Treating osteoporosis by targeting parathyroid hormone to bone.

Osteoporosis is a major public health problem despite widespread use of bisphosphonate therapy. PTH(1-34) is a more effective treatment; but its use has been limited by side effects (hypercalcemia, tumor risk) and inconvenient dosing (daily injection). Long-acting forms of PTH are also effective but cause severe hypercalcemia, presumably from effects in kidney. We hypothesized that targeted delivery of PTH to bone using a collagen binding domain (PTH-CBD) could reduce hypercalcemia. PTH-CBD is cleared from serum within 12hours after subcutaneous administration. In ovariectomized rats, monthly administration of PTH-CBD increased spinal BMD by 14.2% with no associated hypercalcemia. Such bone-targeted anabolic agents may ultimately allow the superior efficacy of anabolic therapy to be obtained with the dosing convenience of bisphosphonates.

Prevention of chemotherapy-induced osteoporosis by cyclophosphamide with a long-acting form of parathyroid hormone.

BACKGROUND: Most chemotherapeutics reduce bone mineral density (BMD) and increase risk for fractures by causing gonadal suppression, which in turn increases bone removal. Cyclophosphamide (CYP) also has a direct effect of inhibiting bone formation and removal, making the resulting bone loss particularly difficult to treat with antiresorptive therapy. AIM: We tested whether a single dose of the anabolic agent PTH linked to a collagen binding domain (PTHCBD) could prevent the effects of CYP-induced bone loss. METHODS: Mice received either buffer alone, CYP, or CYP+ PTH-CBD. BMD and alkaline phosphatase were measured every 2 weeks for a total of 8 weeks. RESULTS: After 6 weeks, mice treated with CYP showed expected reductions in BMD (increase from baseline: 7.4 ± 6.9 vs 24.35 ± 4.86% in mice without chemotherapy, p<0.05) and decrease in alkaline phosphatase levels (42.78 ± 6.06 vs 60.62 ± 6.23 IU/l in mice without chemotherapy, p<0.05), consistent with osteoporosis from impaired bone formation. Administration of a single dose of PTH-CBD (320 µg/kg ip) prior to CYP treatment improved BMD (change from baseline: 23.4 ± 5.4 vs 7.4 ± 6.9%, CYP treatment alone, p<0.05) and increased alkaline phosphatase levels (50.14 ± 4.86 vs 42.78 ± 6.06 IU/l in CYP treatment alone, p<0.05). BMD values and alkaline phosphatase levels were restored to those seen in mice not receiving chemotherapy. CONCLUSIONS: A single dose of PTHCBD prior to chemotherapy reversed CYP-induced suppression of bone formation and prevented CYP-induced bone loss in mice.

Monthly administration of a novel PTH-collagen binding domain fusion protein is anabolic in mice.

We synthesized fusion proteins of parathyroid hormone (PTH) (1-33) and the collagen binding domain of ColH (CBD) and tested them for anabolic bone activity in mice. Two fusion proteins were synthesized, linking the carboxy terminus of PTH(1-33) either directly to the amino terminal of the CBD or to the CBD through an adjacent ColH domain (PTH-PKD-CBD). Both PTH-CBD and PTH-PKD-CBD increased cAMP accumulation in cells stably transfected with the PTH/PTHrP receptor, and both peptides bound to type 1 collagen in flow-through assays. Distribution studies indicated that the PTH-CBD was concentrated in the bone and skin, tissues with abundant collagen and blood flow. Administration of 320 µg/kg PTH-CBD either weekly (for 8 weeks) or monthly (for 6 months) to 7-week-old C57BL/6J mice resulted in a sustained increase in bone mineral density (BMD) (15% for weekly studies, 13% for monthly studies; P < 0.05). PTH-PKD-CBD showed only 5% increases in BMD after weekly administration, and, as expected, neither weekly nor monthly PTH(1-34) affected BMD. PTH-CBD increased serum alkaline phosphatase levels. Importantly, there were no significant increases in serum calcium observed. Collectively, the data suggest that PTH-CBD has a sustained anabolic effect in bone with either weekly or monthly administration. This approach of targeted delivery of PTH to bone may show promise for the treatment of disorders of low bone mass, such as postmenopausal osteoporosis.

Identification of determinants of inverse agonism in a constitutively active parathyroid hormone/parathyroid hormone-related peptide receptor by photoaffinity cross-linking and mutational analysis.

