Effects of the rate and composition of fluid replacement on the pharmacokinetics and pharmacodynamics of intravenous azosemide

The effects of differences in the rate and composition of intravenous fluid replacement for urine loss on the pharmacokinetics and pharmacodynamics of azosemide were evaluated using rabbit as the animal model. Each rabbit received a 4h constant intravenous infusion of 1 mg kg-1 azosemide with 0% replacement (treatment I, n = 4), 50% replacement (treatment II, n = 5), and 100% replacement (treatment III, n = 5) with lactated Ringer's solution, as well as with 100% replacement with 5% dextrose in water (D-5-W, treatment IV; n = 5). Renal clearance and urinary excretion rate of the drug in treatment III were considerably higher than those in treatments I, II, and IV. In spite of the similarities in kinetic properties, diuretic and/or natriuretic effects of azosemide were markedly different among the four treatments. For example, the mean 8 h urine output values were 98.2, 178, 733, and 237 mL for treatments I-IV, respectively, and the corresponding values for sodium excretion were 11.1, 19.4, 76.4, and 14.2 mmol, and for chloride 13.4, 23.8, 78.9, and 17.1 mmol. Except for treatment III, diuresis and/or natriuresis were found to be time dependent, generally decreasing with time until reaching a low plateau during the later hours of infusion. The present findings also show that (i) no fluid replacement and 100% replacement with D-5-W both produce the same degree (not significantly different) of severe acute tolerance in natriuresis, indicating the insignificance of water compensation in tolerance development; (ii) in treatment II, where neutral sodium balance was achieved, the development of acute tolerance in diuresis can mainly be attributed to negative water balance under this special condition; and (iii) at steady state the hourly diuresis and natriuresis can differ up to about 6.87- and 5.21-fold between treatments. Some implications for the bioequivalence evaluation of dosage forms of azosemide are discussed.     

A fetal sheep liver extract reverses age-related increments in spontaneous and induced cytokine production by indirect environmental effects

BALB/c, DBA/2 and C57BL/6 mice of different ages (ranging from 8 to 110 weeks of age) were used as spleen cell donors to assay cytokine production from ConA activated spleen and Peyer's Patch (PP) lymphocytes. As reported in an earlier publication, there was an age-related decline in IL-2 production in all strains, with a general increase in IL-4 and IL-10 production with age, this being particularly marked for PP cell preparations. Similar conclusions were reached from independent analysis of CD44hi and CD44lo cell populations in these groups (memory vs. naive cells, respectively). Interestingly, IL-6 production was dramatically increased (some 4-5-fold in the different strains) and significantly increased levels of IL-6 were detected in the serum of aged mice. A previously described sheep fetal liver extract was able to reverse, to varying degrees, these cytokine changes associated with aging. Interestingly, when cells from aged mice were adoptively transferred to lethally irradiated young (8 week) recipients, the cytokine production phenotype of cells harvested from recipient mice 3 weeks later was that of the aged donor, unless recipients were treated continually with extract. Treatment of the donor alone produced minimal changes in cytokine production 3 weeks following adoptive transfer. The effect of extract was reversed in treated aged mice by concomitant daily intravenous infusion of the competitive inhibitor of nitric oxide synthesis (NG-monomethyl-L-arginine (NMMA)), which also decreased the increased serum nitrate levels in mice treated with extract. Our data suggest an important role for reactive nitrogen products, themselves induced by fetal liver extract, in age-associated changes in cytokine production.     

