Inactivation by plasma may be responsible for lack of efficacy of parathyroid hormone antagonists in hypercalcemia of malignancy.

PTH-related protein (PTHrP) has been shown to be a major factor responsible for hypercalcemia of malignancy. PTHrP acts via the PTH/PTHrP receptor, and therefore, PTH antagonists might be expected to reverse the hypercalcemia in malignancy. In the present studies, the PTH antagonists [Tyr34]bovine (b) PTH-(7-34)NH2, [D-Trp12,Tyr34]-bPTH-(7-34)NH2, or PTHrP-(7-34)NH2, were administered to hypercalcemic athymic nude mice bearing a human squamous cell carcinoma of the lung in 60- to 500-fold molar excess of a dose of PTHrP-(1-34) known to produce hypercalcemia. The antagonists had no significant effect on serum calcium levels. In an adenylyl cyclase assay using the ROS 17/2.8 cells, a potent PTH antagonist, [Leu11,D-Trp12]PTHrP-(7-34)NH2 was rapidly inactivated in the presence of rat or human plasma. This inactivation by plasma was not blocked by common inhibitors of proteolysis (aprotinin, soybean trypsin inhibitor, and leupeptin). Preliminary studies demonstrated that inactivation of the PTHrP antagonist was caused by a plasma component with an apparent mol wt of 230,000 daltons. The knowledge of the structure of the PTH/PTHrP receptor combined with the identification of a hormone-inactivating plasma factor should facilitate the design of PTH-antagonists that are effective in vivo.

Effects of kistrin on bone resorption in vitro and serum calcium in vivo.

In many cell systems, cell-cell and cell-matrix interactions are mediated by integrins, a family of cell surface heterodimeric glycoprotein receptors. Osteoclast integrins may play a role in the process of bone resorption. Osteoclasts express the alpha v and beta 3 subunits of the vitronectin receptor and adhere to a wide range of proteins in vitro, all which contain the amino acid sequence Arg-Gly-Asp (RGD), an adhesion site recognition sequence common to many protein ligands that bind to integrins. The effect of kistrin, an RGD-containing snake venom protein, on osteoclast-mediated bone resorption was investigated in vivo and in vitro. When kistrin was infused into normocalcemic and hypercalcemic mice, serum calcium was significantly lowered at 3 and 6 h after the start of infusion, indicating an inhibitory effect on osteoclast activity in vivo. In vitro, kistrin potently inhibited bone resorption by isolated rat osteoclasts cultured on slices of bovine bone, and kistrin also inhibited the attachment of 293 cells expressing recombinant human alpha v beta 3 to fibrinogen (IC50 = 1 nM). These results indicate the potential therapeutic use of RGD-containing molecules for hypercalcemia of malignancy or for other disorders associated with bone loss.

Effect of transforming growth factor alpha, parathyroid hormone-related protein, or their combination on bone histomorphometry.

Transforming growth factor alpha (TGF alpha) has been implicated in the pathogenesis of inhibited bone formation in hypercalcemia of malignancy. We evaluated the effects of infusions of vehicle or TGF alpha (0.25 microgram/h), or parathyroid hormone-related protein (1-34) (PTHrP) (0.025 microgram/h) alone, or a combination of TGF alpha and PTHrP on bone histomorphometry in mice. The peptides were infused for 6 days via Alzet osmotic minipumps. TGF alpha alone, PTHrP alone, or a combination of the two resulted in an increase in bone resorption as well as bone formation parameters. The increase in bone formation with the combination of TGF alpha and PTHrP was significantly greater than that seen with either peptide alone. Therefore, it is unlikely that TGF alpha is the factor responsible for inhibited bone formation in hypercalcemia of malignancy.

Lack of effects of neutralization of parathyroid hormone-related protein on calcium homeostasis in neonatal mice.

Large quantities of parathyroid hormone-related protein (PTHrP) are present in the milk of various species. It has been suggested that PTHrP may play a role in neonatal calcium homeostasis. In the present study we evaluated the effect of neutralization of amino-terminal PTHrP activity by passive immunization in 1-day-old mouse pups. Neutralization of amino-terminal PTHrP activity had no significant effect on serum calcium or whole-body calcium content in the neonatal mice. In additional studies, we demonstrated that subcutaneous administration of PTHrP-(1-34) increased serum calcium, whereas oral administration had no significant effect in 3-day-old pups. The studies therefore demonstrate that the amino terminus of PTHrP may not play a significant role in neonatal calcium homeostasis. Local effects of PTHrP cannot be excluded by the results of the present study.

Parathyroid hormone-related protein and calcium homeostasis in lactating mice.

Parathyroid hormone (PTH)-related protein has been shown to be a factor responsible for hypercalcemia of malignancy. Recent studies have shown the presence of mRNA for PTH-related protein in lactating breast tissue, suggesting a physiological role for this peptide during lactation. In the present study, we evaluated the effect of neutralization of PTH-related protein activity in lactating mice (by passive immunization) on various parameters of maternal and neonatal calcium homeostasis. PTH-related protein bioactivity, as tested in the adenylate cyclase assay, was present in mouse milk, and this activity was completely neutralized by the antisera used in the present study. In lactating mice, the effects of injection of PTH-related protein antisera on maternal serum calcium concentrations, milk calcium and phosphorus concentration, pup growth, dam femur calcium content, and pup calcium content were similar to those of the injection of normal rabbit serum. Therefore, maternal PTH-related protein does not appear to have a role in calcium homeostasis during lactation.

Effects of progesterone on postovariectomy bone loss in aged rats.

The effects of progesterone on oophorectomy-induced bone loss in aged rats were evaluated. Female rats aged 12 months were divided into three groups: (1) sham-operated controls (SHAM); (2) oophorectomized (OVX); (3) OVX rats treated with progesterone (OVX + PROG). After 20 weeks the dry weight, bone ash, and calcium content of femur, tibia, and fourth lumbar vertebra were significantly lower in OVX than in sham rats. These reductions did not occur in OVX rats treated with PROG. There was no difference in the bone composition between the control and progesterone-treated rats. Vertebral bone histomorphometry showed increased bone resorption as well as increased bone formation parameters in OVX rats. Progesterone treatment inhibited the increased resorption indices, but the bone formation remained elevated. The results indicate that progesterone therapy prevents the postovariectomy bone loss in aged rats. The protective effect of progesterone is mediated by inhibition of bone resorption while maintaining the increased bone formation. These findings suggest that progesterone alone may be a valuable agent for management of postmenopausal osteoporosis.