and the skeleton through a common receptor, the PTH/PTHrP receptor (17). As was originally believed for PTH, PTHrP is generally considered to be a skeletal catabolic agent, because patients with HHM develop marked osteoclastic bone resorption and suppression of osteoblastic bone formation, and because the hypercalcemia that occurs in these patients occurs at the expense of the skeleton (18). As is the case with PTH in hyperparathyroidism, this catabolic skeletal effect of PTHrP in HHM occurs in the context of continuous exposure of the skeleton to PTHrP. However, as has been observed for PTH, administration of PTHrP on an intermittent basis to rodents increases bone mass; in one study it is less effective than PTH (19), whereas in another it exceeds the anabolic effect of PTH (20).
Underscoring the importance of PTHrP in skeletogenesis, Karaplis and colleagues (21, 22) have demonstrated that disruption of the PTHrP gene or that of the PTH/PTHrP receptor leads to a severe, lethal skeletal dysplasia associated with accelerated chondrocyte apoptosis and premature skeletal mineralization. Conversely, Schipani and Weir and their colleagues (23, 24) have demonstrated that overexpression of PTHrP or of the constitutively active PTH/PTHrP receptor in the epiphysis under the control of the collagen type II promoter results in dramatic delays in chondrocyte apoptosis and mineralization. In contrast, skeletal abnormalities are not observed in congenital hypoparathyroidism (25). Collectively, these studies could be interpreted to indicate that PTHrP plays a central role in skeletogenesis, that PTHrP is more important than PTH in these processes, and that PTHrP is the physiological ligand for the PTH/PTHrP receptor, at least in the context of skeletogenesis.
PTHrP is initially translated as a prohormone, which then undergoes extensive posttranslational processing (15, 26). Work from our laboratory has shown that one of the mature secretory forms of PTHrP is human (h) PTHrP-(1–36) (26, 27). Recently, we have shown that PTHrP-(1–36) is equipotent to hPTH-(1–34), in terms of renal calcium and phosphorus handling and renal 1,25-dihydroxyvitamin D [1,25-(OH)2D] production when infused iv into humans over an 8-h period at a dose of 8 pmol/kg·h (27). In addition, hPTHrP-(1–36) is active in humans when administered as a single sc dose (28). The half-life of hPTHrP-(1–36) (6–8 min) (28, 29) is similar to that of PTH-(1–34) (30, 31), but the peak plasma level of hPTHrP-(1–36) after sc injection occurs earlier (15 min) (28) than that observed after PTH-(1–34) administration (30–45 min) (32), suggesting that absorption of hPTHrP-(1–36) from sc sites is more rapid than that of PTH-(1–34).
Given that hPTHrP-(1–36) and hPTH-(1–34) are equipotent in humans, that they share a common receptor, that PTH increases bone mass in humans when administered on a once daily schedule, and that PTHrP increases bone mass in rodents, we wondered whether hPTHrP-(1–36) might also be effective as an anabolic skeletal agent in the treatment of osteoporosis in humans. The current study represents a pilot study designed to begin to explore a potential therapeutic role for hPTHrP-(1–36) in the treatment of osteoporosis. The specific questions being asked were the following. 1) Can hPTHrP-(1–36) increase markers of bone turnover in postmenopausal estrogen-deficient women over a 2-week period? 2) If so, what doses would be required? 3) Finally, what adverse effects, if any, would be observed? The results indicate that 2 weeks of hPTHrP-(1–36) treatment in the doses employed herein are not associated with adverse effects. hPTHrP-(1–36) treatment is indeed capable of stimulating bone formation markers. Moreover, instead of the increase in resorption markers encountered with PTH treatment, resorption markers were reduced with hPTHrP-(1–36) treatment; resorption and formation appear to be dissociated or uncoupled in favor of formation.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The subjects in the current study were 13 postmenopausal Caucasian women (mean age ± SEM, 58 ± 6.3 yr; mean ± SEM duration since menopause, 10.9 ± 5.9 yr) recruited primarily from the employee pool of the Yale-New Haven Hospital (New Haven, CT). All were in excellent health. Their mean weight was 72.7 ± 14 kg (mean ± SEM). Six had used estrogen preparations in the past, but none had received estrogen therapy within the past 5 yr. Bone densitometry was not performed. None had a history of disorders of calcium metabolism (e.g. osteoporosis or Paget’s disease) or of disorders associated with skeletal disease (e.g. renal or gastrointestinal disorders), and none was taking medications that influence mineral or skeletal metabolism. None had received bisphosphonate or other therapy directed against skeletal disease. All underwent baseline physical examinations that were normal and had screening testing that indicated that baseline serum calcium, phosphorus, and creatinine values and hematological measures were normal. All provided informed written consent, and the protocol employed was approved by the Yale University School of Medicine human investigation committee (Protocol 8520).
