AK106-001616, a Potent and Selective Inhibitor of Cytosolic Phospholipase A2: In Vivo Efficacy for Inflammation, Neuropathic Pain, and Pulmonary Fibrosis.

3-[3-Amino-4-(indan-2-yloxy)-5-(1-methyl-1H-indazol-5-yl)-phenyl]-propionic acid (AK106-001616) is a novel, potent, and selective inhibitor of the cytosolic phospholipase A2 (cPLA2) enzyme. Unlike traditional nonsteroidal anti-inflammatory drugs and selective cyclooxygenase-2 inhibitors, AK106-001616 reduced prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) production by stimulated cells. The suppression of PGE2 and LTB4 production was also confirmed using an air pouch model in rats administered a single oral dose of AK106-001616. AK106-001616 alleviated paw swelling in a rat adjuvant-induced arthritis (AIA) model. The maximum effect of the inhibitory effect of AK106-001616 was comparable with that of naproxen on paw swelling in a rat AIA model. Meanwhile, the inhibitory effect of AK106-001616 was more effective than that of naproxen in the mouse collagen antibody-induced arthritis model with leukotrienes contributing to the pathogenesis. AK106-001616 dose dependently reversed the decrease in paw withdrawal threshold not only in rat carrageenan-induced hyperalgesia, but also in a rat neuropathic pain model induced by sciatic nerve chronic constriction injury (CCI). However, naproxen and celecoxib did not reverse the decrease in the paw withdrawal threshold in the CCI model. Furthermore, AK106-001616 reduced the disease score of bleomycin-induced lung fibrosis in rats. In addition, AK106-001616 did not enhance aspirin-induced gastric damage in fasted rats, increase blood pressure, or increase the thromboxane A2/ prostaglandin I2 ratio that is thought to be an underlying mechanism of thrombotic cardiovascular events increased by selective cyclooxygenase-2 inhibitors. Taken together, these data demonstrate that oral AK106-001616 may provide valuable effects for wide indications without attendant gastrointestinal and cardiovascular risks.

Early Effects of Single and Low-Frequency Repeated Administration of Teriparatide, hPTH(1-34), on Bone Formation and Resorption in Ovariectomized Rats.

Intermittent repeated administration of teriparatide (TPTD) has potent anabolic effects on bones in vivo. However, TPTD has both anabolic and catabolic effects on osteoblasts in vitro, and the mechanisms underlying its promotion of bone formation are unclear. This study aimed to elucidate the time-dependent changes in bone formation and resorption by examining changes in bone turnover markers and bone tissue over time after TPTD administration with low frequency in ovariectomized rats. The amount of serum osteocalcin, a bone formation marker, was transiently reduced after single TPTD administration, but increased thereafter, remaining increased for several days. In contrast, the amount of excreted urinary C-telopeptide, a bone resorption marker, increased transiently after single TPTD administration, and subsequently returned to control levels on the day after administration. Tissue histomorphometric analyses conducted 8 h after administration showed no changes in bone formation or bone resorption parameters. However, at 48 h, the bone formation parameters OS/BS and Ob.S/BS were increased, while the bone resorption parameter ES/BS was decreased. After repeated TPTD administration for 4 weeks, OS/BS, Ob.S/BS, and MS/BS increased, while Oc.S/BS decreased. Serum osteocalcin at 4 weeks after repeated administration was significantly correlated with OS/BS and Ob.S/BS. These present findings indicate that TPTD has dual, time-dependent effects on bone resorption and bone formation. Immediately after single administration, there was transient promotion of bone resorption and suppression of bone formation. However, sustained stimulation of bone formation occurred thereafter. Furthermore, these data suggest that this sustained bone formation led to anabolic effects with repeated TPTD administration.

Three-times-weekly administration of teriparatide improves vertebral and peripheral bone density, microarchitecture, and mechanical properties without accelerating bone resorption in ovariectomized rats.

