Impaired intrinsic chiral inversion activity of ibuprofen in rats with adjuvant-induced arthritis.

1. The effects of adjuvant-induced arthritis on the chiral inversion of 'profens', a type of non-steroidal anti-inflammatory drug, have hardly been investigated. The authors investigated the effects of adjuvant-induced arthritis on the chiral inversion of ibuprofen using freshly isolated rat hepatocytes. 2. S- or R-ibuprofen was incubated with hepatocytes isolated from control and adjuvant-induced arthritis rats in the absence of the serum. In the hepatocyte system the chiral inversion rate constant of R- to S-ibuprofen and the metabolic rate constants of both enantiomers in adjuvant-induced arthritis rats were significantly decreased to about 64-80% of the corresponding values in control rats. In contrast, the addition of serum from each group to the corresponding hepatocyte medium resulted in no significant differences in these rate constants between control and adjuvant-induced arthritis rats. 3. With regard to chiral inversion enzymes, adjuvant-induced arthritis decreased the messenger RNA levels of acyl-coenzyme A synthetase (ACS) isoforms, but not 2-arylpropionyl-CoA epimerase, compared with control rats. 4. Chiral inversion of R- to S-ibuprofen was inhibited by triacsin C, a specific inhibitor of ACS1. 5. The results suggest that adjuvant-induced arthritis induces down-regulation of ACS enzymes involved in chiral inversion of R- to S-ibuprofen in rats.

Prediction of metabolic clearance of diclofenac in adjuvant-induced arthritis rats using a substrate depletion assay.

1. The purpose of this study was to evaluate drug clearance measured by the metabolic intrinsic clearance (CL(int)) in a substrate depletion assay in comparison with the in vivo clearance (CL(tot)) observed in adjuvant-induced arthritis (AA) rats. 2. After intravenous administration of diclofenac as a model drug, CL(tot) was 2.8-fold higher in AA rats than in control rats. In two different substrate depletion assays with liver microsomes for glucuronidation and hydroxylation, the CL(int) values for glucuronidation was significantly decreased in AA rats to 60% of the value in control rats, whereas the CL(int) values for hydroxylation were similar. The unbound fraction of diclofenac in plasma (f(u, plasma)) was significantly higher (2.8-fold) in AA rats than in control rats. 3. Hepatic clearance predicted from the CL(int) values for both biotransformation pathways and f(u, plasma) was higher in AA rats than in control rats, with good consistency between predicted and observed values. The same results were obtained for experiments using hepatocytes. 4. The plasma protein-binding activities, rather than metabolic clearance, in both types of rats would be a determining factor in the pharmacokinetic behaviour differences between control and AA rats. 5. In summary, substrate depletion assays with liver microsomes and hepatocytes in combination with protein binding assessment can help to predict changes in pharmacokinetics under AA conditions.

Antithrombin inhibits lipopolysaccharide-induced tumor necrosis factor-alpha production by monocytes in vitro through inhibition of Egr-1 expression.

BACKGROUND: Antithrombin (AT) improves the outcome of septic patients with intravascular coagulation. However, the mechanisms underlying the therapeutic benefits of AT are not fully understood. Tumor necrosis factor-alpha (TNF-alpha) plays a critical role in the development of organ failure and intravascular coagulation in sepsis. AIM: This study aimed to elucidate a molecular mechanism by which AT inhibits TNF-alpha production. METHODS: Human peripheral monocyte was stimulated by lipopolysaccharide (LPS) and TNF-alpha concentration in media was measured. Levels of phosphorylation of extracellular signal-regulated protein kinases (ERK) 1/2 and early growth response factor-1 (Egr-1) were estimated by western blotting or by electrophoretic mobility shift assay. RESULTS: Antithrombin (3 U mL(-1)) inhibited TNF-alpha production by monocytes stimulated with LPS. Conversely, chemically modified AT that lacks affinity for heparin did not. AT inhibited the phosphorylation of ERK 1/2 and decreased the expression of Egr-1 in LPS-stimulated monocytes. However, it did not affect the activation of either nuclear factor-kappaB or activator protein-1. Pretreatment with KT5720, a protein kinase A inhibitor, reversed the inhibitory effect of AT on the LPS-induced phosphorylation of ERK1/2. Although 2 U mL(-1) AT slightly inhibited TNF-alpha production by LPS-stimulated monocytes, it significantly inhibited TNF-alpha production in the presence of a low concentration of beraprost, a stable derivative of prostacyclin. CONCLUSIONS: These observations suggest that AT might inhibit LPS-induced production of TNF-alpha by inhibiting the increase in Egr-1 expression in monocytes via interaction with heparin-like substances expressed on the cell surface.

