Splenic marginal zone lymphoma presenting as myelofibrosis associated with bone marrow involvement of lymphoma cells which secrete a large amount of TGF-beta.

We report here on a patient with splenic marginal zone lymphoma presenting diffuse fibrosis of bone marrow and spleen. After splenectomy and chemotherapy, bone marrow biopsy demonstrated an improvement of fibrosis. Plasma concentration of transforming growth factor (TGF)-beta was much higher in this patient than in those of age-matched non-Hodgkin's lymphoma patients ( n=5) at diagnosis, decreasing after resolution of myelofibrosis. Immunostaining with the TGF-beta antibody revealed that the lymphoma cells in bone marrow and spleen were positive for TGF-beta. TGF-beta secreted by tumor cells was thought to stimulate the growth of fibroblasts and synthesize collagen in bone marrow and splenic fibroblasts, and play an important role in the development of marrow and splenic fibrosis in this patient. This is the first report of a patient with splenic marginal zone lymphoma presenting as myelofibrosis associated with bone marrow involvement of lymphoma cells which secrete a large amount of TGF-beta.

S-oxidation of S-methyl-esonarimod by flavin-containing monooxygenases in human liver microsomes.

1. Studies using human liver microsomes and recombinant human cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) were performed to identify the enzymes responsible for the metabolism of S-methyl-esonarimod (M2), an active metabolite of esonarimod (KE-298, a novel antirheumatic drug). 2. S-oxidative activities of M2 significantly correlated with those of methyl p-tolyl sulfide, a specific substrate of FMOs, as tested using 10 different human liver microsomes (r(2) = 0.539, p<0.05). Thermal treatment of microsomes reduced the S-oxidative activity in the absence of the NADPH-generating system at 45 degrees C for 5 min. However, methimazole, a known competitive substrate of FMOs, was a weak inhibitor of the S-oxidation in liver microsomes. 3. Recombinant human FMO1 and FMO5 produced M3 in greater quantities than recombinant human FMO3. The S-oxidation of M2 by recombinant human FMO5 was not appreciably inhibited in the presence of methimazole. In contrast, methimazole was effective in suppressing the catalytic activity of recombinant human FMO1 and FMO3. 4. The apparent K(m) (K(m app)) for the S-oxidation of M2 in human recombinant FMO5 (2.71 microM) was similar to that obtained using human liver microsomes (2.43 microM). 5. The present results suggest that the S-oxidation of S-methyl esonarimod reflects FMO5 activity in the human liver because the recombinant FMO5 data match well with the human liver microsomal experiments.

Localization of [14C]clarithromycin in rat gastric tissue when administered with lansoprazole and amoxicillin.

After oral and intravenous administration of [14C]clarithromycin to rats, c. 60-70% of the radioactivity in the gastric tissue was found to be distributed in the mucosal layer. Co-administration of lansoprazole and amoxicillin had no apparent effect on this distribution pattern of [14C]clarithromycin. The amount of unchanged [14C]clarithromycin in gastric contents increased with co-administration of lansoprazole and amoxicillin. Microautoradiograms of the gastric mucosa showed that [14C]clarithromycin was highly distributed in the mucous layer and in surface epithelial cells following oral administration. Homogeneous distribution of radioactivity was evident in the fundic gland. With iv administration, [14C]clarithromycin seemed to be secreted by both secreting cells in the gland base and surface epithelial cells.

Localization of [14C]amoxicillin in rat gastric tissue when administered with lansoprazole and clarithromycin.

The gastric mucosal distribution of [14C]amoxicillin when administered to rats with or without lansoprazole and clarithromycin was investigated. After oral administration, the amount found in the gastric mucosa was higher than after iv administration. Co-administration of lansoprazole and clarithromycin had no apparent effect on the distribution pattern of [14C]amoxicillin within the deeper stomach layers. About 50-60% of the radioactivity in the gastric tissue was present in the mucosal layer, irrespective of the route of administration. Microautoradiograms of the gastric mucosa indicated that [14C]amoxicillin was distributed in both the mucous layer and surface epithelial cells following oral administration. [14C]amoxicillin was secreted mainly by surface epithelial cells after iv administration, although only in small quantities.

Effects of lansoprazole and amoxicillin on uptake of [(14)C]clarithromycin into gastric tissue in rats.