We have investigated receptor structural components responsible for ligand-dependent inverse agonism in a constitutively active mutant of the human parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP) receptor type 1 (hP1R). This mutant receptor, hP1R-H223R (hP1R(CAM-HR)), was originally identified in Jansen's chondrodysplasia and is altered in transmembrane domain (TM) 2. We utilized the PTHrP analog, [Bpa(2),Ile(5),Trp(23),Tyr(36)]PTHrP-(1-36)-amide (Bpa(2)-PTHrP-(1-36)), which has valine 2 replaced by p-benzoyl-l-phenylalanine (Bpa); this substitution renders the peptide a photoreactive inverse agonist at hP1R(CAM-HR). This analog cross-linked to hP1R(CAM-HR) at two contiguous receptor regions as follows: the principal cross-link site (site A) was between receptor residues Pro(415)-Met(441), spanning the TM6/extracellular loop three boundary; the second cross-link site (site B) was within the TM4/TM5 region. Within the site A interval, substitution of Met(425) to Leu converted Bpa(2)-PTHrP-(1-36) from an inverse agonist to a weak partial agonist; this conversion was accompanied by a relative shift of cross-linking from site A to site B. The functional effect of the M425L mutation was specific for Bpa(2)-containing analogs, as inverse agonism of Bpa(2)-PTH-(1-34) was similarly eliminated, whereas inverse agonism of [Leu(11),d-Trp(12)]PTHrP-(5-36) was not affected. Overall, our data indicate that interactions between residue 2 of the ligand and the extracellular end of TM6 of the hP1R play an important role in modulating the conversion between active and inactive receptor states.

Multiple sites of contact between the carboxyl-terminal binding domain of PTHrP-(1--36) analogs and the amino-terminal extracellular domain of the PTH/PTHrP receptor identified by photoaffinity cross-linking.

The carboxyl-terminal portions of parathyroid hormone (PTH)-(1--34) and PTH-related peptide (PTHrP)-(1-36) are critical for high affinity binding to the PTH/PTHrP receptor (P1R), but the mechanism of receptor interaction for this domain is largely unknown. To identify interaction sites between the carboxyl-terminal region of PTHrP-(1--36) and the P1R, we prepared analogs of [I(5),W(23),Y(36)]PTHrP-(1--36)-amide with individual p-benzoyl-l-phenylalanine (Bpa) substitutions at positions 22--35. When tested with LLC-PK(1) cells stably transfected with human P1R (hP1R), the apparent binding affinity and the EC(50) of agonist-stimulated cAMP accumulation for each analog was, with the exception of the Bpa(24)-substituted analog, similar to that of the parent compound. The radiolabeled Bpa(23)-, Bpa(27)-, Bpa(28)-, and Bpa(33)-substituted compounds affinity-labeled the hP1R sufficiently well to permit subsequent mapping of the cross-linked receptor region. Each of these peptides cross-linked to the amino-terminal extracellular domain of the P1R: [I(5),Bpa(23),Y(36)]PTHrP-(1-36)-amide cross-linked to the extreme end of this domain (residues 33-63); [I(5),W(23),Bpa(27),Y(36)]PTHrP-(1--36)-amide cross-linked to residues 96--102; [I(5),W(23),Bpa(28),Y(36)]PTHrP-(1--36)- amide cross-linked to residues 64--95; and [I(5),W(23), Bpa(33),Y(36)]PTHrP-(1--36)-amide cross-linked to residues 151-172. These data thus predict that residues 23, 27, 28, and 33 of native PTHrP are each near to different regions of the amino-terminal extracellular receptor domain of the P1R. This information helps define sites of proximity between several ligand residues and this large receptor domain, which so far has been largely excluded from models of the hormone-receptor complex.

Selective and nonselective inverse agonists for constitutively active type-1 parathyroid hormone receptors: evidence for altered receptor conformations.

The spontaneous signaling activity of some G protein-coupled receptors and the capacity of certain ligands (inverse agonists) to inhibit such constitutive activity are poorly understood phenomena. We investigated these processes for several analogs of PTH-related peptide (PTHrP) and the constitutively active human PTH/PTHrP receptors (hP1Rcs) hP1Rc-H223R and hP1Rc-T410P. The N-terminally truncated antagonist PTHrP(5-36) functioned as a weak partial/neutral agonist with both mutant receptors but was converted to an inverse agonist for both receptors by the combined substitution of Leu(11) and D-Trp(12). The N-terminally intact analog [Bpa(2)]PTHrP(1-36)-a partial agonist with the wild-type hP1Rc-was a selective inverse agonist, in that it depressed basal cAMP signaling by hP1Rc-H223R but enhanced signaling by hP1Rc-T410P. The ability of [Bpa(2)]PTHrP(1-36) to discriminate between the two receptor mutants suggested that H223R and T410P confer constitutive receptor activity by inducing distinct conformational changes. This hypothesis was confirmed by the observations that: 1) the double mutant receptor hP1Rc-H223R/T410P exhibited basal cAMP levels that were 2-fold higher than those of either single mutant; and 2) hP1Rc-H223R and hP1Rc-T410P internalized (125)I-PTHrP(5-36) to markedly different extents. The overall results thus reveal that two different types of inverse agonists are possible for PTHrP ligands (nonselective and selective) and that constitutively active PTH-1 receptors can access different conformational states.