Usefulness of hepatocytes for evaluating the genetic polymorphism of CYP2D6 substrates.

The usefulness of human hepatocytes for assessing CYP2D6-related genetic polymorphisms was investigated. Propranolol and propafenone, which undergo phase I and II biotransformations, were used as model substrates alongside metoprolol, which is only metabolized via oxidative pathways. The contributions of CYP2D6 to the primary metabolisms of the substrates were estimated from the quinidine-mediated inhibition of their depletion rate constants in human hepatocytes and liver microsomes. The contributions in hepatocytes were 19.2% for propranolol at 0.05 microM and 36.7--76.3% for propafenone at 0.05--1.0 microM, and smaller than the contribution in microsomes, unlike the case for metoprolol. The differences between microsomes and hepatocytes were attributable to conjugate formation. The CYP2D6 contributions in hepatocytes reflected the in vivo data. The relevance of the concentration-dependent involvement of CYP2D6 in propafenone metabolism in hepatocytes to the in vivo polymorphic profile and the applicability of hepatocytes for evaluating these polymorphisms are discussed.

Effect of steroid add-back therapy on the proliferative activity of uterine leiomyoma cells under gonadotropin-releasing hormone agonist therapy.

Short-term treatment with gonadotropin-releasing hormone agonist (GnRHa) is a useful preoperative medical therapy of uterine leiomyomas. However, adverse effects caused by the hypo-estrogen state sometimes appear, suggesting the necessity of add-back therapy. In this study, we investigated effects of three kinds of add-back therapies on the proliferative activity of uterine leiomyoma cells by examining the expression of Ki-67 in leiomyoma cells by immunostaining. Thirty patients who were to undergo hysterectomy or myomectomy were injected with 3.75 mg depot leuprolide acetate every four weeks until the end of the 12th week. Twenty patients underwent add-back therapy from the 5th week to the end of the 12th week, 8 patients receiving 0.625 mg of conjugated equine estrogen (CEE) /day, 6 patients 5.0 mg of medroxyprogesterone acetate (MPA)/day, 6 patients 0.625 mg CEE plus 2.5 mg of MPA /day. The add-back of CEE or CEE plus MPA suppressed decreases in the proliferative activity of leiomyoma cells caused by GnRHa therapy, but that of MPA did not. These results suggest that the add-back therapy with MPA is of use in preventing the adverse effects caused by hypo-estrogen in the preoperative short-term GnRHa therapy.

[A case of papillary adenocarcinoma of the ovary that responded favorably to irinotecan hydrochloride and cisplatin after the administration of paclitaxel and carboplatin].

Recently, the standard treatment for advanced ovarian cancer has changed from CP therapy (cyclophosphamide, cisplatin (CDDP)) to TJ therapy (paclitaxel (TXL), carboplatin (CBDCA)). Irinotecan (CPT-11) is one of the derivatives of camptotecin and has been reported to have a high efficacy for ovarian cancer. In one case of ovarian cancer, chemotherapy was applied with CBDCA and TXL. However, after 2 months of six courses of the chemotherapy, CA-125 was elevated. The elevation of tumor marker levels in serum without the recurrent focus forced us to treat the patient with CPT-11 and CDDP for the second line chemotherapy. Tumor marker levels improved at the beginning of the therapy. In conclusion, CPT-11 and CDDP was effective against the recurrence of ovarian cancer treated with TJ therapy.

Induction of superovulation by inhibin vaccine in cyclic guinea-pigs.

Experiments were conducted to determine whether neutralizing endogenous inhibin affects follicular development and ovulation rate in guinea-pigs. Eighteen female guinea-pigs bearing 4 week progesterone implants were divided into three groups. At 1 week after removal of the progesterone implants, the animals were given a s.c. injection of 1 ml placebo (saline in oil emulsion; control), or 25 or 50 micrograms inhibin vaccine three times at 4 week intervals. Blood samples were collected once a week throughout the experiment for measuring inhibin antibody titres. After the third injection of inhibin vaccine, blood samples and ovaries were collected on the morning of day 8 after the day of oestrus. Inhibin vaccine increased the ovulation rate in a dose-dependent manner (placebo: 4.2 +/- 0.4; 25 micrograms inhibin vaccine: 6.2 +/- 0.9; 50 micrograms inhibin vaccine: 9.8 +/- 0.9) without any effects on the duration of the oestrous cycle. The results also showed that active immunization against inhibin increased the number of atretic follicles of 300-399 microns in diameter on day 8 after ovulation. The present study is the first to show that the active immunization against inhibin may be a useful method for inducing multiple ovulation in guinea-pigs.