Study design
Each of the study subjects underwent baseline screening as described above. Each provided baseline blood and 2-h urine samples on day 1 of the study, and then began a schedule of daily sc injection of hPTHrP-(1–36) injected into abdominal fat at approximately 0800–0900 h each day by the Clinical Research Center staff or by the subjects themselves under CRC staff supervision. The peptide was suspended in 1.0–2.0 mL sterile saline immediately before administration. The subjects received 1.64 µg/kg·day (low dose), 3.28 µg/kg·day (medium dose), or 6.56 µg/kg/day (high dose). Each dose was administered to a total of seven study subjects. Several subjects participated in the study receiving more than one dose, such that seven received only one dose, four received two doses, and two received all three doses. As this was a pilot study, and each subject served as her own control, no attempt was made to randomize the subjects. In cases where a study subject received the test drug on more than one occasion, the lower dose was always administered first, and there was a minimum of a 4-week washout period between doses. In each subject receiving more than one dose, bone turnover markers (see below) had returned to baseline by the end of the washout period.
Repeat bloods and urine samples (2 h) were obtained on days 7 and 14 of the study 22–24 h after the last injection of the study drug.
Analyses
Blood and urine samples were analyzed for serum total and ionized calcium, phosphorus, and creatinine; plasma and urinary cAMP; and plasma 1,25-(OH)2D and intact PTH-(1–84) as described in detail previously (27, 28, 33). Calculations of fractional excretion of Ca, fractional excretion of phosphorus, tubular maximum for phosphorus (TmP/GFR), and nephrogenous cAMP (NcAMP) were performed as reported in detail previously (27, 28, 33). Bone-specific alkaline phosphatase was measured by immunoradiometric assay using the Tandem-R Ostase kit purchased from Hybritech (San Diego, CA). Osteocalcin was measured using a homologous RIA we have described previously in detail (34). N-Telopeptide was measured using the Osteomark kit purchased from Ostex International (Seattle, WA). Deoxypyridinoline cross-links were measured using the Pyrilinks-D kit purchased from Metra Biosystems (Mountain View, CA).
hPTHrP-(1–36) peptide
This peptide was synthesized at the William Keck Peptide Synthesis Facility at Yale University (New Haven, CT). The composition and content of the peptide were confirmed by mass spectroscopy and amino acid analysis, and the purity was documented using reverse phase high performance liquid chromatography as described in detail previously (27, 28). The immunoreactivity and bioactivity were also documented as described in detail previously (27, 28). The lot of peptide used in the current study was the same as that employed in our prior studies (27, 28). The peptide was sterile filtered and aliquoted into sterile vials, and the vials were tested for pyrogen and sterility as described previously in detail (27, 28). The use of the peptide for these studies was approved by the FDA (Investigational New Drug 49,175).
Statistical analysis
Statistical analysis was performed using ANOVA in which each parameter measured, as shown in the figures, was compared on days 7 and 14 to the baseline, day 0, value in the same group of subjects receiving the same PTHrP dose. Differences from the baseline value were considered significant if P < 0.03. ANOVA was performed using the SigmaStat 1.0 for Windows software.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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. As is clear
from the figure, there were no changes in serum calcium. There were no
changes in ionized serum calcium (not shown). Similarly, as shown in
Fig. 2, there were no significant changes
in serum phosphorus or the fractional excretion of phosphorus over the
2-week period. Interestingly, in contrast to the short term reduction
in the TmP/GFR reported over a 2- to 8-h period after hPTHrP-(1–36)
administration (27, 28), there was an increase in the TmP/GFR
determined in subjects receiving the highest dose of PTHrP. There were
no significant changes in the fractional excretion of calcium.
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. As can be seen from the
figure, subjects receiving the highest dose of PTHrP-(1–36) displayed
a significant decline in both endogenous plasma PTH-(1–84)
concentrations and NcAMP excretion. In contrast to the decline in these
two measurements, plasma 1,25-(OH)2D rose during the first
week of PTHrP administration and then returned to baseline.
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, bone-specific alkaline phosphatase concentrations appeared to rise in
subjects receiving the two highest doses, but these changes did not
achieve statistical significance. In a parallel fashion, osteocalcin
values progressively rose in study subjects receiving both the medium
and the highest dose, and these changes were highly significant in
statistical terms.
