Semisolid Enteral Nutrients Alter the Pharmacokinetics of Orally Administered Levetiracetam in Rats.

Enteral nutrients (ENs) affect the plasma drug concentration of orally co-administered drugs, particularly those of antiepileptic drugs, such as phenytoin and carbamazepine. However, few studies have reported the interactions of levetiracetam (LEV), an upcoming antiepileptic drug, with ENs. In this study we aimed to investigate the pharmacokinetics of LEV in 55 rats after oral co-administration of LEV with liquid or semisolid ENs. Compared with the control group, co-administration with Terumeal ® Soft significantly decreased the plasma LEV concentration at 0.5, 1, and 2 h and area under the plasma concentration-time curve from 0 to 3 h (AUC0→3h) (P < 0.01). However, the AUC0→3h of LEV remained unchanged following the administration of Terumeal ® Soft 2 h after the initial LEV administration. Moreover, co-administration with semisolid Racol® NF delayed the absorption of LEV without decreasing the AUC0→3h, whereas liquid Racol ® NF did not alter LEV pharmacokinetics. Thus, co-administration of LEV with Terumeal® Soft reduced the absorption of LEV from the gastrointestinal tract, which was prevented by administering Terumeal ® Soft 2 h after LEV administration. Semisolid Racol ® NF altered LEV pharmacokinetics without decreasing its gastrointestinal absorption. Our findings suggested that careful monitoring of the plasma LEV levels is necessary when co-administering LEV with Terumeal ® Soft, semisolid Racol ® NF, or any other semisolid ENs, to prevent the inadvertent effects of the interaction between LEV and ENs.

Interaction between phenytoin and enteral nutrients and its influence on gastrointestinal absorption.

The gastrointestinal absorption of phenytoin (PHT), an antiepileptic drug, is often affected by its interaction with co-administered enteral nutrients through a nasogastric (NG) tube, resulting in decreased plasma PHT concentration. In this study, we measured the recovery rate (%) of PHT (Aleviatin® powder) passed through an NG tube when co-administered with distilled water or enteral nutrients (F2α®, Racol® NF, Ensure Liquid® and Renalen® LP). We also measured plasma PHT levels in rats, after oral co-administration of PHT with enteral nutrients. We demonstrate that PHT recovery rate was close to 100 % in all cases after passage through the NG tube. In the rat study, the AUC0→∞ of PHT concentration after oral administration significantly decreased when it was co-administered with F2α® and Racol® NF compared to distilled water. However, the AUC0→∞ of PHT was unchanged when co-administered with F2α® 2 h after initial PHT administration. We therefore conclude that the co-administration of PHT with F2α® and Racol® NF caused a reduction in the absorption of PHT from the gastrointestinal tract to the blood, without adsorption to the NG tube. The administration of enteral nutrients 2 h after PHT is one clear way to prevent a decrease in plasma PHT concentration.

Risk factors increasing blood pressure in Japanese colorectal cancer patients treated with bevacizumab.

Bevacizumab has been reported to increase blood pressure. However, the factors, including patient characteristics and laboratory data contributing to this side effect remain unclear. Therefore, we investigated the relationships between increased blood pressure and bevacizumab administration, patient characteristics, and laboratory data. Between April 2007 and January 2018, factor analysis was retrospectively conducted by monitoring increases in blood pressure, the status of bevacizumab administration, patient characteristics, and laboratory data before the first administration in Japanese patients with colorectal cancer who satisfied the criteria for this study. Sixty-seven patients were included, 34 of whom (50.7%) had an increase in blood pressure after bevacizumab administration. On univariate analysis, liver metastasis, antihypertensive drug use, systolic blood pressure at rest before the first bevacizumab administration, body mass index, creatinine, and blood platelet count were significantly different between the two groups. Multivariate analysis was conducted using increased blood pressure as an objective variable and the factors extracted by the univariate analysis as explanatory variables. The results suggested that liver metastasis, antihypertensive drugs, systolic blood pressure at rest before the first bevacizumab administration, and creatinine were associated with the increase in blood pressure. Furthermore, a log-rank test performed based on Kaplan-Meier curves demonstrated that liver metastasis in patients not taking antihypertensive drugs and antihypertensive drug use in patients without liver metastasis were significantly associated with increased blood pressure. Additionally, liver metastasis in patients with antihypertensive drug use was significantly associated with increased blood pressure. Our findings suggest that liver metastasis and antihypertensive drug use, which was previously reported, are risk factors for increased blood pressure.

Molecular species of prostaglandins involved in modulating luteinising hormone pulses of female rats under infectious stress conditions.