Primary leiomyosarcoma of the heart subsequent to double carcinomas of the thyroid and lung.

A 63-year-old woman underwent surgical operations for left lower lung cancer and for thyroid cancer. Nine months later, a third cancer developed in her heart and this tumor was removed by open heart surgery. A pathologic study revealed that the tumor was primary leiomyosarcoma of the heart and thus independent from the previous lung and thyroid carcinomas. This case was regarded as a triple carcinoma including a primary leiomyosarcoma arising from the left atrium. Reports in the literature on primary malignant tumors of the heart are reviewed briefly.

Rapid diagnosis of adenovirus respiratory tract infections by immunochromatography.

A one-step diagnostic test based on an immunochromatographic (IC) assay for adenovirus was evaluated with purified adenovirus and clinical specimens. According to five clinically common serotypes of purified adenovirus tested, the IC test was more sensitive than two commercially available enzyme immunoassay (EIA) test kits. For tonsilopharyngeal specimens from 63 febrile pediatric patients with suspected adenoviral upper respiratory tract infection, the sensitivity and specificity of the IC test against viral isolation by cell culture was 88.5% (23/26) and 100% (37/37), respectively. The IC test, which is quicker and easier to perform than EIA test kits, is very useful in the rapid diagnosis of adenoviral upper respiratory tract infection of pediatric patients.

Secretion of ovarian inhibin and its physiologic roles in the regulation of follicle-stimulating hormone secretion during the estrous cycle of the female guinea pig.

To characterize inhibin secretion during the estrous cycle in guinea pigs, the concentrations of plasma inhibin, estradiol, progesterone, and FSH were determined. A significant positive correlation was observed between inhibin and estradiol throughout the estrous cycle. Plasma inhibin and estradiol started to increase a few days before ovulation (Day 0 = day of estimated ovulation), and decreased after ovulation. These two hormones remained low during the luteal phase. The immunoreactivity of inhibin alpha, betaA, and betaB subunits was colocalized in the granulosa cells of one or two healthy large follicles in the ovary before ovulation. There was no positive reaction of inhibin alpha and beta subunits in the corpora lutea or other follicles. Ovariectomy resulted in an abrupt decrease in plasma inhibin and a significant increase in plasma FSH. Injection of anti-inhibin serum into adult female guinea pigs induced an elevation in plasma FSH in a dose-dependent manner. This report presents the first description of sequential changes in plasma inhibin and estradiol during the estrous cycle of guinea pigs. Results suggest that inhibin is secreted mainly by granulosa cells of a few healthy large follicles in the ovary and that it plays an important role in the regulation of FSH secretion during the estrous cycle in guinea pigs.

[Chlamydia pneumoniae infection and Mycoplasma pneumoniae infection in pediatric patients].

We evaluated a total of 1104 pediatric patients with acute lower respiratory tract infection for C. pneumoniae infection and M. pneumoniae infection by serology during July 1995 to December 1998. A microimmunofluorescence test was used for diagnosis of C. pneumoniae infection and a high density particle agglutination test for that of M. pneumoniae infection. Acute C. pneumoniae infection was found in 149 patients (13.5%), acute M. pneumoniae infection in 118 patients (10.7%), and dual infection in 27 patients (2.4%). Among 305 patients with pneumonia, M. pneumoniae infection (83 patients, 27.2%) was more common than C. pneumoniae infection (47 patients, 15.4%). However among 799 patients with bronchitis. C. pneumoniae infection (102 patients, 12.8%) was more common than M. pneumoniae infection (35 patients, 4.4%). Patients with C. pneumoniae infection were more younger and more frequently wheezing than patients with M. pneumoniae infection. These findings demonstrate that C. pneumoniae infection in very common pathogen of pediatric lower respiratory tract infection as M. pneumoniae infection in Japan.

Contributions of endogenous inhibin and estradiol to the regulation of follicle-stimulating hormone and luteinizing hormone secretion in the pregnant rat.

