Characterization of Transporters in the Hepatic Uptake of TAK-475 M-I, a Squalene Synthase Inhibitor, in Rats and Humans.

TAK-475 (lapaquistat acetate) is a squalene synthase inhibitor and M-I is a pharmacologically active metabolite of TAK-475. Preclinical pharmacokinetic studies have demonstrated that most of the dosed TAK-475 was hydrolyzed to M-I during the absorption process and the concentrations of M-I in the liver, the main organ of cholesterol biosynthesis, were much higher than those in the plasma after oral administration to rats. In the present study, the mechanism of the hepatic uptake of M-I was investigated.The uptake studies of (14)C-labeled M-I into rat and human hepatocytes indicated that the uptakes of M-I were concentrative, temperature-dependent and saturable in both species with Km values of 4.7 and 2.8 µmol/L, respectively. M-I uptake was also inhibited by cyclosporin A, an inhibitor for hepatic uptake transporters including organic anion transporting polypeptide (OATP). In the human hepatocytes, M-I uptake was hardly inhibited by estrone 3-sulfate as an inhibitor for OATP1B1, and most of the M-I uptake was Na(+)-independent. Uptake studies using human transporter-expressing cells revealed the saturable uptake of M-I for OATP1B3 with a Km of 2.13 µmol/L. No obvious uptake of M-I was observed in the OATP1B1-expressing cells.These results indicated that M-I was taken up into hepatocytes via transporters in both rats and humans. OATP1B3 would be mainly involved in the hepatic uptake of M-I in humans. These findings suggested that hepatic uptake transporters might contribute to the liver-selective inhibition of cholesterol synthesis by TAK-475. This is the first to clarify a carrier-mediated hepatic uptake mechanism for squalene synthase inhibitors.

Rewarming following accidental hypothermia in patients with acute subdural hematoma: case report.

A 57-year-old man was admitted to the Emergency and Critical Care Department with accidental hypothermia (31.5 degrees C) after resuscitation from cardiopulmonary arrest (CPA). Brain CT revealed an acute subdural hematoma. Active core rewarming to 33 degrees C was performed using an intravenous infusion of warm crystalloid. The patient underwent craniotomy soon after admission, with bladder temperature maintained at 33 to 34 degrees C throughout the surgery. Therapeutic hypothermia (34 degrees C) was continued for 2 days, followed by gradual rewarming. After rehabilitation, the patient was able to continue daily life with assistance. Traumatic brain injury (TBI) following CPA is associated with extremely unfavorable outcomes. Very few patients with acute subdural hematomas presenting with accidental hypothermia and CPA have been reported to recover. No suitable strategies have been clearly established for the rewarming performed following accidental hypothermia in patients with TBI. Our experience with this patient suggests that therapeutic hypothermia might improve the outcome in some patients with severe brain injury. It also appears that the method used for rewarming might play an important role in the therapy for TBI with accidental hypothermia.

Alterations in large and small proteoglycans in bleomycin-induced pulmonary fibrosis in rats.

In bleomycin (BM)-induced lung fibrosis, alterations have been shown in the expression and deposition of small proteoglycans (PGs). Less, however, is known about changes in large PGs. We investigated changes in large aggregating (versican [VS]), basement membrane (heparan sulfate proteoglycan [HSPG]), and small (biglycan and fibromodulin) PGs during the development of BM-induced pulmonary fibrosis. BM (1.5 U) was instilled intratracheally into male Sprague-Dawley rats. Control rats received saline. At 7, 14, and 28 d after administration of BM, lungs were excised; one lung was fixed in formalin and 5-microm sections were cut and stained with hematoxylin-eosin. The other lung was used for PG extraction. PGs were extracted with guanidine and were separated through composite gel polyacrylamide gel electrophoresis (PAGE) (large PGs) and sodium dodecylsulfate-PAGE (small PGs). Gels were either stained or electrophoretically transferred and probed with antibodies to VS, HSPG, biglycan, and fibromodulin. Histologic samples showed prominent inflammation, with abundant proteinaceous material, most evident in the samples obtained at 7 and 14 d after administration of BM. By 28 d after BM, much of the inflammatory response had resolved, and heterogeneous distribution of fibrosis was observed. Immunoblots showed a relative abundance of VS at 7 and 14 d. Control lungs stained minimally for VS. Levels of HSPG, biglycan, and fibromodulin were increased maximally at 14 d after administration of BM. Immunocytochemistry showed intense immunostaining of biglycan and fibromodulin in the areas of injured lung tissue from rats 14 and 28 d after BM administration. Control lungs revealed minimal staining for small PGs. Our findings indicate that changes in all subclasses of PGs occur during the development of BM-induced pulmonary fibrosis.