Tuberoinfundibular peptide 39 binds to the parathyroid hormone (PTH)/PTH-related peptide receptor, but functions as an antagonist.

The tuberoinfundibular peptide TIP39 [TIP-(1-39)], which exhibits only limited amino acid sequence homology with PTH and PTH-related peptide (PTHrP), stimulates cAMP accumulation in cells expressing the PTH2 receptor (PTH2R), but it is inactive at the PTH/PTHrP receptor (PTH1R). However, when using either (125)I-labeled rat [Nle(8,21),Tyr(34)]PTH-(1-34)amide (rPTH) or (125)I-labeled human [Tyr(36)]PTHrP-(1-36)amide [PTHrP-(1-36)] for radioreceptor studies, TIP-(1-39) bound to LLCPK(1) cells stably expressing the PTH1R (HKrk-B7 cells), albeit with weak apparent affinity (243 +/- 52 and 210 +/- 64 nM, respectively). In comparison to the parent peptide, the apparent binding affinity of TIP-(3-39) was about 3-fold higher, and that of TIP-(9-39) was about 5.5-fold higher. However, despite their improved IC(50) values at the PTH1R, both truncated peptides failed to stimulate cAMP accumulation in HKrk-B7 cells. In contrast, the chimeric peptide PTHrP-(1-20)/TIP-(23-39) bound to HKrk-B7 cells with affinities of 31 +/- 8.2 and 11 +/- 4.0 nM when using radiolabeled rPTH and PTHrP-(1-36), respectively, and it stimulated cAMP accumulation in HKrk-B7 and SaOS-2 cells with potencies (EC(50), 1.40 +/- 0.3 and 0.38 +/- 0.12 nM, respectively) and efficacies (maximum levels, 39 +/- 8 and 31 +/- 3 pmol/well, respectively) similar to those of PTH-(1-34) and PTHrP-(1-36). In both cell lines, TIP(9-39) and, to a lesser extent, TIP-(1-39) inhibited the actions of the three agonists with efficiencies similar to those of [Leu(11),D-Trp(12),Trp(23),Tyr(36)]PTHrP-(7-36)amide, an established PTH1R antagonist. Taken together, the currently available data suggest that the carboxyl-terminal portion of TIP-(1-39) interacts efficiently with the PTH1R, at sites identical to or closely overlapping those used by PTH-(1-34) and PTHrP-(1-36). The amino-terminal residues of TIP-(1-39), however, are unable to interact productively with the PTH1R, thus enabling TIP-(1-39) and some of its truncated analogs to function as an antagonist at this receptor.

Homologous up-regulation of vitamin D receptors is tissue specific in the rat.