Mammalian reproductive function is controlled by the hypothalamic-pituitary-gonadal (HPG) axis, which is suppressed under infectious stress conditions. By analysing the pulsatility of luteinising hormone (LH), we have previously demonstrated that prostaglandins (PGs) in the central nervous system mediate infectious stress to suppress the activity of the HPG axis. The present study aimed to characterise the types of PGs responsible for suppression of the HPG axis. We focused on three major types of PGs: PGE2 , PGD2 and PGF2α . We used female rats overiectomised bilaterally 1 week before the experiments. Lipopolysaccharide (100 µg kg-1 ) suppressed LH pulses at the same time as enhancing the concentration of all three PGs in the cerebrospinal fluid, which was restored by indomethacin (10 mg kg-1 ). Subsequently, we observed LH pulsatility after a single injection of each PG and after co-injection of PGE2 with PGF2α into the third cerebral ventricle. A single injection of PGE2 dose-dependently induced a transient increase in mean LH concentration and LH pulse amplitude, and PGD2 significantly increased the amplitude of LH pulses, wereas PGF2α did not affect LH pulsatility. On the other hand, co-injection of PGE2 and PGF2α induced a significant suppression of both the frequency and amplitude of LH pulses. These results suggest that PGE2 and PGF2α can represent two of the mediators that suppress the HPG axis in situations of infectious stress. Moreover, the results imply that there are two contradictory effects of PGE2 on LH pulsatility: (i) enhancive when working alone and (ii) suppressive when working together with PGF2α .

Main metabolic pathways of TAK-802, a novel drug candidate for voiding dysfunction, in humans: the involvement of carbonyl reduction by 11ß-hydroxysteroid dehydrogenase 1.

To investigate species differences in the metabolism of TAK-802, in vitro and in vivo metabolic profiles were compared between humans and animals. TAK-802 was mainly metabolized to M-I, M-II, M-III and M-IV in human and animal liver microsomes. Especially the M-IV formation in humans was greater than that in animals. Likewise, M-IV was detected to a lower extent in the plasma and excreta of animals administered with TAK-802, whereas the AUC0-48 h of M-IV was approximately five-fold higher than that of TAK-802 in human plasma. These results indicate that the in vitro metabolic profile reflects the in vivo condition. Thus, to identify the metabolic pathway of TAK-802 in humans, the responsible enzyme to form M-IV was elucidated in vitro. Since M-IV is a reductive metabolite formed in microsomes, the possibility of involvement of 11ß-hydroxysteroid dehydrogenase (11ß-HSD), a carbonyl reductase located in microsomes, was first investigated. Consequently, M-IV formation was confirmed by incubation with human 11ß-HSD1-expressing microsomes and was concentration-dependently inhibited by glycyrrhetinic acid, an inhibitor for 11ß-HSD enzymes, indicating the involvement of 11ß-HSD1 in the M-IV formation. In contrast, little M-IV formation was observed using rat 11ß-HSD1, suggesting species differences between humans and rats. In addition, M-II was formed via M-IV, not via M-I and the CYP identification studies revealed that both M-I formation from TAK-802 and M-II formation from M-IV were mainly catalyzed by CYP3A4. In conclusion, 11ß-HSD1 and CYP3A4 are principally responsible for the metabolism of TAK-802 in humans and 11ß-HSD1 may be responsible for the observed species difference.

Increased lysosomal biogenesis in activated microglia and exacerbated neuronal damage after traumatic brain injury in progranulin-deficient mice.

Progranulin (PGRN) is known to play a role in the pathogenesis of neurodegenerative diseases. Recently, it has been demonstrated that patients with the homozygous mutation in the GRN gene present with neuronal ceroid lipofuscinosis, and there is growing evidence that PGRN is related to lysosomal function. In the present study, we investigated the possible role of PGRN in the lysosomes of activated microglia in the cerebral cortex after traumatic brain injury (TBI). We showed that the mouse GRN gene has two possible coordinated lysosomal expression and regulation (CLEAR) sequences that bind to transcription factor EB (TFEB), a master regulator of lysosomal genes. PGRN was colocalized with Lamp1, a lysosomal marker, and Lamp1-positive areas in GRN-deficient (KO) mice were significantly expanded compared with wild-type (WT) mice after TBI. Expression of all the lysosome-related genes examined in KO mice was significantly higher than that in WT mice. The number of activated microglia with TFEB localized to the nucleus was also significantly increased in KO as compared with WT mice. Since the TFEB translocation is regulated by the mammalian target of rapamycin complex 1 (mTORC1) activity in the lysosome, we compared ribosomal S6 kinase 1 (S6K1) phosphorylation that reflects mTORC1 activity. S6K1 phosphorylation in KO mice was significantly lower than that in WT mice. In addition, the number of nissl-positive and fluoro-jade B-positive cells around the injury was significantly decreased and increased, respectively, in KO as compared with WT mice. These results suggest that PGRN localized in the lysosome is involved in the activation of mTORC1, and its deficiency leads to increased TFEB nuclear translocation with a resultant increase in lysosomal biogenesis in activated microglia and exacerbated neuronal damage in the cerebral cortex after TBI.