Daily and weekly administration of teriparatide (PTH1-34) reduces the risk of osteoporotic bone fractures. However, their effects on markers of bone formation and bone resorption differ. These results indicate that the dosing frequency of teriparatide may affect bone metabolism and bone structure, with different effects on bone strength. In the present study, to evaluate the dose-related effects of a low administration frequency of teriparatide on bone status, we investigated the effects of three-times-weekly administration of teriparatide (1.1, 5.6, or 28.2 µg/kg) for 12 months on bone parameters, including bone metabolism markers, bone mineral density (BMD), micro-computed tomography, and bone strength, using 6-month-old ovariectomized (OVX) rats. Three-times-weekly administration of teriparatide dose-dependently increased the BMD of the lumbar vertebra and femur in OVX rats, and increased serum osteocalcin (a marker of bone formation), but not type I collagen C-telopeptide (a marker of bone resorption). The trabecular number and thickness increased in the vertebrae and femur, as in prior reports of daily teriparatide administration in OVX rats. Cortical thickness increased only toward the endocortical side of the femur, unlike with daily administration. Bone strength of the vertebrae and proximal and shaft of the femur was correlated with the changes in BMD and bone structure. These results demonstrate the effects of low frequency, intermittent administration of teriparatide on the biomechanical, and microstructural properties of bone in OVX rats.

5-Hydroxytryptamine-induced bladder hyperactivity via the 5-HT2A receptor in partial bladder outlet obstruction in rats.

We investigated the effects of partial bladder outlet obstruction (BOO) on the function and gene expression of 5-hydroxytryptamine (5-HT) receptor subtypes in rat bladder. Isometric contractions of the isolated bladders from sham-operated control and BOO rats were examined. The contractile responses to 5-HT were significantly increased in BOO rat bladder strips, while the responses to KCl, carbachol, or phenylephrine were not different from the control. The 5-HT-induced hypercontraction in BOO rat bladder strips was inhibited by ketanserin, a 5-HT(2A) receptor antagonist. The contractile responses to 5-HT in bladder strips were not affected by urothelium removal from the intact bladder. The gene expression of 5-HT receptor subtypes in the bladders was analyzed by RT-PCR. The mRNA expression of the 5-HT(2A), 5-HT(2B), 5-HT(2C), 5-HT(4), and 5-HT(7) receptors was detected in both the control and BOO rat bladders. Quantitative RT-PCR analysis showed there was a significant increase of 5-HT(2A) receptor mRNA in the BOO rat bladder compared with the control bladder. On the other hand, the gene expression of the 5-HT(4) receptor was not changed in the BOO rat bladder. These results suggest that the increased contractile responses to 5-HT in BOO rat bladder may be partly caused by 5-HT(2A) receptor upregulation in the detrusor smooth muscles.

Anti-hyperalgesic effects of calcitonin on neuropathic pain interacting with its peripheral receptors.

BACKGROUND: The polypeptide hormone calcitonin is clinically well known for its ability to relieve neuropathic pain such as spinal canal stenosis, diabetic neuropathy and complex regional pain syndrome. Mechanisms for its analgesic effect, however, remain unclear. Here we investigated the mechanism of anti-hyperalgesic action of calcitonin in a neuropathic pain model in rats. RESULTS: Subcutaneous injection of elcatonin, a synthetic derivative of eel calcitonin, relieved hyperalgesia induced by chronic constriction injury (CCI). Real-time reverse transcriptase-polymerase chain reaction analysis revealed that the CCI provoked the upregulation of tetrodotoxin (TTX)-sensitive Nav.1.3 mRNA and downregulation of TTX-resistant Nav1.8 and Nav1.9 mRNA on the ipsilateral dorsal root ganglion (DRG), which would consequently increase the excitability of peripheral nerves. These changes were reversed by elcatonin. In addition, the gene expression of the calcitonin receptor and binding site of 125I-calcitonin was increased at the constricted peripheral nerve tissue but not at the DRG. The anti-hyperalgesic effect and normalization of sodium channel mRNA by elcatonin was parallel to the change of the calcitonin receptor expression. Elcatonin, however, did not affect the sensitivity of nociception or gene expression of sodium channel, while it suppressed calcitonin receptor mRNA under normal conditions. CONCLUSIONS: These results suggest that the anti-hyperalgesic action of calcitonin on CCI rats could be attributable to the normalization of the sodium channel expression, which might be exerted by an unknown signal produced at the peripheral nerve tissue but not by DRG neurons through the activation of the calcitonin receptor. Calcitonin signals were silent in the normal condition and nerve injury may be one of triggers for conversion of a silent to an active signal.