Triple therapy consisting of clarithromycin (CLR), lansoprazole (LPZ), and amoxicillin (AMZ) is effective as eradication therapy for patients with peptic ulcer disease and Helicobacter pylori infection. We evaluated the effects of LPZ and AMZ on the uptake of [(14)C]CLR into the gastric tissue of rats. After administration of [(14)C]CLR alone or in combination with LPZ and AMZ, the distributions of [(14)C]CLR in the main organs and gastrointestinal tissues were compared. LPZ and AMZ had no effect on the distribution of [(14)C]CLR in any tissue except gastric tissue. The concentration of radioactivity in gastric tissue was several times higher when [(14)C]CLR was administered orally together with LPZ than when it was administered alone. The gastric emptying of [(14)C]CLR became smaller in the case of the coadministration of LPZ. AMZ had no apparent influence on the disposition of [(14)C]CLR. After the intravenous administration of [(14)C]CLR, no effects of drug coadministration were evident. In vitro uptake of [(14)C]CLR into gastric tissue was enhanced in the case of a high-pH environment. The uptake was not influenced by the concurrent presence of LPZ and AMZ. These results suggest that the penetration of [(14)C]CLR possibly depends on elevated gastric pH, as gastric acid secretion was inhibited by LPZ, and this may be a primary factor in explaining why the concentration of [(14)C]CLR at the target site, gastric tissue, was enhanced by the coadministration of LPZ.

Stereoselectivity and species difference in plasma protein binding of KE-298 and its metabolites.

In vitro protein binding of KE-298 and its plasma metabolites, deacetyl-KE-298 (M-1) and S-methyl-KE-298 (M-2), was high in rat (>97%), dog (>89%) and human plasma (>99%), respectively. Human serum albumin (>93%) was the main protein involved in the binding to plasma proteins, while the binding to human serum globulins was low (16-33%). The binding of KE-298 and its metabolites in all species of plasma was stereoselective. The (+)-(S)-enantiomers of these compounds bound rat, dog and human plasma proteins to a greater extent than did the (-)-(R)-enantiomers, except that the case of KE-298 was opposite in rat plasma. The stereoselective plasma levels of these compounds in rats, dogs, or humans would likely be due to stereoselective differences in binding to plasma albumin. The protein binding of M-1 in adjuvant-induced arthritis rat plasma was >97%, and the stereoselectivity was similar to the case of normal rat plasma. KE-298 and its metabolites remarkably displaced [14C]warfarin, which bound on albumin in a solution of diluted rat serum albumin. Similarly, there was a displacement of [14C]warfarin in solutions of dog and human serum albumin, and concomitantly the displacement of [14C]diazepam. [3H]Digitoxin was not displaced by any of the enantiomers in each albumin solution. No stereoselectivity was found in displacement by enantiomers of the three compounds. These results suggest that stereoselective protein binding can be attributed to quantitative differences in binding to albumin rather than to the different binding sites.

Effects of lansoprazole, clarithromycin and pH gradient on uptake of [14C]amoxycillin into rat gastric tissue.

The effect of lansoprazole and clarithromycin on the uptake of [(14)C]amoxycillin into rat gastric tissue was investigated. After oral administration of [(14)C]amoxycillin, the levels of radioactivity in gastrointestinal tissue were two to 15 times higher than those in plasma. The level of radioactivity in glandular stomach was significantly higher when lansoprazole and [(14)C]amoxycillin were administered together. After intravenous administration of [(14)C]amoxycillin, there was less radioactivity in gastric tissue than after oral administration, and co-administration of lansoprazole and clarithromycin had no obvious effect. The gastric emptying rate of [(14)C]amoxycillin was not apparently affected by the co-administration of lansoprazole and clarithromycin. In vitro uptake of [(14)C]amoxycillin into gastric tissue depended on the pH, with uptake at pH 7.4 being four times greater than that at pH 4.0. The apparent synergic effects of lansoprazole are due to enhanced penetration of amoxycillin in gastric mucus and tissue by increasing intragastric pH and play an important role in the eradication of H. pylori.

Development of a homogeneous time-resolved fluorescence assay for high throughput screening to identify Lck inhibitors: comparison with scintillation proximity assay and streptavidin-coated plate assay.