To examine the contributions of endogenous inhibin and estradiol to the regulation of FSH and LH secretion in the pregnant rat, some rats were passively immunized against inhibin and/or estradiol, and others were ovariectomized, on Days 5, 10, 15, and 20 of pregnancy. Ovarian and uterine venous blood was collected separately to confirm the sources of inhibin and steroid hormones during pregnancy. Immunoreactivity of inhibin in the placenta was also examined by RIA. Levels of inhibin in ovarian venous plasma were significantly higher than those in peripheral plasma during pregnancy. No difference was observed between the levels of inhibin in uterine venous plasma and peripheral plasma. No immunoreactivity of inhibin was detected in placental homogenate from rats at Days 10, 15, and 20. FSH secretion significantly increased after immunoneutralization of inhibin during pregnancy. A marked increase in FSH secretion was noted on Days 5 and 20, and the smallest increase was observed on Day 15. Administration of estradiol antiserum (AS) alone did not induce a significant increase in FSH secretion on any day of pregnancy. However, a synergistic effect of estradiol AS and inhibin AS was observed on Day 20. On Days 5, 10, and 20, administration of inhibin AS or estradiol AS induced a significant increase in LH secretion. A synergistic effect of inhibin AS and estradiol AS on LH secretion was observed on Day 5. On Days 5 and 10, significantly high LH secretion was noted in ovariectomized rats as compared with that in rats treated with both inhibin AS and estradiol AS, indicating that other ovarian hormones such as progesterone may be involved in the suppression of LH secretion in these stages of pregnancy. These data indicate that both inhibin and estradiol, predominantly secreted from the ovary, are involved in the regulation of gonadotropin secretion during pregnancy as during the estrous cycle in the rat.

Involvement of autocrine mechanism of transforming growth factor-beta in the functional differentiation of pregnant mouse mammary gland.

We studied the presence and possible role of the autocrine mechanism of transforming growth factor-beta (TGF-beta) in pregnant mouse mammary gland. Northern blot analysis revealed the expression of the TGF-beta 1 gene transcript at 2.5 kb in mammary epithelial cells isolated from virgin and mid-pregnant mice. The TGF-beta activity was higher in the conditioned medium from mid-pregnant mouse mammary explants than that from virgin explants by a bioassay system using mink lung epithelial cells. A binding study using [125I]TGF-beta 1 as a ligand showed that pregnant mouse mammary epithelial cells possessed a single class of high-affinity TGF-beta 1 binding sites (Kd = 28.0 pmol/l, 1.2 x 10(4) sites per cell). These results suggested the presence of a TGF-beta autocrine mechanism in pregnant mouse mammary epithelial cells. Next, we examined the effect of TGF-beta 1 on the functional differentiation of pregnant mouse mammary gland. Transforming growth factor-beta 1 inhibited alpha-lactalbumin production in cultured mammary explants from mid-pregnant mice in a dose-dependent manner without inhibiting DNA synthesis. All these results suggest that TGF-beta may play a role in regulating the functional differentiation of mouse mammary glands during pregnancy.

Effect of prostacyclin on platelet intracellular free calcium concentration of hypertensive and normotensive humans.

Platelet intracellular free calcium concentration ([Ca2+]i) has been reported to be increased in essential hypertensive patients (EHT) as compared with normotensive controls (NT). Prostacyclin (PGI2), which influences cellular Ca2+, has been reported to be reduced in EHT. This study tested the hypothesis that the resting level of platelet [Ca2+]i in humans is influenced by PGI2. We also investigated the role of PGI2 in regulating platelet [Ca2+]i of 28 EHT subjects compared to 28 NT controls. Platelet [Ca2+]i was measured using the fluorescent Ca2+ probe fura-2 under control conditions and a 10-min preincubation with PGI2. Simultaneous measurement of platelet cyclic-adenosine 3':5'-monophosphate (cAMP) was performed by radioimmunoassay. The resting level of platelet [Ca2+]i was significantly higher in EHT than in NT (32.7 +/- 1.4 v 28.3 +/- 0.9 nmol/L; P < .01). PGI2 from 30 nM to 1 mumol/L lowered the resting level of platelet [Ca2+]i in a dose-dependent manner (EHT -22.2 +/- 2.4, NT -22.9 +/- 2.3%, 1 mumol/L PGI2); however, no significant difference in platelet [Ca2+]i was observed between NT and EHT. While prostacyclin induced a transient rise in platelet cAMP, the magnitude of PGI2-induced cAMP level was similar between the two groups. These results do not support the hypothesis that endogenous PGI2 activity contributes to the increased level of platelet [Ca2+]i in EHT, although PGI2 incubation lowered the resting level of platelet [Ca2+]i.