Type I collagen is a non-adhesive extracellular matrix for macrophages.

Macrophages adhere to a variety of substrata including plastic, glass or an extracellular matrix either in a highly specific manner or through less specific mechanisms. We investigated the effect of type I collagen, the most abundant protein in animal tissues, on the adhesion of macrophages derived from a human monoblastic cell line U937. Macrophages were observed to adhere very weakly to type I collagen and aggregate, whereas they adhered firmly and spread on plastic, bovine serum albumin or fibronectin. On the adhesive substratum, the lower surface of the macrophages was flat and closely apposed to the substratum. In contrast, macrophages adhered on type I collagen at the tip of cell processes. The adhesion of macrophages to plastic, bovine serum albumin or fibronectin was associated with the induction of tyrosine phosphorylation of a variety of proteins including a major protein band at 66 kDa. In contrast, the induction of tyrosine phosphorylation was markedly reduced when the macrophages were cultured on type I collagen. Two members of the src family, Lyn and Hck, were tyrosine phosphorylated in firmly adhered macrophages but not in macrophages cultured on type I collagen. These results suggest that the adhesion of macrophages is associated with the tyrosine phosphorylation of a variety of proteins including Lyn and Hck, and that type I collagen serves as a non-adhesive substratum for macrophages, resulting in an altered signal transduction.

[Shift to home treatment and the function of pharmacies].

A crisis point has been reached in the medical health system in Japan, where there has been a rapid aging of the population. The method of providing health system benefits is being reconsidered. As a result, medical treatment has been classified into inpatient, outpatient, and home care systems according to treatment functions. In concrete terms, the insurance system now classifies medical treatment and care separately, payment for medical fees has changed from payment at piece rates to a fixed amount system, and the number of days patients stay in hospital has been reduced. It is predicted that hospital treatment will increasingly rely on early discharge, and patients highly reliant on medical treatment will be shifted to home treatment. For this shift to home treatment, therefore, supplies of injectable agents such as used in total parenteral nutrition, and equipment by specialist pharmacies have become necessary. However, to dispense injectable agents, "standards of compounded sterile pharmaceuticals by licensed pharmacies" is applied, meaning there are very few pharmacies that can meet these standards. In the present survey, we found that 40 pharmacies have been recognized as meeting these standards.

[Role of the pharmacy for home cancer patients].

Otsukadai Pharmacy compounded morphine chloride for injection in 32 cancer patients. In 22 cases we compounded morphine into a TPN bag. However we recommend using an infusion pump to control the patient's pain at home. We think that we need a system with a pharmacy for cancer patients before they leave the hospital for home care.

Percutaneous dye image cardioscopy for detection of endocardial lesions.

Endocardial lesions are caused not only by inflammatory processes but also by myocardial ischemia, resulting in endocardial thrombosis and cerebral embolism. We deviced a method for direct visualization of endocardial damages by a novel dye image cardioscopy with Evans blue and examined its feasibility in patients with heart disease. The dye was injected into the left ventricle before and after endomyocardial biopsy. Endocardial surface was stained in dark blue in 63% of patients with angina pectoris before biopsy. After biopsy, the biopsied portions were stained in blue in all. The results indicate that endocardium is damaged even in apparently intact LV in patients with ischemic heart disease and that endomyocardial biopsy causes severe endocardial damages.

Site-directed lipid modification of IgG-binding protein by intracellular bacterial lipoprotein process.

IgG-binding protein was genetically expressed and lipid-modified in a site-directed manner in Escherichia coli. The DNA sequence encoding the signal peptide and the nine N-terminal amino acid residues of the major lipoprotein of E. coli (lpp) was fused to the sequence of B-domain which was one of the IgG binding domains of Staphylococcal Protein A (SpA). The N-terminal cysteine residue of the resulting protein was enzymatically linked with lipids in the bacterial membrane. The lipid-modified protein was translocated at the bacterial membrane in a manner similar to native bacterial lipoprotein, and it was purified with IgG-Sepharose by affinity chromatography. The lipid modified proteins (lppB1 and lppB5) showed a similar IgG binding activity to unmodified proteins, which was estimated by competitive ELISA. Proteoliposomes of lipid modified proteins were prepared in an elegant fashion so that the IgG binding site should be properly oriented on the surface of an individual liposome by anchoring the lipid-tail into the hydrophobic layer of the liposome membrane. As compared with the unmodified one, the lipid modified protein incorporated into the proteoliposome exhibited higher IgG binding activity.