Compensatory increase in fatty acid synthesis in adipose tissue of mice with conditional deficiency of SCAP in liver.

The escort protein SCAP transports SREBPs from ER to Golgi where the active domains are released to activate genes for fatty acid (FA) and cholesterol synthesis. Mice with conditional SCAP deficiency in liver (L-Scap-) manifest marked reductions in hepatic lipid synthesis. Here, we show that the decreased FA synthesis in liver is balanced by an equal increase in nonhepatic tissues, primarily adipose tissue. Extrahepatic synthesis of FAs preserves adipose mass, even when L-Scap- mice consume a fat-free diet. This compensatory response disappears upon fasting, implicating a role for insulin, the major hormonal activator of FA synthesis. This response is mediated by an insulin-dependent increase in adipocyte SREBP-1c and its target mRNAs. In epididymal fat of L-Scap- mice, phosphorylated Akt, Glut-4 mRNA, and glucose uptake are also increased, indicating insulin hypersensitivity. Plasma VLDL triglycerides are dramatically reduced in L-Scap- mice, underscoring the benefits of synthesizing FAs in fat rather than liver.

Schoenheimer effect explained--feedback regulation of cholesterol synthesis in mice mediated by Insig proteins.

End-product feedback inhibition of cholesterol synthesis was first demonstrated in living animals by Schoenheimer 72 years ago. Current studies define Insig proteins as essential elements of this feedback system in mouse liver. In cultured cells, Insig proteins are required for sterol-mediated inhibition of the processing of sterol regulatory element-binding proteins (SREBPs) to their nuclear forms. We produced mice with germline disruption of the Insig2 gene and Cre-mediated disruption of the Insig1 gene in liver. On a chow diet, these double-knockout mice overaccumulated cholesterol and triglycerides in liver. Despite this accumulation, levels of nuclear SREBPs and mRNAs for SREBP target genes in lipogenic pathways were not reduced. Whereas cholesterol feeding reduced nuclear SREBPs and lipogenic mRNAs in wild-type mice, this feedback response was severely blunted in the double-knockout mice, and synthesis of cholesterol and fatty acids was not repressed. The amount of HMG-CoA reductase protein was elevated out of proportion to the mRNA in the double-knockout mice, apparently owing to the failure of cholesterol to accelerate degradation of the enzyme. These studies indicate that the essential elements of the regulatory pathway for lipid synthesis function in liver as they do in cultured cells.

Overexpression of Insig-1 in the livers of transgenic mice inhibits SREBP processing and reduces insulin-stimulated lipogenesis.

In the current studies we generated transgenic mice that overexpress human Insig-1 in the liver under a constitutive promoter. In cultured cells Insig-1 and Insig-2 have been shown to block lipid synthesis in a cholesterol-dependent fashion by inhibiting proteolytic processing of sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors that activate lipid synthesis. Insig's exert this action in the ER by binding SREBP cleavage-activating protein (SCAP) and preventing it from escorting SREBPs to the Golgi apparatus where the SREBPs are processed to their active forms. In the livers of Insig-1 transgenic mice, the content of all nuclear SREBPs (nSREBPs) was reduced and declined further upon feeding of dietary cholesterol. The nuclear content of the insulin-induced SREBP isoform, SREBP-1c, failed to increase to a normal extent upon refeeding on a high-carbohydrate diet. The nSREBP deficiency produced a marked reduction in the levels of mRNAs encoding enzymes required for synthesis of cholesterol, fatty acids, and triglycerides. Plasma cholesterol levels were strongly reduced, and plasma triglycerides did not exhibit their normal rise after refeeding. These results provide in vivo support for the hypothesis that nSREBPs are essential for high levels of lipid synthesis in the liver and indicate that Insig's modulate nSREBP levels by binding and retaining SCAP in the ER.

Complete regression of xenografted human carcinomas by a paclitaxel-carboxymethyl dextran conjugate (AZ10992).