Pharmacokinetic analysis of enterohepatic circulation of 4-[2-(4-isopropylbenzamido)ethoxy]benzoic acid. Effect of intramolecular rearrangement of its acyl glucuronide.

The enterohepatic circulation of 4-[2-(4-isopropylbenzamido)ethoxy]benzoic acid (PBAB) was studied in rats after an iv administration of 30 mg/kg. After the bolus injection, the PBAB concentrations in the plasma decreased rapidly, then increased to a peak concentration at 4 hr. Over a 6-hr period, 52% of the dose (Fe) was excreted in the bile as 1 beta-O-acyl glucuronide of PBAB (1 beta-PG). Elimination of PBAB from the plasma of bile duct-cannulated rats was more rapid than for the sham-operated rats. These results suggest that PBAB undergoes enterohepatic circulation. The equation for an enterohepatic circulation model was fitted to the plasma PBAB concentrations for intact rats using the program MULTI (FILT) to estimate the single circulating fraction (Fc) (bile----intestine----systemic circulation). The Fc value was 0.072, which means that 7.2% of the dose was reabsorbed to systemic circulation during the first cycle. The fraction (Fa) reabsorbed from small intestine to systemic circulation during first cycle can be estimated by the formula Fa = Fo/Fa. The Fa value obtained was 14% of the dose, which was smaller than the Fa' (26% of the dose) standing for systemic availability of PBAB after oral dosing. When 1 beta-PG was incubated with bile for 2 hr, 79% was transformed to beta-glucuronidase-resistant isomers by intramolecular acyl migration. We consider that the acyl migration of 1 beta-PG in the small intestine or bile causes the difference between Fa and Fa' values, and decreases the enterohepatic circulation of PBAB.

[Effects of intravenously infused magnesium on renal calcium metabolism and plasma parathyroid hormone in patients with essential hypertension].

This study aimed to elucidate the effects of intravenously infused magnesium on renal calcium and sodium metabolism in patients with essential hypertension. Mean arterial pressure (MAP), heart rate (HR), urine volume (UV), endogenous creatinine clearance (Ccr), urinary excretion of calcium (UCaV) and sodium (UNaV), fractional excretion of calcium (FECa) and sodium (FENa), plasma ionized calcium (pCa2+) and parathyroid hormone(PTH) were measured before and after intravenous infusion of 10% magnesium sulfate (initial dose: Mg 13.5mg/m2.BSA/15 min.: maintenance dose: Mg 2.7mg/m2.BSA/105min) in 6 normotensive subjects (NT) and 13 mild-to-moderate essential hypertensives (EHT). After the magnesium infusion, significant increases of UV, UCaV, UNaV, FECa and FENa, and a significant decrease of PTH were observed in both NT and EHT while MAP and HR did not change in either group. PCa2+ significantly decreased and Ccr tended to increase only in EHT. Although no significant difference was found in the change in Ccr (delta Ccr) or PTH (delta PTH) between NT and EHT, the changes of UCaV (delta UCaV), UNaV (delta UNaV), FECa (delta FECa) and FENa (delta FENa) were greater in EHT than each in NT. A positive correlation was found between delta UCaV and delta FECa, as well as delta UCaV and delta Ccr, but the former was more remarkable in both groups. In addition, delta UCaV was positively correlated with delta FENa in EHT, but not in NT. No significant relationship was observed between delta UCaV and delta PTH in either group.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the conformational aspects of inulin oligomers.

The conformational studies of inulin oligomers from G-F2 to G-F9, which isolated from Platycodon grandiflorum, suggested a plausible conformational change between G-F7 and G-F8 from the trends in their chemical shift patterns and molecular rotation; the oligomers higher than G-F8 would form some secondary conformations more rigid than shorter oligomers. On the other hand, spin-lattice relaxation (T1) studies of the protons proposed through-space interactions of 2- and 4-H's of glucose moiety in G-F5, presumably with some atom(s) of the terminal fructose moiety. This would reflect that the inulin molecule adopts a 5/1 helix.