Protein kinase inhibitor H7 blocks the induction of immediate-early genes zif268 and c-fos by a mechanism unrelated to inhibition of protein kinase C but possibly related to inhibition of phosphorylation of RNA polymerase II.

1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H7) has often been used in combination with protein kinase inhibitor (N-(2-guanidinoethyl)-5-isoquinolinesulfonamide) (HA1004) to assess the contribution of protein kinase C (PKC) to cellular processes, including the induction of gene expression. This use of H7 and HA1004 is based upon the fact that H7 inhibits PKC more potently than HA1004 in in vitro assays. Thus, although both compounds are broad spectrum protein kinase inhibitors, inhibition by H7, but not by HA1004, has often been interpreted as evidence for the involvement of PKC in the cellular process under study. Here we describe experiments that show that this interpretation is not correct with regard to the induction of two immediate-early genes, zif268 and c-fos, in PC12D cells. In these studies we confirmed that H7, but not HA1004, potently blocks the induction of zif268 and c-fos mRNA by nerve growth factor, carbachol, phorbol ester, Ca2+ ionophore, or forskolin. Surprisingly, however, H7 has no effect on the ability of these agents to activate mitogen-activated protein kinase (MAPK), an upstream activator of zif268 and c-fos gene expression. H7 also does not inhibit preactivated MAPK in vitro. Taken together, these results suggest that H7 blocks gene expression by acting at a site downstream from MAPK. H7 has previously been shown to block transcription in vitro by blocking the phosphorylation of the carboxyl-terminal domain of RNA polymerase II (Yankulov, K., Yamashita, K., Roy, R., Egly, J.-M., and Bentley, D. L.(1995) J. Biol. Chem. 270, 23922-23925). In this study, we show that pretreating PC12D cells with H7, but not with HA1004, significantly reduces levels of phosphorylated RNA polymerase II in vivo. These results suggest that H7 blocks gene expression by inhibiting the phosphorylation of RNA polymerase II, a step required for progression from transcription initiation to mRNA chain elongation.

Nerve growth factor induces zif268 gene expression via MAPK-dependent and -independent pathways in PC12D cells.

In this study we examined the contribution of MAPK1 and 2 [also known as extracellular signal-regulated kinases (ERK)-1 and 2] to the induction of zif268 mRNA in PC12D cells by using two methods to block the activation of these kinases. In one set of experiments, we inhibited the activation of MAPK by pretreating cells with PD098059, a specific inhibitor of MEK (MAPKK), the immediate upstream activator of MAPK. In the second set of experiments, we blocked the activation of MAPK by overexpressing N17Ras, a dominant-negative form of Ha-Ras. These two approaches yielded similar results and showed that inhibition of MAPK blocks less than half of the induction of zif268 mRNA by NGF. Much of the residual induction of zif268 mRNA is blocked by low concentrations of wortmannin, an inhibitor of phosphatidylinositol (PI) 3-kinase. Since PI 3-kinase was previously shown to function upstream in epidermal growth factor (EGF)-mediated activation of c-Jun N-terminal kinase (JNK), and JNK is known to phosphorylate and activate transcription factors that regulate the expression of zif268, we investigated the role of JNK in the induction of zif268 mRNA by NGF. Stimulation of PC12D cells with NGF weakly activates JNK, but this activation is enhanced rather than inhibited by pretreatment with wortmannin, suggesting that JNK does not function downstream of PI 3-kinase in the induction of zif268 mRNA. A role for JNK in the induction of the zif268 gene is indicated, however, by the fact that cotransfection of expression vectors encoding JIP-1 or the JNK binding domain of JIP-1, which act as dominant-negative inhibitors of JNK, partially blocks the NGF-mediated induction of a luciferase reporter gene linked to the zif268 promoter. Together, these results suggest that MAPK, PI-3 kinase and JNK each play a role in the induction of zif268 gene expression by NGF in PC12D cells.

Effect of 5-hydroxytryptamine on intracellular calcium dynamics in cultured rat vascular smooth muscle cells.