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No subject experienced adverse effects during the study. Subjects were specifically asked to report vasomotor, allergic, and gastrointestinal adverse effects and were encouraged before and at the conclusion of the study to report any other type of adverse effect. None were experienced. As evidence of the lack of adverse effects, more than half of the subjects requested that they be allowed to receive more than one dose of the study drug, as outlined in Materials and Methods.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Treatment of humans with sc PTHrP-(1–36) in the doses employed in this
study produced no change in serum calcium over a 6- to 10-h period
(28), but produced prompt (within 2–8 h) changes in serum phosphorus,
renal phosphorus excretion, renal calcium excretion, NcAMP excretion,
and plasma 1,25-(OH)2D concentrations (28). These changes
returned to baseline within 12 h of administration of PTHrP (28).
It is important, therefore, in the interpretation of the results shown
in
Figs. 1–3
to bear in mind that the results reflect values obtained
22–24 h after the last administration of PTHrP, when changes in these
parameters would be expected to have returned to baseline; the values
were obtained either before the administration of PTHrP-(1–36) on day
0 or 24 h after the last administration of PTHrP-(1–36) on days 7
and 14.
As expected, therefore, no changes were observed in serum total or ionized calcium, serum phosphorus, fractional excretion of phosphorus, or calcium over the 2 weeks of the study. Interestingly and in contrast to what might have been expected, in the subjects receiving the highest dose of the peptide, there was a small increase in the TmP/GFR at the end of the first week, and this had corrected itself by the end of the second week. The lack of significant change in serum and urinary mineral chemistries were exactly what had been desired in the design of the study and the selection of the doses of hPTHrP-(1–36), as this is what one would like to see in subjects treated for osteoporosis. There were no important changes in circulating 1,25-(OH)2D during the study.
Statistically and quantitatively significant declines were observed in endogenous PTH-(1–84) concentrations and NcAMP excretion. Again, this is not likely to be artifactual because the declines in endogenous PTH-(1–84), as assessed using a two-site immunoradiometric assay, and the declines in NcAMP, the biological readout for PTH, were quantitatively and directionally similar. These changes were only observed in subjects receiving the highest dose of hPTHrP-(1–36). Why this occurred is uncertain, but several possibilities come to mind, including a direct inhibitory effect of PTHrP administration on PTH secretion (36), or an unmeasurably small, but significant, increase in serum calcium concentration. Importantly, the declines in PTH and NcAMP may in part contribute to the decline in markers of bone resorption observed, as detailed further below.
In the current study, hPTHrP-(1–36) administration in the two higher doses led to an elevation in osteocalcin, a marker of bone formation. Bone-specific alkaline phosphatase, although not achieving statistical significance, appeared to be rising during the study in subjects receiving the two higher doses. The increment in bone formation markers is in accord quantitatively with prior studies using PTH. In one study by Hodsman and colleagues (39), the formation markers alkaline phosphatase, procollogen-1 carboxyterminal peptide (P1CP), and osteocalcin were beginning to rise after 2 weeks of PTH-(1–34) therapy at 800 U/day, although the increments were not yet statistically significant by 2 weeks of therapy. In the longer term studies of bone formation markers after PTH administration reported by Lindsay, Finkelstein, and Hodsman et al. (11, 12, 13), 2 week values were not reported, but by interpolation of their data, the changes observed at 2 weeks might have been of similar magnitude to those reported herein. Whether the early increase in bone formation observed in the current study would continue over the longer term with continued hPTHrP-(1–36) therapy is unknown, but there seems no reason to presume that it would not, given the experience with PTH in humans (1, 2, 3, 11, 12, 13) and PTHrP in animals (1, 2, 3, 7, 8, 9, 10).
The most surprising finding in the current study was that both markers of bone resorption employed, N-telopeptide and deoxypyridinoline cross-links, declined during PTHrP treatment. This is counterintuitive for several reasons. First, PTHrP was initially identified through its role as a potent bone-resorbing factor in patients with HHM (15, 18). Second, continuous administration of PTHrP to laboratory animals by miniosmotic pump stimulates bone resorption (37, 38). Third, osteoclastic and osteoblastic activities are generally coupled, and agents that stimulate bone formation have generally also stimulated bone resorption. In particular, this is the case for PTH given sc to humans for the treatment of osteoporosis. In subjects treated with PTH peptides, bone resorption markers increase with (although perhaps with a delay and to a lesser extent than) the increase in formation markers (11, 12, 13), even at early time points such as 14 days (39). On the other hand, the doses employed in the high dose group in the current study were higher than those employed for PTH in human studies on osteoporosis. It is, therefore, formally possible that PTH administered at higher doses could have this resorption-lowering effect.