This study details the development of a homogeneous time-resolved fluorescence (HTRF) high throughput screening assay to identify inhibitors of Lck. HTRF was compared with scintillation proximity and streptavidin-coated plate assays. Because of the differences in the sensitivity of detection of phosphotyrosine among the three assays, different amounts of enzyme were used. However, the concentrations of the other assay components were standardized. When using similar assay conditions, the calculated IC(50) values of inhibitory compounds were independent of assay format. Furthermore, filtration experiments revealed that phosphorylation of a biotinyl poly-Glu,Ala, Tyr peptide substrate was less than autophosphorylation of the Lck enzyme; this was due to the low K(m) value for biotinyl poly-Glu,Ala,Tyr. In the HTRF assay, small amounts of enzyme and high concentrations of ATP could be used, thereby minimizing the effects of autophosphorylation. Higher ATP concentration would also minimize the effect of ATP competitors. Using this technology, it may be possible to find novel kinase inhibitors that do not act at the ATP binding site of protein tyrosine kinases.

Stereoselective disposition and chiral inversion of KE-298, a new antirheumatic drug, in rats.

The present study was an attempt to elucidate the relationship between stereoselective pharmacokinetics and protein binding of KE-298 and its active metabolites, deacetyl-KE-298 (M-1) and S-methyl-KE-298 (M-2). Metabolic chiral inversion was also investigated. The levels of unchanged KE-298 in plasma after oral administration of (+)-(S)-KE-298 to rats were lower than those of (-)-(R)-KE-298, whereas the levels of M-1 and M-2 after administration of (+)-(S)-KE-298 were higher than after (-)-(R)-KE-298. In vitro, rat plasma protein binding of (+)-(S)-KE-298 was lower than that of (-)-(R)-KE-298. In contrast, the binding of (+)-(S)-M-1 and (+)-(S)-M-2 was higher than that of (-)-(R)-M-1 and (-)-(R)-M-2. Displacement studies revealed that the (+)-(S) and (-)-(R)- enantiomers of KE-298 and their metabolites bound to the warfarin binding site on rat serum albumin. These results suggested that the stereoselective plasma levels in KE-298 and its metabolites were closely related to enantiomeric differences in protein binding attributed to quantitative differences in binding to albumin rather than to the different binding sites. Unidirectional chiral inversion was detected after oral administration of either (-)-(R)-KE-298 or (-)-(R)-M-2 to rats both yielding (+)-(S)-M-2.

Pharmacological profile of a novel, orally active leukotriene B4 antagonist, SM-15178.

SM-15178, a new hydroxyacetophenone derivative, was evaluated to determine its antiinflammatory activity and antagonistic activity against leukotriene B4 (LTB4). SM-15178 inhibited [3H]LTB4 binding to its receptors on human neutrophils (IC50 = 0.30 microM). It inhibited LTB4-induced chemotaxis of human neutrophils (IC50 = 0.72 microM) with little inhibitory effect against C5a or FMLP-induced chemotaxis at concentrations up to 30 microM. The compound alone did not cause human neutrophil chemotaxis at concentrations up to 10 microM. LTB4-induced chemotaxis of mouse and rat neutrophils and guinea pig eosinophils was also inhibited by the compound, with IC50 values of 0.55, 0.52, and 0.58 microM, respectively. In an in vivo study, SM-15178, given orally, significantly prevented LTB4-induced transient leukopenia. It also suppressed LTB4-induced bronchoconstriction in the guinea pig almost completely when given orally at a dose of 40 mg/kg. Furthermore, orally given SM-15178 suppressed arachidonic acid-induced neutrophil infiltration in mouse ears and Arthus reaction-induced paw edema in the mouse in a dose-dependent manner. These results suggest that SM-15178 is a selective and orally active LTB4 antagonist and that it might be effective for the treatment of some types of inflammatory diseases.

[A case of advanced mediastinal choriocarcinoma showing complete remission for two years by high-dose chemotherapy with peripheral blood stem cell transplantation].

A twenty-six-year-old male patient with advanced choriocarcinoma originated from mediastinum was treated by high-dose chemotherapy with peripheral blood stem cell (PBSC) transplantation. He had massive tumors in the cervical, mediastinal and bilateral lung fields. After 3 kur of PVeBV therapy (CDDP 60 mg x day 1-3, etoposide 160 mg x day 1-3, BLM 30 mg x day 1, VBL 12 mg x day 1), he obtained complete remission. PBSC was collected at the time of hematopoietic recovery and high-dose chemotherapy with PBSC transplantation was conducted because of the high possibility of recurrence. The hematopoietic recovery was rapid, and the patient remained well with no sign of recurrence for 24 months.