The present study elucidated the precise mechanism of 5-hydroxytryptamine (5-HT)-induced increase of intracellular Ca2+ concentration ([Ca2+]i) in cultured vascular smooth muscle cells isolated from rat aortic media. [Ca2+]i was measured using fluorescent Ca2+ indicator, fura-2. 5-HT caused a dose-dependent increase in [Ca2+]i, which was completely inhibited by ketanserin. alpha-Methyl-5-HT had an equipotent effect to 5-HT. Diltiazem at 10 microM partially suppressed the 5-HT-induced increase in [Ca2+]i. 5-HT also augmented Mn2+ influx, when monitored by Mn2+ quenching of fura-2 fluorescence. When extracellular Ca2+ (1.3 mM) was removed, a decrease in resting level and a small, transient increase in [Ca2+]i were observed. 5-HT stimulation also induced an increase in the production of inositol triphosphate. 5-HT-induced increase in [Ca2+]i was significantly, but partially inhibited by staurosporin and H-7. Phorbol 12-myristate 13-acetate induced an increase in [Ca2+]i, which was abolished by removal of extracellular Ca2+. 5-HT-induced increase in [Ca2+]i was not affected by the pretreatment with pertussis toxin (PTX), and was not accompanied by a change in cyclic AMP content. These results suggest that, in cultured rat aortic smooth muscle cells, 5-HT increases [Ca2+]i via 5-HT2 receptor subtype by inducing influx of extracellular Ca2+ partially through L-type voltage-dependent Ca2+ channel, as well as by mobilizing Ca2+ from its intracellular stores. Activation of protein kinase C may be positively involved in the regulatory mechanism of Ca2+ influx, but PTX-sensitive G protein and cyclic AMP seem to be not involved.

Muscarinic acetylcholine receptor-mediated induction of zif268 mRNA in PC12D cells requires protein kinase C and the influx of extracellular calcium.

The immediate-early gene zif268 (egr-1, NGFI-A, krox-24) encodes a transcription factor that has been proposed to play a role in differentiation and neuronal plasticity. zif268 mRNA is undetectable in unstimulated PC12D cells, a subline of PC12 characterized by accelerated differentiation in the presence of nerve growth factor, but is rapidly and robustly induced following exposure to muscarinic acetylcholine receptor (mAChR) agonists. Although PC12D cells express mRNAs for both m1 and m4 mAChR subtypes, induction of zif268 mRNA by mAChR agonists is apparently mediated exclusively by the m1 subtype because the induction is completely blocked by Dendroaspis angusticeps m1 toxin. Pretreatment of the cells with the specific protein kinase C (PKC) inhibitor GF109203X partially inhibits mAChR-mediated induction of zif268 mRNA. The remaining induction is blocked by chelation of extracellular calcium with EGTA. EGTA prevents the influx of calcium without blocking mAChR-mediated release of calcium from internal stores. These data indicate that both PKC and the influx of extracellular calcium play a role in the induction of zif268 mRNA following activation of the m1 mAChR. Transient increases in intracellular calcium levels resulting from the release of calcium from internal stores alone are not sufficient to induce zif268 mRNA. Rather, induction requires a prolonged elevation of intracellular calcium levels, which is sustained by the influx of extracellular calcium.

Role of substance P in cough.

The sensory neuropeptide, substance P (SP), is present in human airway nerves, beneath and within the epithelium where the condensed localization of neutral endopeptidase (NEP), the major enzyme degrading SP, is observed. To test the hypothesis whether SP stimulates the cough reflex and NEP modifies the cough reflex, we studied the cough response to various stimuli in awake guinea-pigs. Inhibition of NEP with phosphoramidon caused cough, which was inhibited by systemic capsaicin treatment and by aerosols of a specific NK1 receptor antagonist FK 888. Aerosols of FK 888 also inhibited cough induced by bronchoconstricting agents such as acetylcholine and histamine in non-sensitized animals and by ovalbumin antigen in animals sensitized to ovalbumin. The number of coughs induced by histamine aerosols was inhibited by systemic capsaicin treatment and enhanced by pretreatment with a NEP inhibitor phosphoramidon. Likewise, FK 888 inhibited the augmented cough response to aerosolized capsaicin in female guinea-pigs treated with a long-term medication of an angiotensin-converting enzyme inhibitor, cilazapril. In humans, aerosols of SP did not cause cough in normal subjects, whereas it did in patients with common colds. The SP fragment a major metabolite of SP produced by NEP, was less effective compared with SP in these patients, suggesting that damaged epithelium may facilitate the penetration of SP. These findings suggest that SP released from sensory nerves in response to stimuli may mediate cough and NEP may have a role in modulating SP-induced effects.

Pemphigus: from immunofluorescence to molecular biology.