Clinically available taxanes, such as paclitaxel and docetaxel, represent one of the most promising classes of anticancer agents, despite their toxicity. To improve their pharmacological profiles, AZ10992 was synthesized based on the concept that a rational design of a polymer-drug conjugate would increase the efficacy of the parent drug. This prodrug is a paclitaxel-carboxymethyl dextran conjugate (molecular weight 150,000 g/mol) via a gly-gly-phe-gly linker. The in vivo antitumor study using AZ10992 against colon26 carcinoma cells, resistant to paclitaxel, supported this concept. Additionally, the comparative efficacy studies of AZ10992 and paclitaxel using a panel of human tumor xenografts in nude mice showed the advantages of drug-polymer conjugation. The maximum tolerated dose of AZ10992 was more than twice as high as the MTD of paclitaxel. A repeated intravenous administration of AZ10992 at 30 mg/kg/day (five injections for 4-days) showed complete regression of MX-1 mammary carcinoma xenografts. Also, HT-29 colorectal tumor xenografts, which are highly refractory to paclitaxel, showed complete regression after AZ10992 administered at 30 mg/kg/day (seven injections for 4-days). Pharmacokinetic studies showed that there were significant increases in the amount and the exposure time of total paclitaxel in the tumors after intravenous administration of AZ10992, which explains the enhanced efficacy of AZ10992.

Adipocyte-derived plasma protein adiponectin acts as a platelet-derived growth factor-BB-binding protein and regulates growth factor-induced common postreceptor signal in vascular smooth muscle cell.

BACKGROUND: Vascular smooth muscle cell proliferation plays an important role in the development of atherosclerosis. We previously reported that adiponectin, an adipocyte-specific plasma protein, accumulated in the human injured artery and suppressed endothelial inflammatory response as well as macrophage-to-foam cell transformation. The present study investigated the effects of adiponectin on proliferation and migration of human aortic smooth muscle cells (HASMCs). Methods and Results- HASMC proliferation was estimated by [(3)H] thymidine uptake and cell number. Cell migration assay was performed using a Boyden chamber. Physiological concentrations of adiponectin significantly suppressed both proliferation and migration of HASMCs stimulated with platelet-derived growth factor (PDGF)-BB. Adiponectin specifically bound to (125)I-PDGF-BB and significantly inhibited the association of (125)I-PDGF-BB with HASMCs, but no effects were observed on the binding of (125)I-PDGF-AA or (125)I-heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) to HASMCs. Adiponectin strongly and dose-dependently suppressed PDGF-BB-induced p42/44 extracellular signal-related kinase (ERK) phosphorylation and PDGF beta-receptor autophosphorylation analyzed by immunoblot. Adiponectin also reduced PDGF-AA-stimulated or HB-EGF-stimulated ERK phosphorylation in a dose-dependent manner without affecting autophosphorylation of PDGF alpha-receptor or EGF receptor. CONCLUSIONS: The adipocyte-derived plasma protein adiponectin strongly suppressed HASMC proliferation and migration through direct binding with PDGF-BB and generally inhibited growth factor-stimulated ERK signal in HASMCs, suggesting that adiponectin acts as a modulator for vascular remodeling.

Coordinated regulation of fat-specific and liver-specific glycerol channels, aquaporin adipose and aquaporin 9.

Plasma glycerol is a major substrate for hepatic gluconeogenesis. Aquaporin adipose (AQPap/7), an adipose-specific glycerol channel, provides fat-derived glycerol into plasma. In the present study, we cloned the coding and promoter regions of mouse aquaporin 9 (AQP9), a liver-specific glycerol channel. Fasting and refeeding of mice increased and decreased hepatic AQP9 mRNA levels, respectively. Insulin deficiency induced by streptozotocin resulted in increased hepatic AQP9 mRNA. These changes in hepatic AQP9 mRNA were accompanied by those of hepatic gluconeogenic mRNAs and plasma glycerol levels. In cultured hepatocytes, insulin downregulated AQP9 mRNA. The AQP9 promoter contained the negative insulin response element TGTTTTC at -496/-502, similar to the promoter of the AQPap/7 gene. In contrast, in insulin-resistant db+/db+ mice, AQPap/7 mRNA in fat and AQP9 mRNA in liver were increased, despite hyperinsulinemia, with high plasma glycerol and glucose levels. Glycerol infusion in the db+/db+ mice augmented hepatic glucose output. Our results indicate that coordinated regulations of fat-specific AQPap/7 and liver-specific AQP9 should be crucial to determine glucose metabolism in physiology and insulin resistance.