Antibody against neurofibromatosis type 1 gene product reacts with a triton-insoluble GTPase activating protein toward ras p21.

Cellular fractionation of GTPase activating protein (GAP) activity using bovine cerebral cortex revealed that about half of GAP activity was found in membrane fraction. GAP activity of membrane was not solubilized with 0.5% (v/v) triton X-100 and was immunoprecipitated with antibody against carboxy-terminus of neurofibromatosis type 1 (NF1) gene product. In contrast, soluble GAP activity was precipitated with antibody against GAP but not with anti-NF1. These results suggest that NF1 gene product is a GTPase activating protein toward ras p21 with completely different intracellular distribution from that of GAP.

Affinity labeling of ras oncogene product p21 with guanosine diphospho- and triphosphopyridoxals.

Purified v-rasH p21 overproduced in Escherichia coli was treated with guanosine diphospho- and triphosphopyridoxals (GP2- and GP3-PL), affinity labeling reagents specific to a lysyl residue located in the guanine nucleotide binding site. GP2-PL and GP3-PL inhibited [3H]GDP binding to p21 competitively. Incubation of p21 with GP2-PL and GP3-PL followed by reduction with NaBH4 resulted in 40 and 50% loss of [3H]GDP binding activity, respectively, whereas the addition of excess GDP completely protected p21 from the inactivation. The tryptic digest of p21 which was modified with GP2-PL or GP3-PL in the presence or absence of protective GDP and subsequently reduced by NaBH4 was analyzed by reverse phase high performance liquid chromatography. The profile of the effluent monitored by the fluorescence from the pyridoxyl moiety showed the existence of peptides which were specifically labeled only in the absence of GDP. Structural analyses of these peptides allowed us to identify the labeled residue as Lys-16. These results suggest that Lys-16 is located in the guanine nucleotide binding site, close to the beta- or gamma-phosphate group of the nucleotide.

A prospective study of urinary ligandin in patients at risk of renal tubular injury.

The urinary excretion of ligandin, a proximal tubular enzyme and binding protein, was measured by radioimmunoassay in eight normals, six patients receiving radiocontrast media, and six patients in a critical care unit who were considered at high risk for acute renal failure. Ligandinuria was found to occur normally at rates under 5 micrograms/hr. In the patients receiving radiocontrast media, abnormal rates of ligandinuria were found in four patients. In 102 ligandin measurements in the critically ill patients, rates of ligandinuria exceeded normal only once (after contrast media exposure) despite 13 identifiable episodes of potentially nephrotoxic circumstances and two episodes of acute renal failure. Ligandinuria appears more sensitive as a marker for tubular injury from contrast media than from other renal insults.

Hepatic bilirubin uptake in the isolated perfused rat liver is not facilitated by albumin binding.

Bilirubin uptake by the liver is a rapid process of high specificity that has kinetic characteristics which suggest carrier-mediation. In the circulation, bilirubin is readily bound to albumin, from which it is extracted by the liver. Although several studies suggested that it is the small, unbound fraction of bilirubin which interacts with hepatocytes and is removed from the circulation, recent experiments have been interpreted as suggesting that binding to albumin facilitates ligand uptake. A liver cell surface receptor for albumin has been postulated. The present study was designed to examine directly whether albumin facilitates the hepatic uptake of bilirubin and whether uptake of bilirubin depends on binding to albumin. Rat liver was perfused with a protein-free fluorocarbon medium, and single-pass uptake of 1, 10, or 200 nmol of [3H]bilirubin was determined after injection as an equimolar complex with 125I-albumin, with 125I-ligandin, or free with only a [14C]sucrose reference. Uptake of 10 nmol of [3H]bilirubin was 67.5 +/- 3.7% of the dose when injected with 125I-albumin, 67.4 +/- 6.5% when injected with 125I-ligandin, and 74.9 +/- 2.4% when injected with [14C]sucrose (P greater than 0.1). At 200 nmol, uptake fell to 46.4 +/- 3.1% (125I-albumin) and 63.3 +/- 3.4% [( 14C]sucrose) of injected [3H]bilirubin (P less than 0.01), which suggests saturation of the uptake mechanism. When influx was quantitated by the model of Goresky, similar results were obtained. When [3H]bilirubin was injected simultaneously with equimolar 125I-albumin and a [14C]sucrose reference, there was no delay in 125I-albumin transit as compared with that of [14C]sucrose. This suggested that the off-rate of albumin from a putative hepatocyte receptor would have to be very rapid, which is unusual for high affinity receptor-ligand interaction. There was no evidence for facilitation of bilirubin uptake by binding to albumin or for interaction of albumin with a liver cell surface receptor. These results suggest that the hepatic bilirubin uptake mechanism is one of high affinity which can extract bilirubin from circulating carriers such as albumin, ligandin, or fluorocarbon.