Exacerbated inflammatory responses related to activated microglia after traumatic brain injury in progranulin-deficient mice.

Progranulin (PGRN), a multifunctional growth factor, appears to play a role in neurodegenerative diseases accompanied by neuroinflammation. In this study, we investigated the role of PGRN in neuroinflammation, especially in the activation of microglia, by means of experimental traumatic brain injury (TBI) in the cerebral cortex of mice. The expression of GRN mRNA was increased in association with neuroinflammation after TBI. Double-immunohistochemical study showed that PGRN-immunoreactive (-IR) cells were mainly overlapped with CD68-IR cells, suggesting that the main source of PGRN was CD68-positive activated microglia. To investigate the role of PGRN in inflammatory responses related to activated microglia, we compared the immunoreactivity and expression of ionized calcium-binding adaptor molecule 1 (Iba1), CD68, and CD11b as markers for activated microglia between wild-type (WT) and GRN-deficient (KO) mice. The number of Iba1- and CD11b-IR cells and gene expression of Iba1 and CD11b were not significantly different between WT and KO mice, while the number of CD68-IR cells and CD68 expression in KO mice were significantly greater than those in WT mice. Double-immunohistochemical study showed that CD68-IR microglia were also IR for TGFß1, and TGFß1 expression and Smad3 phosphorylation in KO mice were elevated compared to WT mice. Moreover, double-immunostaining between phospho-Smad3 and glial fibrillary acidic protein suggested increased TGFß1-Smad3 signal mainly by astrocytes. The levels of protein carbonyl groups, which reflect protein oxidation, and laminin immunoreactivity, which is associated with angiogenesis, were also significantly increased in KO mice compared to WT mice. These results suggest that PGRN is produced in CD68-positive microglia and suppresses excessive inflammatory responses related to activated microglia after TBI in mice.

Two-week cast immobilization induced chronic widespread hyperalgesia in rats.

It has been postulated that physical immobilization is an essential factor in developing chronic pain after trauma or surgery in an extremity. However, the mechanisms of sustained immobilization-induced chronic pain remain poorly understood. The present study, therefore, aimed to develop a rat model for chronic post-cast pain (CPCP) and to clarify the mechanism(s) underlying CPCP. To investigate the effects of cast immobilization on pain behaviours in rats, one hindlimb was immobilized for 2 weeks with a cast and remobilization was conducted for 10 weeks. Cast immobilization induced muscle atrophy and inflammatory changes in the immobilized hindlimb that began 2 h after cast removal and continued for 1 week. Spontaneous pain-related behaviours (licking and reduction in weight bearing) in the immobilized hindlimb were observed for 2 weeks, and widespread mechanical hyperalgesia in bilateral calves, hindpaws and tail all continued for 5-10 weeks after cast removal. A sciatic nerve block with lidocaine 24 h after cast removal transitorily abolished bilateral mechanical hyperalgesia in CPCP rats, suggesting that sensory inputs originating in the immobilized hindlimb contribute to the mechanism of both ipsilateral and contralateral hyperalgesia. Intraperitoneal injection of the free radical scavengers 4-hydroxy-2,2,6,6-tetramethylpiperydine-1-oxy1 or N-acetylcysteine 24 h after cast removal clearly inhibited mechanical hyperalgesia in bilateral calves and hindpaws in CPCP rats. These results suggest that cast immobilization induces ischaemia/reperfusion injury in the hindlimb and consequent production of oxygen free radicals, which may be involved in the mechanism of widespread hyperalgesia in CPCP rats.

Progranulin enhances neural progenitor cell proliferation through glycogen synthase kinase 3ß phosphorylation.