Androgens decrease plasma adiponectin, an insulin-sensitizing adipocyte-derived protein.

Adiponectin, an adipose-specific secretory protein, exhibits antidiabetic and antiatherogenic properties. In the present study, we examined the effects of sex hormones on the regulation of adiponectin production. Plasma adiponectin concentrations were significantly lower in 442 men (age, 52.6 +/- 11.9 years [mean +/- SD]) than in 137 women (53.2 +/- 12.0 years) but not different between pre- and postmenopausal women. In mice, ovariectomy did not alter plasma adiponectin levels. In contrast, high levels of plasma adiponectin were found in castrated mice. Testosterone treatment reduced plasma adiponectin concentration in both sham-operated and castrated mice. In 3T3-L1 adipocytes, testosterone reduced adiponectin secretion into the culture media, using pulse-chase study. Castration-induced increase in plasma adiponectin was associated with a significant improvement of insulin sensitivity. Our results indicate that androgens decrease plasma adiponectin and that androgen-induced hypoadiponectinemia may be related to the high risks of insulin resistance and atherosclerosis in men.

Regulation of branched-chain, and sulfur-containing amino acid metabolism by glutathione during ultradian metabolic oscillation of Saccharomyces cerevisiae.

Autonomous ultradian metabolic oscillation (T approximately or =50 min) was detected in an aerobic chemostat culture of Saccharomyces cerevisiae. A pulse injection of GSH (a reduced form of glutathione) into the culture induced a perturbation in metabolic oscillation, with respiratory inhibition caused by H2S burst production. As the production of H2S in the culture was controlled by different amino acids, we attempted to characterize the effects of GSH on amino acid metabolism, particularly with regard to branched chain and sulfur-containing amino acids. During stable metabolic oscillation, concentrations of intracellular glutamate, aspartate, threonine, valine, leucine, isoleucine, and cysteine were observed to oscillate with the same periods of dissolved O2 oscillation, although the oscillation amplitudes and maximal phases were shown to differ. The methionine concentration was stably maintained at 0.05 mM. When GSH (100 microM) was injected into the culture, cellular levels of branched chain amino acids increased dramatically with continuous H2S production, whereas the cysteine and methionine concentrations were noticeably reduced. These results indicate that GSH-dependent perturbation occurs as the result of the promotion of branched chain amino acid synthesis and an attenuation of cysteine and methionine synthesis, both of which activate the generation of H2S. In a low sulfate medium containing 2.5 mM sulfate, the GSH injections did not result in perturbations of dissolved O2, NAD(P)H redox oscillations without burst H2 production. This suggests that GSH-dependent perturbation is intimately linked with the metabolism of branched-chain amino acids and H2 generation, rather than with direct GSH-GSSG redox control.

Involvement of oxidative stress in the regulation of H(2)S production during ultradian metabolic oscillation of Saccharomyces cerevisiae.

Periodic evolution of H(2)S during aerobic chemostat culture of Saccharomyces cerevisiae resulted in ultradian metabolic oscillation via periodic inhibition of respiratory activity. To understand the nature of periodic H(2)S evolution, we investigated whether oxidative stress is associated with H(2)S production. The cellular oxidative states represented by intracellular level of lipid peroxides oscillated out of phase with the oscillation of dissolved O(2). Pulse addition of antioxidant, oxidative agent or inhibitor of antioxidation enzymes perturbed metabolic oscillation producing changes in H(2)S evolution. Analysis of H(2)S production profiles during perturbation of oscillation revealed that the amount of H(2)S production is closely linked with cellular oxidative states. Based on these results and our previous reports, we suggest that oxidative stresses result in periodic depletion of glutathione and cysteine, which in turn causes stimulation of the sulfate assimilation pathway and H(2)S production.

Naftopidil inhibits 5-hydroxytryptamine-induced bladder contraction in rats.

