For over three decades, renal physiology has sought a putative natriuretic hormone (third factor) that might control the body's pool of extracellular fluid, an important determinant in hypertension, congestive heart failure, and cirrhosis. In our search for this hormone, we have isolated several pure natriuretic factors from human uremic urine that would appear, alone or in combination, to mark a cluster of phenomena previously presumed to be that of a single "natriuretic hormone." This paper reports the purification, chemical structure, and total synthesis of the first of these compounds, LLU-a, which proved to be 2,7,8-trimethyl-2-(pcarboxyethyl)-6-hydroxychroman, presumably a metabolite of y-tocopherol. Both natural LLU-a and synthetic material are identical (except for optical activity) with respect to structure and biological activity. It appears that the natriuretic activity of LLU-a is mediated by inhibition of the 70 pS K+ channel in the apical membrane of the thick ascending limb of the kidney.As a result of salt-induced plasma-volume expansion in mammals, three concurrent events have been observed: sustained natriuresis, rising plasma concentration of a Na+ transport inhibitor, and pressor activity. It has been presumed that these effects are due to elaboration of a low-molecular-weight "natriuretic hormone," the putative controller of extracellular fluid (1). Over 30 years of effort have failed to characterize the putative hormone (2, 3). Atrial natriuretic factor, when infused, produces a natriuresis and a decrease in blood pressure that are short-lived (4, 5) and whose physiological role is still undefined (6, 7).The approach of essentially all other workers has been to investigate mammalian-derived isolates that inhibit the Na+ pump (2,8). From these studies, digoxin (9) and "iso-ouabain" (10-13) have been isolated; however, digoxin and ouabain lead to kaliuresis (4,(14)(15)(16)(17). Therefore, we hypothesized that this search tool is an inadequate marker for natriuresis. Consequently, a natriuretic assay has been developed in which the in vivo physiological events, urine volume, K+ and Na+ concentrations, and mean arterial pressure are measured (18).In this paper, we report the isolation of pure LLU-a, the determination of its structure by spectroscopy, its synthesis in racemic form, and its biological characterization. Given its structure, we infer that it is the product of in vivo oxidative metabolism of y-tocopherol, a member of the vitamin E complex. MATERIALS AND METHODSPurification of LLU-a. Human uremic urine (800 liters) was collected, processed by ultrafiltration (3 kDa) and lyophilization, and then chromatographed on Sephadex G-25 to obtain the post-salt fraction as described (18 Step 1. Reversed-phase HPLC was performed by gradient elution with 0.2 M pyridinium acetate, pH 5.5, and methanol as described (4). Based on bioassay data, fractions 50-80 were combined for further purification.Step 2. The next column (5 pm; 10 x 250 mm) was eluted at 2 ml/min with a gradient formed from...

Growth factors are endogenous proteins capable of stimulating new bone formation, but their clinical benefit for systemic stimulation of bone mass has not been demonstrated. The critical challenge is to deliver a significant dose of the proteins to bone after intravenous injection. This challenge may be overcome by derivatizing proteins with ligands that exhibit a high bone affinity (e.g., bisphosphonates). To demonstrate the feasibility of this approach, 1-amino-1,1-diphosphonate methane (aminoBP) was conjugated to a model protein, albumin. The conjugation was performed by (1) converting the amino group of aminoBP to a thiol group using 2-iminothiolane, (2) derivatizing the albumin amino groups with a thiol-reactive sulfosuccinimidyl-4-(N-maleimidomethyl)-1-cyclohexane carboxylate, and (3) reacting the derivatized albumin with thiolated aminoBP. Typically, 1-4 aminoBP molecules per albumin were obtained. The conjugated albumin exhibited a high affinity to hydroxyapatite that was proportional to the extent of conjugation. The conjugates were shown to exhibit a high affinity to bone matrix in vitro in a serum-containing medium. Once bound to bone matrix, the conjugates were found to desorb more slowly than the unmodified albumin, especially from bone whose organic matrix was removed by ashing. In conclusion, conjugation of bisphosphonates to albumin was shown to impart a high bone affinity to the protein, and such conjugates can be potentially targeted to bone.

No abstract

No abstract

No abstract

Growth factors capable of stimulating bone formation are potential therapeutic agents for osteoporosis treatment. It is essential, however, that a targeting mechanism is incorporated into the growth factors to deposit them at osseous tissue with minimal distribution to extraskeletal sites. To this end, a strategy has been developed in which a bone-seeking molecule, 1-amino-1,1-diphosphonate methane (aminoBP), was chemically conjugated to a model protein, bovine serum albumin (BSA). This study was carried out to assess the bone affinity of the conjugates in a tibia injection model. Using ovariectomized (OVX) rats, initial (3 h) retention of BSA and aminoBP-BSA were found to be equivalent when injected into the medullary cavity of tibia. After 1 day, an 8- and 12-fold higher tibiae retention of the protein was obtained in normal and OVX rats as a result of aminoBP conjugation. A similar result ( approximately 12-fold difference) was also obtained in OVX rats after 3 days. We concluded that aminoBP conjugation to BSA imparted a high bone affinity and enhanced bone retention of proteins in normal and OVX rats.

Information about the potential and extent of bioinversion of chiral drugs in laboratory animal species and humans is critical to the interpretation of preclinical pharm-tox studies with these drugs. Unlike in the dog, guinea pig, and rabbit, in humans the 2-arylpropionic acid (APA) R-flurbiprofen (R-FB) undergoes very little bioinversion to S-flurbiprofen. The primary objective of this research was to identify laboratory animal species with an R- to S-bioinversion profile similar to humans. Detailed evaluations of the pharmacokinetics parameters of R-flurbiprofen in male and female rats and mice, and male nude rats and monkeys demonstrated R- to S-bioinversion of 30% (average) in monkeys, 15-24% in mice, and approximately 4% in rats. To date, no laboratory animal species has been identified with an R-flurbiprofen bioinversion profile identical to humans. However, the rat has a bioinversion profile sufficiently similar to humans to be useful for preclinical.

Nonsteroidal antiinflammatory drugs (NSAIDs) are recognized for inhibiting growth of colon tumors in animal models, and for reducing the risk of colon cancer in humans. The mechanisms involved have not been established, but are thought to be related to reduced prostaglandin biosynthesis. The present study investigates the effect of COX-inhibiting and non-COX-inhibiting enantiomers of flurbiprofen on rat colonocyte proliferation. Intestinal ulceration was used as a surrogate indicator of COX inhibition. Sprague Dawley rats were treated orally with 6.3 mg/kg of R- or s-flurbiprofen or vehicle. Colonocyte labeling index and small bowel ulcer index were measured. R-flurbiprofen and S-flurbiprofen significantly reduced colonocyte labeling index, by 34% and 23% respectively, compared with vehicle. R-flurbiprofen caused minimal ulcer formation (4.48 mm2) compared with S-flurbiprofen (94.4 mm2). These findings suggest that R-flurbiprofen-mediated control of colonocyte proliferation is independent of prostaglandin biosynthesis.