A procedure is described which permits the rapid isolation of large amounts of elastase and cathepsin G from purulent sputum. This procedure involves: (1) digestion of sputum with DNase, (2) extraction of the insoluble residue that remains with 1 M NaCl, pH 8, (3) affinity chromatography on Sepharose-bound Trasylol, and (4) separation of the two enzymes by chromatogrphy on CM-Sephadex. Starting with 500 g of sputum it was possible to isolate 175 mg of each of these two enzymes within 7 to 10 days. Active site titration indicated both enzymes to be at least 97% pure. Disc gel electrophoresis in the presence and absence of SDS and amino acid sequence of the N-terminal region support the conclusion that the elastase and cathepsin G isolated from sputum are identical to the same enzymes isolated directly from the leukocytes of human blood.

Summary. A transplantable nonmetastasizingLeydig cell tumor, which occurs spontaneously in the aged Fischer rat, was examined both in vitro and in vivo. Animals carrying this tumor were found to have the syndrome recently called the humoral hypercalcemia of malignancy, characterized by hypercalcemia, hypercalciuria, hypophosphatemia, renal phosphate wasting, increased urinary 3'5'-cyclic monophosphate (cyclic AMP) excretion, and suppressed circulating parathyroid hormone (PTH) concentrations. The changes in urinary cyclic adenosine monophosphate (cAMP) excretion occurred simultaneously with hypercalcemia in most animals. In one animal, the primary tumor was excised and this was followed by an immediate fall in serum calcium and urine cAMP excretion. Hypercalcemia was due to increased bone resorption. This was shown by bone histology, which demonstrated an increase in osteoclast number and activity on trabecular bone surfaces associated with bone loss in tumor-bearing animals. No tumor cells were seen adjacent to the osteoclasts. There was no evidence of metastatic disease as assessed by bone-seeking isotopes. Urinary hydroxyproline excretion was increased in tumor-bearing hypercalcemic animals, indicating an increase in bone turnover. The tumor cells were established in culture and found to produce a bone-resorbing factor in vitro using a bioassay for bone resorption based on the release of previously incorporated 4~Ca from fetal rat long bones in culture. This model of the humoral hypercalcemia of malignancy should make it possible to determine the nature of the boneresorbing factor produced by the cultured tumor cells which is responsible for the hypercalcemia, and the relationship of hypercalcemia and the production of the bone-resorbing factor to the other Send offprint requests to G. R. Mundy at the above address.parameters of the syndrome, namely, renal phosphate wasting and increased urinary cAMP excretion.

Summary. There is a high frequency of Leydig cell tumors associated with hypercalcemia in the aged Fischer 344 rat. We studied a transplantable tumor cell line (Rice D-6) which is associated with hypercalcemia, hypercalciuria, hypophosphatemia, renal phosphate wasting, increased urinary cyclic adenosine monophosphate (AMP) excretion, absence of bone metastases, increased osteoclastic bone resorption, and suppressed immunoreactive parathyroid hormone (iPTH) concentrations. We examined the ability of dichloromethylene diphosphonate (CI2MDP) to lower serum calcium and decrease the parameters of increased bone resorption. We used this drug also as a pharmacologic tool to determine the relationship of hypercalcemia and increased bone resorption to the abnormalities in renal tubular function associated with the humoral hypercalcemia of malignancy.Daily administration of CI.,MDP before development of hypercalcemia, in doses from 2.5-40 mg/kg body weight subcutaneously, delayed and suppressed both the hypercalcemia and hypercalciuria. There was an increase in bone mass and decrease in both osteoclast number and activity compared with bones from untreated tumor-bearing animals. The urinary hydroxyproline excretion in treated animals declined towards the normal range. There were no significant effects on serum phosphorus, urine phosphorus, or urine cyclic AMP excretion.These data suggest that C12MDP reverses the increased bone resorption that occurs in the humoral hypercalcemia of malignancy, and confirms that diphosphonates are effective agents in the prevention and treatment of increased bone resorption associated with malignant disease. They also suggest Send offprint requests to Dr. Martodam at the above address.that renal phosphate wasting and increased urinary cyclic AMP excretion are not directly related to the hypercalcemia.

Methods are described for the covalent attachment of succinoyl-Ala-Ala-Pro-ValCH2Cl, an active sitedirected inhibitor of human leukocyte elastase (EC 3.4.21.11), to microspheres of human albumin. (EC 3.4.21.11), an enzyme that has been implicated in lung tissue injury resulting in the development of emphysema (6, 7). Although these inhibitors would appear to offer promise for the treatment of emphysema, their therapeutic effectiveness would be considerably enhanced if they could be targeted directly to the lungs. As a step in this direction we report here the manner in which the succinoyl peptide chloromethyl ketone (SPCK) succinoyl (Suc)-AlaAla-Pro-ValCH2Cl, which has been shown to be one of the more potent inhibitors of human leukocyte elastase (5), may be covalently attached to HAM with the retention of significant inhibitory activity towards this enzyme. Also reported here are the results of preliminary experiments in vio that show that the carrier-borne inhibitor is in fact targeted primarily to the lungs, where about half of the injected dose has a half-life of approximately 17 days. (8) and is currently available in kit form as a carrier of radioactive tracers for diagnostic lung scanning (2-4), circulatory studies (9), and cardiac output (10). SPCK was synthesized as described (5). Ethylenediamine, 5,5'-dithiobis-(2-nitrobenzoic acid), and hexanediamine were purchased from Aldrich; 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-acetylglycine from Sigma; ethyleneimine from Pierce; N-acetyl-DL-homocysteine thiolactone from Schwarz/Mann; and iodo[1-'4C]acetic acid and Na125I (carrier-free) from New England Nuclear. All other chemicals were reagent grade available from commercial sources. All aqueous reagents which were used during the course of the derivatization of HAM contained 0.025% Pluronic F-68 (BASF, Wyandotte, MI) in order to prevent the adherence of microspheres to the sides of reaction vessels and to facilitate uniform suspension. MATERIALS AND METHODSThe sequence of reactions involved in the covalent attachment of SPCK to HAM with extended side chains is described in detail in the legend accompanying Fig. 1. Briefly, the exposed carboxyl groups of HAM were first aminoethylated (step 1), and the amino groups so introduced (I), plus any amino groups already exposed on the surface of HAM, were thiolated with N-acetyl-DL-homocysteine thiolactone (step 2). The thiolated derivative (II) was again aminoethylated with ethyleneimine (step 3) to give the amino derivative (III), or with iodoacetate (step 4) to give the carboxyl derivative (IV). The side chain of IV could be further elongated by condensation with hexanediamine (step 5), yielding V. By virtue of the succinoyl group at the NH2 terminus of the peptide, the latter could be condensed with the amino groups of I, III, and V (step 6), thus yielding HAM-inhibitor derivatives denoted as Ii, IIIi, and Vi, respectively.Assay for Enzyme Activity. Human leukocyte elastase was prepared from purulent sputum as described (12) Step 1: 1...

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