Immunohistochemical staining of ovarian granulosa cell tumors with monoclonal antibody against inhibin

Inhibin is a peptide hormone produced by ovarian granulosa cells and by granulosa cell tumors. Serum inhibin measurements have been used as a biochemical marker of the presence or progression of ovarian granulosa cell tumors and their metastases. In the current study, an antibody against the alpha-subunit of human inhibin was used to stain 16 cases of ovarian adult granulosa cell tumors, 15 cases of other ovarian sex cord-stromal tumors, and 51 cases of a range of ovarian and extraovarian neoplasms, many of which may mimic granulosa cell tumor. There was diffuse strong cytoplasmic staining of all cases of adult granulosa cell tumor. Diffuse positive staining also was observed in all Leydig cell tumors, and there was focal staining in a proportion of fibrothecomas. There was focal weak staining of one case of ovarian clear cell carcinoma but no staining of other ovarian and extraovarian neoplasms. Immunohistochemical staining with antibodies against inhibin is of value in the diagnosis of granulosa cell tumor and in the distinction of this neoplasm from others that may mimic it. The antibody also may be useful for the confirmation of late metastasis of granulosa cell tumor, especially when the previous history is not known.     

Inhibition of neutral endopeptidase causes vasoconstriction of human resistance vessels in vivo

BACKGROUND: Neutral endopeptidase (NEP) degrades vasoactive peptides, including the natriuretic peptides, angiotensin II, and endothelin-1. Systemic inhibition of NEP does not consistently lower blood pressure, even though it increases natriuretic peptide concentrations and causes natriuresis and diuresis. We therefore investigated the direct effects of local inhibition of NEP on forearm resistance vessel tone. METHODS AND RESULTS: Four separate studies were performed, each with 90-minute drug infusions. In the first study, 10 healthy subjects received a brachial artery infusion of the NEP inhibitor candoxatrilat (125 nmol/min), which caused a slowly progressive forearm vasoconstriction (12+/-2%; P=0.001). In a second two-phase study, 6 healthy subjects received, 4 hours after enalapril (20 mg) or placebo, an intra-arterial infusion of the NEP inhibitor thiorphan (30 nmol/min). Thiorphan caused similar degrees of local forearm vasoconstriction (P=0.6) after pretreatment with both placebo (13+/-1%, P=0.006) and enalapril (17+/-6%, P=0.05). In a third three-phase study, 8 healthy subjects received intra-arterial thiorphan (30 nmol/min), the endothelin ETA antagonist BQ-123 (100 nmol/min), and both combined. Thiorphan caused local forearm vasoconstriction (13+/-1%, P=0.0001); BQ-123 caused local vasodilatation (33+/-3%, P=0.0001). Combined thiorphan and BQ-123 caused vasodilatation (32+/-1%, P=0.0001) similar to BQ-123 alone (P=0.98). In a fourth study, 6 hypertensive patients (blood pressure >160/100 mm Hg) received intra-arterial thiorphan (30 nmol/min). Thiorphan caused a slowly progressive forearm vasoconstriction (10+/-2%, P=0.0001). CONCLUSIONS: Inhibition of local NEP causes vasoconstriction in forearm resistance vessels of both healthy volunteers and patients with hypertension. The lack of effect of ACE inhibition on the vasoconstriction produced by thiorphan and its absence during concomitant ETA receptor blockade suggest that it is mediated by endothelin-1 and not angiotensin II. These findings may help to explain the failure of systemic NEP inhibition to lower blood pressure.     

Affinity chromatography of human plasma and platelet factor XIII on organomercurial agarose

A method for affinity chromatography of plasma and platelet factor XIII has been developed, based on known structural characteristics of these molecules. Plasma factor XIII is composed of a and b subunits which are held together by noncovalent interactions; platelet factor XIII has only a subunits. a subunit contains free sulfhydryl groups, while in b subunit all the cystines form disulfide bonds. The affinity gel is an organomercurial agarose with p-chloromercuribenzoate as the reactive group. Both the zymogen and activated forms of a subunit reversibly bind to the ligand by forming covalent mercaptide bonds and are eluted by reducing agents. b subunit does not bind to the affinity gel and is held to it only through interaction with a subunit. Affinity chromatography can be used to purify plasma and platelet factor XIII and to study interactions of the subunits. Experiments on the affinity chromatography of purified plasma factor XIII in several stages of activation agree with earlier observations that activation is a two-step procedure in which b subunit is not quantitatively released from the complex until the final stage of activation by Ca2+.