Naftopidil is an α(1D) and α(1A) subtype-selective α(1)-adrenoceptor antagonist that has been used to treat lower urinary tract symptoms of benign prostatic hyperplasia. In this study, we investigated the effects of naftopidil on 5-hydroxytryptamine (5-HT)-induced rat bladder contraction (10(-8)-10(-4) M). Naftopidil (0.3, 1, and 3 µM) inhibited 5-HT-induced bladder contraction in a concentration-dependent manner. On the other hand, other α(1)-adrenoceptor antagonists, tamsulosin, silodosin or prazosin, did not inhibit 5-HT-induced bladder contraction. The 5-HT-induced bladder contraction was inhibited by both ketanserin and 4-(4-fluoronaphthalen-1-yl)-6-propan-2-ylpyrimidin-2-amine (RS127445), serotonin 5-HT(2A) and 5-HT(2B) receptor antagonists, respectively. In addition, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and α-methyl-5-HT, 5-HT(2A) and 5-HT(2) receptor agonists, respectively, induced bladder contraction. The 5-HT-induced bladder contraction was not inhibited by N-[2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl]-N-pyridin-2-yl-cyclohexanecarboxamide (WAY-100635), [1-[2[(methylsulfonyl)amino]ethyl]-4-piperidinyl]methyl-1-methyl-1H-indole-3-carboxylate (GR113808) or (R)-3-[2-[2-(4-methylpiperidin-1-yl)ethyl]pyrrolidine-1-sulphonyl]phenol (SB269970), 5-HT(1A), 5-HT(4) and 5-HT(7) receptor antagonists, respectively. Naftopidil inhibited both the 5-HT(2A) and 5-HT(2) receptor agonists-induced bladder contractions. Naftopidil binds to the human 5-HT(2A) and 5-HT(2B) receptors with pKi values of 6.55 and 7.82, respectively. These results suggest that naftopidil inhibits 5-HT-induced bladder contraction via blockade of the 5-HT(2A) and 5-HT(2B) receptors in rats. Furthermore, 5-HT-induced bladder contraction was enhanced in bladder strips obtained from bladder outlet obstructed rats, with this contraction inhibited by naftopidil. The beneficial effects of naftopidil on storage symptoms such as urinary frequency and nocturia in patients with benign prostatic hyperplasia may be due, in part, to the blockade of the 5-HT(2A) and 5-HT(2B) receptors in the bladder.

Calcitonin ameliorates enhanced arterial contractility after chronic constriction injury of the sciatic nerve in rats.

In addition to its regulatory effect on bone mass, calcitonin has been shown to relieve pain and alleviate peripheral circulatory disturbance in patients with Raynaud's syndrome and complex regional pain syndrome. In the present study, we investigated whether calcitonin ameliorates diminished blood flow and enhanced arterial contraction in response to noradrenaline in chronic constriction injury (CCI) of the sciatic nerve in rats. Following surgically induced CCI, laser Doppler flowmetry studies showed a significant decrease in plantar skin blood flow of the ipsilateral hind paw compared to the contralateral side. A subcutaneous bolus injection of elcatonin (20 U/kg), a synthetic derivative of eel calcitonin, significantly improved decreased skin blood flow in the ipsilateral side. In vitro analysis of plantar arteries isolated from the ipsilateral hind paw 7-13 days after the CCI procedure showed higher sensitivity to noradrenaline than the plantar arteries from the contralateral side. Elcatonin (0.1-10 nm) significantly reduced noradrenaline-induced contraction in the arteries of the ipsilateral side, whereas it had little effect on those of the contralateral side. These results suggest that calcitonin selectively ameliorates enhanced arterial contractility in CCI neuropathic rats, thus leading to its alleviating effect on peripheral circulatory disturbance.

Correspondence between neurological deficit, cerebral infarct size, and Rho-kinase activity in a rat cerebral thrombosis model.