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) receptors (VDR) are expressed in multiple tissues within the body. VDR levels are increased by 1,25(OH)2D3 in intestine and kidney and in numerous cell models. The ability of 1,25(OH)2D3 to affect VDR levels in other target tissues in vivo was studied by assessing VDR levels by the 3H-1,25(OH)2D3 binding assay under varied physiological conditions in the rat. When compared with vitamin D-deficient (-D) controls, rats raised on a normal vitamin D-sufficient (+D) diet showed elevated VDR levels in kidney (391 +/- 53 vs. 913 +/- 76 fmol/g of tissue;p < 0.05), but not in testis, heart, or lung. Up-regulation of the VDR also occurred in kidney of +D rats 1 day after a single 100-ng dose of 1,25(OH)2D3 (454 +/- 43 vs. 746 +/- 113 fmol/mg of DNA; p < 0.05), but no changes were seen in intestine, testis, or lung. Because 1,25(OH)2D3-induced hypercalcemia may independently affect VDR regulation, 1,25(OH)2D3 was infused into -D rats, and normocalcemia was maintained by reduced dietary calcium intake. In this model, the renal VDR was again up-regulated (446 +/- 115 vs. 778 +/- 58 fmol/mg of DNA; p < 0.05), but VDR levels in testis and lung were unaffected. Scatchard analysis and tests of 1,25(OH)2D3 dose (1-100 ng/day for 7 days) and temporal (100 ng/day for 1-7 days) responsiveness further supported the tissue-specific nature of the homologous VDR regulation. Assay of VDR levels by L-1-tosylamido-2-phenylethyl chloromethyl ketone-3H-1,25(OH)2D3 exchange assay ruled out differences in endogenous 1,25(OH)2D3 occupancy as the basis for the observed differences in VDR regulation. Finally, coidentity of the VDR-like sites in kidney versus testis was confirmed by competitive binding analysis comparing their relative affinities for 25(OH)D3 versus 1,25(OH)2D3 (30.5 +/- 6.4 vs. 35.6 +/- 3.6 in kidney and testis, respectively) and by immunoblot analysis using a highly specific monoclonal anti-rat VDR antibody. Thus, under a wide variety of experimental conditions, homologous up-regulation of the VDR occurs in the rat kidney in vivo, but not in several other target tissues which do not regulate plasma calcium homeostasis. Moreover, this differential VDR regulation did not result from secondary changes in plasma calcium, from differential 1,25(OH)2D3 responsiveness in the various tissues, nor from differences in endogenous 1,25(OH)2D3 occupancy of the VDR. These studies thus establish that, in contrast to observations in vitro, the widely described phenomenon of homologous VDR up-regulation in kidney and intestine is not a universal property of 1,25(OH)2D3 target tissues in vivo in the rat.

Dexamethasone downregulates vitamin D receptors in rat kidney, unmasking a high affinity binding site.

Dexamethasone exerted tissue-specific effects on rat vitamin D receptor levels: upregulation of receptors in intestine, downregulation in kidney, and no effect on receptors in testis, heart, and lung. Scatchard analysis showed selective downregulation of the low affinity receptor site (Kd = 0.9 nM) in kidney, thus unmasking a high affinity binding site (Kd = 0.05 nM). The single low affinity site in intestine (Kd = 0.3-0.7 nM) was upregulated, while the single high affinity site in testis (Kd = 0.07-0.09 nM) was not changed. These results demonstrate the existence of two types of nuclear binding sites in rat kidney and establish that there are similarities between the high affinity binding site in the rat kidney and the single high affinity receptor site in the testis.

Evidence for two classes of 1,25-dihydroxyvitamin D3 binding sites in classical vs. nonclassical target tissues.

Possible differences in 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] binding sites in classical and nonclassical target tissues were tested by Scatchard analysis of [3H]1,25(OH)2D3 binding in parallel chromatin preparations of rat kidney vs. testis. Two distinct binding components were resolved in kidney (p less than 0.005). Moreover, the single binding site in testis exhibited a 10-fold lower Kd (p less than 0.05) than did the principal binding site in kidney (50 +/- 4 vs. 405 +/- 142 pM). Secondly, regulation of [3H]1,25(OH)2D3 binding sites also differed. 1,25(OH)2D3 injection resulted in increased 1,25(OH)2D3 binding (p less than 0.05) in kidney (92%) and intestine (415%), but not in testis, lung or heart. These results suggest that the principal 1,25(OH)2D3 binding sites in classical targets kidney and intestine may be intrinsically different from those in at least some nonclassical targets.

Arterial pressure effects on preglomerular microvasculature of juxtamedullary nephrons.

Videomicroscopic and micropuncture techniques were utilized to determine segmental microvascular responses of in vitro blood-perfused juxtamedullary nephrons to step changes in renal arterial perfusion pressure (PP). At a PP of 104 +/- 2 mmHg, inside diameters of arcuate arteries (ARC), interlobular arteries (ILA), and afferent arterioles (AFF) averaged 68.6 +/- 6.4, 35.7 +/- 1.5, and 20.4 +/- 2.3 microns, respectively. Variations in PP within the range of 70-180 mmHg elicited alterations in microvessel diameters with the following slopes: ARC, -0.15 micron/mmHg; ILA, -0.13 micron/mmHg; and AFF, -0.14 micron/mmHg. In other experiments, intravascular pressures were measured during changes in PP. Glomerular capillary pressure was well regulated (slope = 0.19 +/- 0.03 mmHg/mmHg), and mid-AFF pressure was partially regulated (slope = 0.60 +/- 0.17 mmHg/mmHg); however, pressure measured at the ILA-AFF branch point responded passively to changes in PP (slope = 0.95 +/- 0.06 mmHg/mmHg). These observations reveal that, although the entire preglomerular vasculature of juxtamedullary nephrons is capable of actively responding to changes in PP, afferent arterioles are responsible for the predominant resistance adjustment throughout the normal autoregulatory range.