leupeptins and Hypercalcemia

The process of bone resorption by osteoclasts involves the dissolution of mineral salts and enzymatic degradation of the mainly collagenous extracellular matrix. Cysteine proteinases, which can efficiently degrade collagen at acidic pH, have been suggested to play an important role in the bone resorptive process. The cysteine proteinase cathepsin L is secreted by osteoclasts, and inhibitors of this enzyme can prevent bone resorption in vitro. The activity of acetyl-leu-leu-norleucinol (ALLN), a selective inhibitor of cathepsin L, was investigated in two models of bone resorption in vivo. In the first study, the ability of ALLN to inhibit bone resorption was investigated in Ro-13-6298 (arotinoid)-treated thyroparathyroidectomized (TPTX) rats. ALLN [100 mg/kg, intraperitoneally (i.p.)] inhibited hypercalcemia by 62.8% acutely (p < 0.001), compared to 94.9% (p < 0.001) inhibition by salmon calcitonin (sCT) (10 IU/kg, subcutaneously). In rats treated for 3 days with ALLN, arotinoid-induced reduction in cortical bone mineral density measured by peripheral quantitative computed tomography (pQCT) was inhibited by 86.4% (p < 0.05) in rats treated with ALLN 100 mg/kg, i.p., and by 82% in rats treated with 50 mg/kg, i.p. (p < 0.05). In a second study, the efficacy of ALLN was tested in a longitudinal study in ovariectomized (ovx) rats. Bone loss, measured by pQCT, was unaffected by treatment with ALLN. The bisphosphonate alendronate, however, inhibited bone loss in this model. These data demonstrate the ability of a cathepsin L inhibitor to inhibit bone resorption in arotinoid-treated TPTX rats, a process which may be dependent on the activity of cathepsin L-like cysteine proteinases. In contrast to its effects in TPTX rats, ALLN had no inhibitory activity on bone resorption in ovx rats. It is possible that in chronic bone resorption in ovx rats, the activity of other enzymes such as cathepsins OC-2 or K allows the process of resorption to continue even when cathepsin L is inhibited by ALLN. Further studies are required to determine why the activity of ALLN varies between different animal models. These data indicate that there may be variations in the effects of drugs in different animal models of bone resorption which should be considered when investigating novel antiresorptive therapies.

Topics: Animals; Benzoates; Bone Resorption; Cathepsin L; Cathepsins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Disease Models, Animal; Endopeptidases; Female; Humans; Hypercalcemia; Leupeptins; Male; Osteoporosis; Ovariectomy; Ovary; Parathyroid Glands; Parathyroidectomy; Rats; Rats, Wistar; Retinoids; Thyroid Gland; Thyroidectomy

We describe a systematic comparison of the effects of anticoagulants, protease inhibitors and conditions of sample handling on the in vitro stability of endogenous parathyroid hormone-related protein (PTHrP) in blood from patients with hypercalcaemia of malignancy (HM). When blood was separated within 15 min of collection, PTHrP1-86 levels measured by two-site immunoradiometric assay in serum and heparinized plasma were significantly lower than in ethylenediaminetetraacetic acid (EDTA) plasma (P < 0.02). PTHrP was unstable in blood kept at 20 degrees C for 4 h and inclusion of protease inhibitors reduced, but failed to abolish, this instability. In blood collected in the presence of EDTA, inclusion of leupeptin either alone or in combination with pepstatin and aprotinin increased the mean half-time of disappearance from 3.9 to 10.1 and 11.2 h, respectively (P < 0.05). In contrast, when blood containing EDTA was separated within 15 min, PTHrP was stable in plasma at 20 degrees C for at least 4 h. As a result of the instability of PTHrP1-86 immunoreactivity in whole blood at ambient temperatures we advise that for our immunoradiometric assay (IRMA) blood collected in EDTA should be separated within 15 min, and the plasma frozen until assay.

Topics: Anticoagulants; Aprotinin; Blood Specimen Collection; Edetic Acid; Heparin; Humans; Hypercalcemia; Immunoradiometric Assay; In Vitro Techniques; Infant; Leupeptins; Neoplasms; Parathyroid Hormone-Related Protein; Pepstatins; Peptide Fragments; Peptides; Protease Inhibitors; Temperature

We have developed a sensitive, specific solid-phase immunoradiometric assay (IRMA) of parathyroid hormone-related protein (PTH-RP) with use of affinity-purified polyclonal immunoglobulins. Antibodies recognizing PTH-RP(37-74) are immobilized to a polystyrene bead to "capture" analytes from the sample; antibodies to epitopes within the 1-36 amino acid region of PTH-RP are labeled with 125I. This IRMA recognizes PTH-RP(1-74) and PTH-RP(1-86) equivalently, but does not detect N-terminal or C-terminal fragments of PTH-RP, intact human parathyrin (PTH), or fragments of PTH. PTH-RP is not stable in plasma at 3-5 degrees C or room temperature, but a mixture of aprotinin (500 kallikrein units/L) and leupeptin (2.5 mg/L) improves PTH-RP stability in blood samples. In plasma collected in the presence of these protease inhibitors from normal volunteers and patients with various disorders of calcium metabolism, PTH-RP concentrations were above normal (greater than 1.5 pmol/L) in 91% (42 of 46) of patients with hypercalcemia associated with nonhematological malignancy. In plasma from patients with other hypercalcemic conditions (e.g., primary hyperparathyroidism, sarcoidosis, and vitamin D excess), PTH-RP was undetectable. Above-normal concentrations of PTH-RP and total calcium decreased to normal in a patient with an ovarian cyst adenocarcinoma after surgical removal of the tumor. We conclude that PTH-RP is related to and probably the causative agent of hypercalcemia in most patients with cancer, and that measurements of PTH-RP are useful in the diagnosis and management of patients with tumor-associated hypercalcemia.

Topics: Aprotinin; Diagnosis, Differential; Drug Stability; Humans; Hypercalcemia; Immunoradiometric Assay; Leupeptins; Neoplasms; Parathyroid Hormone-Related Protein; Peptide Fragments; Proteins