Whether Rho-kinase activity is really associated with the pathogenesis of cerebral infarction remains unclear. To consider this question, we investigated correspondences between severity of neurological deficit, infarct size, amount of various marker proteins, and Rho-kinase activity in a rat cerebral infarction model. Sodium laurate was injected into the left internal carotid artery, inducing cerebral infarction in the ipsilateral hemisphere in rats. We prepared rats with various severities of neurological deficit (mild to severe) 3 days after injection of laurate, then measured infarct size and amount of various marker proteins, phosphorylation of substrates of Rho-kinase, myosin-binding subunit (MBS), myosin light chain (MLC), ezrin/radixin/moesin (ERM), and adducin using Western blot methods. First, infarct size increased corresponding to the severity of neurological deficit. Second, amounts of activating transcription factor 3, nestin, CD68, proliferating cell nuclear antigen, and heat shock protein 70 were increased, whereas neurofilament and myelin-associated glycoprotein were decreased corresponding to the severity of neurological deficit and infarct size. Finally, Rho-kinase activity (phospho-MBS/MBS, phospho-MLC/MLC, phospho-ERM/ERM, and phospho-adducin/adducin) was increased corresponding to the severity of neurological deficit and infarct size. Rho-kinase thus appears to play a crucial role in the pathogenesis of cerebral infarction.

Carrier effects on antitumor activity and neurotoxicity of AZ10992, a paclitaxel-carboxymethyl dextran conjugate, in a mouse model.

AZ10992 is a novel paclitaxel-carboxymethyl (CM) dextran conjugate via a Gly-Gly-Phe-Gly linker with the molecular weight (MW) of 150 kDa. Our previous studies demonstrated that AZ10992 exerts strong antitumor activity against the human tumor xenografts that are highly refractory to paclitaxel, attributable to passive tumor targeting of released paclitaxel. This study examines the effects of carrier MW, anionic charge and drug-contents on the antitumor effects of AZ10992. To study antitumor effects, colon26 carcinoma-bearing BALB/c female mice received repeated (3 injections administered with 7 d intervals) intravenous administration of non-polymer-bound paclitaxel or paclitaxel-CM dextran conjugates. The results indicated that the conjugate comprising dextran T-110 (MW 110 kDa) with the degree of substitution (DS) value for the CM group of 0.50-0.55 per glucose residue and the drug contents of 5.5-6.5% (w/w) would be appropriate for AZ10992 regarding antitumor activity. Maximal tolerated dose (MTD) of AZ10992 was more than twice of non-polymer-bound paclitaxel. Furthermore, normal BALB/c female mice were treated with repeated (3 injections administered with 2 d intervals) intravenous administration of non-polymer-bound paclitaxel or AZ10992 at 50 mg/kg/d (based on the amount of paclitaxel to CM dextran) to study neurotoxicity. AZ10992 did not induce degeneration of myelin or swelling of Schwann cells in sciatic nerves.

The perinatal transition of the hepatobiliary cyst size provides information about the condition of bile flow in biliary cystic malformation cases.

PURPOSE: The aim of this study was to understand the level of bile flow by perinatal transitions of the hepatobiliary cyst size in biliary cystic malformation (BCM) cases and to examine the association between the transitions of the cyst size, cholangiographic images of the intrahepatic bile ducts (IHBDs), and histological features of the liver specimens in BCM. METHODS: We measured perinatal size of the hepatobiliary cyst in 7 BCM cases, composed of 1 case with choledochal cyst and 6 cases with biliary atresia (BA). Bile excretion was inferred from the transition of cyst size and the postoperative course. The IHBD cholangiographic images were also examined. In addition, histology of liver specimens obtained at the time of initial surgery was evaluated for IHBD maturity and fibrosis. RESULTS: The size of the small cyst in 2 BA cases remained almost unchanged throughout gestation. These cases required longer postoperative period for the clearance of jaundice. Their IHBD images were indistinct, and histology showed severe fibrosis. The ratio of bile ducts to portal tracts was 0.5 in 1 of the cases. However, in other BCM cases where the cysts grew large perinatally, bile excretion was good postoperatively, and their jaundice cleared in shorter period. Their cholangiographic images demonstrated clearer periphery of the IHBDs, and their histology showed mild or moderate fibrosis, if any. CONCLUSIONS: The perinatal transition of the cyst size in BCM cases can be helpful in assessing the level of bile flow. BCM with a small cyst during gestation can include some BAs with poorer bile flow, vaguer IHBD images, and prominent liver fibrosis. For the treatment of BCM cases, especially patients with a small biliary cyst, prompt response may be required after birth.