Progranulin (PGRN) is an estrogen-inducible growth factor thought to affect multiple processes in the CNS, including brain sexual differentiation, adult neurogenesis in the hippocampus, and development of neurodegenerative diseases. However, the precise physiological functions of PGRN in individual nerve cells are not fully understood. The aim of the present study was to enhance the understanding of PGRN function in the CNS by investigating the effects of PGRN on neural progenitor cells (NPCs). We found that significant amounts of endogenous PGRN were secreted from isolated NPCs in cultures. To assess the bioactivities of endogenous and exogenous PGRN, we studied NPCs derived from wild-type mice (WT-NPCs) and PGRN-deficient mice (KO-NPCs). We found that proliferation of KO-NPCs was significantly enhanced by PGRN treatment; however, PGRN treatment apparently did not affect proliferation of WT-NPCs perhaps because of the high levels of endogenous PGRN expression. NPC death and asymmetric cellular division of KO-NPCs and WT-NPCs, which results in production of neural stem cells, astrocytes, or oligodendrocytes, were not affected by PGRN treatment. We also investigated the signaling mechanism(s) that mediate PGRN-induced NPC proliferation and found that phosphorylation of serine 9 (S9) of glycogen synthase kinase 3-beta (GSK3ß), which was dependent on phosphatidylinositol 3-kinase (PI3K) activity, was induced by PGRN treatment. In addition, a GSK3ß-specific inhibitor enhanced NPC proliferation. Taken together, our observations indicate that PGRN enhanced NPC proliferation, at least in part, via inducing GSK3ß phosphorylation.

Hemodynamic studies of intracranial dural arteriovenous fistulas using arterial spin-labeling MR imaging.

Arterial spin-labeling (ASL) magnetic resonance imaging (MRI) enables non-invasive acquisition of the brain perfusion information in cerebrovascular disease. We investigated hemodynamic changes in intracranial dural arteriovenous fistulas (DAVFs) using ASL-MRI. ASL-MRI by a Q2TIPS sequence on a 3.0-Tesla MRI was performed for three patients with Cognard's IIa+b type of DAVFs before and after treatment. Perfusion images obtained by ASL-MRI (ASL images) before treatment were visually compared with those by single-photon emission computed tomography images (SPECT images). Increasing rates of temporal changes of regional perfusion values in ASL images (ASL values) before and after treatment were also calculated. In all three patients, ASL images before treatment demonstrated high perfusion in regions around the shunting areas, where normal or low perfusion were detected on SPECT images; thus, ASL images might have demonstrated the abundant arterial shunting flow via the fistulas. On days eight to 20 after treatment, ASL values around the shunt areas remained the same or decreased, and those in the regions other than the shunt areas increased in all three patients. This might have been due to a combination of the following: a decrease in shunt flow volume, an amelioration of venous congestion, and a sustained an upward shift in the autoregulation of the brain perfusion pressure. All regional ASL values decreased on days 112 and 120 after treatment in two patients, which possibly reflects a reduction in the upward shift in autoregulation. ASL-MRI might be useful for identifying the hemodynamic behavior of DAVFs before and after treatment.

Possible involvement of microglia containing cyclooxygenase-1 in the accumulation of gonadotrophin-releasing hormone in the preoptic area in female rats.

Prostaglandins (PGs), especially PGE(2), are involved in the hypothalamic control of gonadotrophin-releasing hormone (GnRH) release, acting at least in part on the terminal of GnRH axons in the median eminence. The present study aimed: (i) to clarify the role of PG(s) in regulating GnRH cell function at the level of the perikarya in the preoptic area; (ii) to determine the cyclooxygenase (COX) isozyme responsible for producing PG(s) that regulates GnRH perikarya; and (iii) to identify cell types that contain the responsible COX isozyme in female rats. A surge of luteinising hormone (LH) secretion was induced by oestrogen and progesterone in ovariectomised rats. Treatment of the rat before the LH surge with indomethacin, a nonselective COX inhibitor, or NS-398, a selective COX-2 inhibitor, did not interfere with the surge. However, treatment with indomethacin or flurbiprofen, a selective COX-1 inhibitor, significantly reduced the number of GnRH-immunoreactive cells in the preoptic area at the time of peak LH secretion during the surge. NS-398 did not affect the GnRH immunoreactivity. Double-labelled immunofluorescent histochemistry revealed COX-1 immunoreactivity in the vicinity of, but not within, GnRH containing neurones in the preoptic area. COX-2 immunoreactivity was not found in the same area. The COX-1 immunoreactivity was almost entirely localised in microglia in the preoptic area, but not in neurones or astrocytes. These results suggest that microglia in the preoptic area containing COX-1 are responsible for producing PG(s), which, in turn, facilitates the accumulation of GnRH during the gonadotrophin surge in female rats.

High expression of interleukin-1beta in the corneal epithelium of MRL/lpr mice is under the control of their genetic background.