Since the discovery of autoantibodies in patients with pemphigus, pemphigus has been intensively studied by dermatologists and cutaneous or cellular biologists by means of various techniques including immunofluorescence, immunoelectron microscopy, immunoprecipitation, immunoblotting, and molecular biology. In this article, up-dated topics on pemphigus obtained by each individual technique are reviewed. In the course of immunofluorescence studies on unusual cases of blistering diseases, a new entity characterized by immunoglobulin A (IgA)-type autoantibodies directed against keratinocyte cell surfaces has been discovered. Immunoelectron microscopy using low temperature post-embedding gold labeling enabled us to quantitate binding sites of pemphigus autoantibodies within desmosomes at different levels of epidermis. Immunoprecipitation and immunoblot analyses allowed us to characterize antigen complexes in paraneoplastic pemphigus. Finally, approaches using molecular biology not only have given us a fundamental insight that pemphigus autoantigen is a cadherin-type cell adhesion molecule both in pemphigus vulgaris and pemphigus foliaceus, but also provided tools to develop novel diagnostic and therapeutic strategies.

Angiotensin-converting enzyme inhibitor and danazol increase sensitivity of cough reflex in female guinea pigs.

To examine the mechanisms of an angiotensin-converting enzyme (ACE) inhibitor-induced cough in perimenopausal and postmenopausal women, cough responses to aerosols of capsaicin and citric acid were examined in four groups of female guinea pigs treated orally with danazol (D) (an agent decreasing plasma estrogen levels), cilazapril (C) (an inhibitor of ACE), both danazol and cilazapril (C+D), or without either drug (control group) for 4 to 5 wk. Capsaicin caused dose-dependent increases in the number of coughs in all four groups. C or D alone shifted dose-response curves to capsaicin (from 10(-7) M to 10(-3) M) to lower concentrations compared with the control, and C+D further shifted them. Likewise, the number of coughs induced by citric acid (3 x 10(-1) M; 2 min) was highest in animals treated with C+D and significantly higher in animals treated with C or D than in the control group. Aerosols of a selective substance P (SP) receptor antagonist FK 888 (10(-5) M; 2 min) inhibited capsaicin-induced cough in all four groups, and dose-response curves to capsaicin did not differ significantly at any concentrations among the four groups in the presence of FK 888 (p > 0.10). D decreased cyclic AMP levels in the trachea, irrespective of the combination of C. A beta 2-adrenoceptor agonist, procaterol, which is thought to inhibit SP release by elevation of cyclic AMP in sensory nerves, dose-dependently inhibited the number of coughs induced by capsaicin (10(-3) M) in animals treated with D. The present study suggests that SP is a common mechanism mediating increases in the sensitivity of cough reflex induced by both ACE inhibition and a decrease in plasma estrogen levels, and the additive effects of the two events may explain the high incidence of cough during ACE inhibitor therapy in perimenopausal and postmenopausal women.

[Problems and solutions of home care for a terminal cancer patient--cooperation as a team approach].

In order to have a terminal cancer patient spending fulfilling remaining of his life, the role of home care is important. Caremark clinical team, consists of nurses, pharmacists and clinical coordinators (CC), provides home care service for patients. We have faced many problems regarding providing care for our cancer patients, especially terminal patients, and solved the problems in cooperation with the physicians. The main problems which we have faced with care for terminal cancer patients are as follows: (1) For emergency case, such as sudden changes in patient condition, if a hospital where the patient used to stay can not accept his return, our clinical coordinator makes a contact with closer hospital and asks them to hospitalize the patient. (2) Concerns of a patient and his family increase when his pain is getting severe. For this case, our nurse gives his caregiver training on treatment method, and normal pain management care for the patient. Also our pharmacist talks with the patient and his family and try to reduce the pain by using more suitable drugs at each conditions. (3) A patient with serious conditions which requires long term home care, his family and caregivers have more burdens. For this problem we suggests them to use high touch home care providers, and also our nurse provides psychological care (counseling). We have solved other problems in cooperation with each specialists, and we are confident that we are able to provide better home care service for our patients.

Unusual acantholytic bullous dermatosis associated with neoplasia and IgG and IgA antibodies against bovine desmocollins I and II.

There are unusual cases of pemphigus that have antibodies nonreactive with either pemphigus vulgaris or pemphigus foliaceus antigens. We describe a patient with an acantholytic bullous dermatosis and lung cancer with intercellular IgG and IgA antibodies that differed in specificity from those of pemphigus vulgaris and pemphigus foliaceus and reacted with bovine desmocollins I and II and recombinant desmocollin protein.