Uncoupling of formation from resorption is characteristic of animals and humans with HHM. Thus, there is some precedent for uncoupling of bone cell responses to PTHrP. In that situation, however, the uncoupling is directionally opposite to that observed herein, with resorption far exceeding formation (15, 18). It is not clear why this uncoupling of formation and resorption occurs in humoral hypercalcemia of malignancy, and it is no clearer why uncoupling of formation and resorption markers was observed in the current study. One could speculate that the brief (30–180 min) appearance of hPTHrP-(1–36) in the circulation after sc injection is sufficient to chronically activate osteoblastic bone formation, but inadequate to activate osteoclastic or osteocytic calcium mobilization more than momentarily. This, of course, would require that the plasma appearance of PTHrP be different from that observed for PTH after sc administration, and this has, in fact, been demonstrated. hPTHrP-(1–36) is more rapidly absorbed than hPTH-(1–34) from abdominal fat after sc administration. hPTHrP-(1–36) reaches a peak in plasma 15 min after sc injection (28), whereas the peak in plasma PTH-(1–34) after sc administration occurs in 30–45 min (32). The half-life of both peptides in humans, once peak plasma levels have occurred, is similar, in the range of 5–8 min (28, 30, 31). Thus, different pharmacokinetics for the two peptides seem a plausible, although unsubstantiated, explanation for the uncoupling of formation and resorption observed after hPTHrP-(1–36) administration.
Another explanation, not mutually exclusive with the first, may lie in the suppression of endogenous PTH-(1–84). Perhaps the transient exposure to hPTHrP-(1–36) is sufficient to activate osteoblastic bone formation over the long term, but has no effect on osteoclastic activity, and the mean decline in PTH over 24 h results in a fall in daily integrated bone resorption rates. Again, there is no direct experimental support for this possibility. Finally, it is possible that PTHrP may in some unknown fashion directly inhibit bone resorption, or PTHrP may indirectly activate some inhibitor of osteoclastic bone resorption or osteoclastic differentiation within the marrow compartment, although, to our knowledge, there is no evidence to support either of these possibilities.
From a pragmatic standpoint, however, the directionally opposite affects of hPTHrP-(1–36) on formation and resorption are precisely those that would be sought in an ideal antiosteoporosis drug; these are the features of a pure skeletal anabolic agent. If these findings prove to be reproducible, to continue over the long term, and to be unassociated with toxicities, it is plausible to consider that the anabolic skeletal effects of PTHrP-(1–36) might exceed those of PTH or other known skeletal anabolic agents. Whether this proves to be the case will, of course, require further study.
PTHrP is a hemodynamically active peptide in rodents (40) and, in much larger doses than those employed herein, in humans (35). No hemodynamic changes were observed in any of our subjects despite monitoring and despite questioning about side-effects such as dizziness or flushing. Moreover, no other adverse effects were observed in any subject. Indeed, half of the subjects volunteered to receive a second course of treatment after having participated in one 2-week course.
This study has a number of important limitations. First, it was a small study, with only seven participants per dose. Nonetheless, even with this small number of participants, the results were highly significant. Second, it was neither blinded nor placebo controlled. Again, this was a pilot study, and it should be clear that each subject was her own control, and that knowledge of the doses of peptide received could not have influenced any of the measurements performed, nor could knowledge of the dose received yielded the dose-related responses observed in the turnover markers. Third, the design was not optimal, with several subjects receiving more than one course of treatment. It is highly unlikely that the repeated use of some subjects could have influenced the results, however, because each subject had returned to baseline in terms of turnover markers by the next treatment period of the protocol, and because of the mandatory washout period of the protocol. In subsequent studies, of course, this design issue needs to be specifically addressed. Fourth, it would have been optimal in retrospect to have included a hPTH-(1–34) treatment arm of this study to determine whether PTH-(1–34) therapy over 2 weeks would also have dissociated resorption from formation. As this result was unanticipated, such an arm was not included. Finally, this was a very short trial. This was intentional for three reasons: 1) we were reluctant to administer a potentially catabolic skeletal agent for more that 2 weeks to postmenopausal women without preliminary data; 2) we were unsure whether adverse events might occur if the peptide were administered for more than a single dose; and 3) FDA approval for this initial trial was limited to a 2-week trial period.
In conclusion, hPTHrP-(1–36) administered over 2 weeks as a daily sc injection is safe and well tolerated. hPTHrP-(1–36) activates bone formation as expected. Surprisingly, it has an unexpected ability to reduce bone resorption over a 2-week time frame, which is not explained, but which, if sustained over a period of months to years, would in theory be advantageous in terms of increasing bone mass. If it were to continue, hPTHrP-(1–36) would have a theoretical advantage over PTH analogs as an anabolic skeletal agent for the treatment of osteoporosis, because PTH peptides stimulate both formation and resorption, and the resulting increment in bone mass reflects the marginal increase in formation over resorption. Clarifying these issues must await longer term preclinical trials in animals and certainly merits further investigation.
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Acknowledgments |
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Footnotes |
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Received February 2, 1998.
Revised May 7, 1998.
Accepted May 12, 1998.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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