MRL/Mp mice bearing the Fas deletion mutant gene, lpr (MRL/lpr), spontaneously develop polyarthritis, sialoadenitis and dacryoadenitis, resembling rheumatoid arthritis (RA), and also corneal involvement such as keratopathy and scleritis, which is a major complication in RA patients. In this study, we found that the expression levels of IL-1beta and MMP-1 mRNAs in cornea were high in both MRL/lpr and MRL/Mp-+/+ strains of mice at an age younger than when they develop any inflammatory lesions. This was not true of other inbred strains, even those bearing the lpr gene, and also not of (NZB x NZW) F1 lupus mice. There was no significant difference in the expression of IL-1alpha and TGFbeta in cornea in these strains. Using crosses between MRL/lpr and C3H/HeJ-lpr/lpr (C3H/lpr) mice, at least the expression of IL-1beta was found to be under the control of the MRL genetic background, likely with a recessive mode of inheritance. Considering that IL-1beta in cornea was detected particularly in the epithelial layer, the high expression of IL-1beta in cornea is most likely involved in the genetic predisposition for corneal involvement and possibly also for arthritis in an MRL strain of mice.

Correlation between spontaneous feeding behavior and neuropeptide Y profile in the third ventricular cerebrospinal fluid of goats.

Feeding behavior is regulated by neural signals in the hypothalamus, but secretory activities of these signals in vivo and their relationship with spontaneous feeding remain to be solved. In the present study, we investigated the correlation between neuropeptide Y (NPY) and somatostatin (SRIF) profiles in cerebrospinal fluid (CSF) and spontaneous feeding behavior in goats. CSF samples were collected every 15 min for 8 h from the third ventricle and feeding behavior was observed throughout the experimental period. The spontaneous feeding behavior, the mean duration of which was 58 min, occurred with an interval of 146 min. NPY in the CSF fluctuated in an episodic fashion with a 145 min interval. Each NPY episode was followed by spontaneous feeding with a time lag of 24 min. SRIF levels in CSF changed more frequently in a pulsatile manner and were related to neither NPY profiles nor feeding behavior. These results suggest that NPY, but not SRIF, is a physiological signal to drive feeding in goats.

Successful laparoscopic Ladd's procedure and appendectomy for intestinal malrotation with appendicitis.

We successfully performed a laparoscopic Ladd's procedure and an appendectomy in a 15-year-old girl with intestinal malrotation and appendicitis. She had tenderness and rebound pain in the umbilicus and left lower abdominal quadrant. Blood analysis revealed a moderate inflammatory response. Enhanced computerized tomography (CT) scanning revealed a whirl-like pattern and a superior mesentric vein (SMV) rotation sign in the mesentry of the small intestine. A swollen appendix was seen just below the umbilicus. An upper gastrointestinal (GI) radiological series confirmed agenesis of Treitz's arch. The patient was diagnosed as having a nonrotation type of malformation accompanied by acute appendicitis. She underwent a laparoscopic Ladd's procedure, an appendectomy, peritoneal lavage, and drainage. The technique for this procedure and its effectiveness are briefly discussed.

Production of 5,8,11-eicosatrienoic acid by a delta5 and delta6 desaturation activity-enhanced mutant derived from a delta12 desaturation activity-defective mutant of Mortierella alpina 1S-4.

Enhanced production of 5,8,11-eicosatrienoic acid (Mead acid, 20:3omega9) was attained with a mutant fungus, Mortierella alpina JT-180, derived from delta12 desaturation activity-defective and delta6 desaturation activity-enhanced M. alpina M209-7. Production of 20:3omega9 by JT-180 was 1.4 times greater than that of the parent strain M209-7. This is thought to be due to its enhanced Delta5 desaturation activity, which was 3.3 times higher than that of M209-7. In both strains, 78.5-80.4% of the total lipids comprised triacylglycerol (TG), and 76.6-79.0% of 20:3omega9 was present in TG. Comparing the fatty acid compositions among various lipid species, the highest percentages (24.1-37.6%) of 20:3omega9 in total lipids were found in phosphatidylcholine. For optimization of 20:3omega9 production by JT-180, a glucose concentration of 4% in the culture medium and shifting of the growth temperature from 28 degrees C to 20 degrees C on the 2nd day were shown to be effective. Under optimal conditions, 20:3omega9 production by JT-180 reached 1.92 g/l culture medium in a 10-l jar fermentor (corresponding to 81.5 mg/g dry mycelia and 18.3% of total fatty acids), which is greater than that reported previously from M209-7 (1.65 g/l).