Hepatocellular ligandin during N-2-fluorenylacetamide carcinogenesis.

Ligandin was decreased by 75% as determined immunologically and by glutathione-S-transferase or steroid isomerase activities in rat hepatocellular carcinomas induced by exposure to N-2-fluorenylacetamide. Minor variable differences in ligandin levels were noted between the putative, premalignant nodules induced by this regimen and normal liver.

Binding of 3'-methyl-N,N-dimethyl-4-aminoazobenzene metabolites to rat liver cytosol proteins and ligandin subunits.

Twenty min after i.p. administration of 3'-[14C]methyl-N,N-dimethyl-4-aminoazobenzene in corn oil to rats, 0.73% of administered radioactivity was present in the liver. Only 0.45% of radioactivity present in liver was recovered in the nuclear fraction, whereas 25% was present in the cytosol fraction. Twenty-seven % of cytosolic radioactivity was trichloroacetic acid precipitable, and 2% was immunoprecipitable with monospecific anti-rat liver ligandin immunoglobulin G. After 3 hr of administration, 3.2% of administered radioactivity was present in the liver, 40% of which was in the cytosol. Although 59% of radioactivity present in liver cytosol was trichloroacetic acid precipitable as compared to 27% at 20 min, the radioactivity precipitated by anti-ligandin immunoglobulin G was still 2%. When liver cytosol obtained from rats after 20 min of 3'-[14C]methyl-N,N-dimethyl-4-aminoazobenzene administration was fractionated on a Sephadex G-75 column, three peaks of radioactivity were observed. When cytosol was subjected to sodium dodecyl sulfate gel electrophoresis and fluorography, radioactivity was mainly associated with 5 proteins with molecular weights of 88,000, 47,000, 41,000, 31,000, and 22,000. When the immunoprecipitate obtained from cytosol with anti-ligandin immunoglobulin G was subjected to sodium dodecyl sulfate gel electrophoresis and fluorography, radioactivity was exclusively associated with the subunit of ligandin with a molecular weight of 22,000. Approximately 90% of the radioactivity in the immunoprecipitate was covalently associated with this subunit. These studies reveal that 3'-methyl-N,N-dimethyl-4-aminoazobenzene or its metabolites are selectively bound to the subunit of ligandin with a molecular weight of 22,000 and four other cytosol proteins in vivo.

Ligandinemia in primary liver cell cancer in rat and man.

Ligandin was quantitated by radioimmunoassay in serum and, when possible, in tumors from patients with primary hepatocellular carcinoma, massive hepatic metastasis or nonhepatic primary neoplasms, and in rats and athymic (nu/nu) mice bearing transplantable ligandin-containing or nonligandin-containing rat hepatocellular carcinomas. Following transplantation of a ligandin-containing rat hepatocellular carcinoma in rats or athymic (nu/nu) mice, mean serum ligandin concentrations progressively increased and, within 4 months, exceeded normal serum ligandin concentrations by 10-fold. In 11 of 15 HBsAg negative patients with primary hepatocellular carcinoma, serum ligandin concentrations ranged from 82 to 551 (mean: 298) ng per ml; the mean ligandin concentration in the hepatic neoplasm was 32% of the ligandin concentration in normal liver. In 19 of 22 patients with extensive hepatic metastasis and in each of 20 patients with primary carcinomas without hepatic metastasis, serum ligandin concentrations were normal (7.0 +/- 4.0 ng per ml).