PKN1 kinase-negative knock-in mice develop splenomegaly and leukopenia at advanced age without obvious autoimmune-like phenotypes.

Protein kinase N1 (PKN1) knockout (KO) mice spontaneously form germinal centers (GCs) and develop an autoimmune-like disease with age. Here, we investigated the function of PKN1 kinase activity in vivo using aged mice deficient in kinase activity resulting from the introduction of a point mutation (T778A) in the activation loop of the enzyme. PKN1[T778A] mice reached adulthood without external abnormalities; however, the average spleen size and weight of aged PKN1[T778A] mice increased significantly compared to aged wild type (WT) mice. Histologic examination and Southern blot analyses of spleens showed extramedullary hematopoiesis and/or lymphomagenesis in some cases, although without significantly different incidences between PKN1[T778A] and WT mice. Additionally, flow cytometry revealed increased numbers in B220+, CD3+, Gr1+ and CD193+ leukocytes in the spleen of aged PKN1[T778A] mice, whereas the number of lymphocytes, neutrophils, eosinophils, and monocytes was reduced in the peripheral blood, suggesting an advanced impairment of leukocyte trafficking with age. Moreover, aged PKN1[T778A] mice showed no obvious GC formation nor autoimmune-like phenotypes, such as glomerulonephritis or increased anti-dsDNA antibody titer, in peripheral blood. Our results showing phenotypic differences between aged Pkn1-KO and PKN1[T778A] mice may provide insight into the importance of PKN1-specific kinase-independent functions in vivo.

Retention and molecular-recognition mechanisms of molecularly imprinted polymers for promazine derivatives.

Monodisperse molecularly imprinted polymers (MIPs) for promazine derivatives [promazine (PZ), methylpromazine (MPZ), chlorpromazine (CPZ) and bromopromazine (BPZ)], MIPPZ, MIPMPZ, MIPCPZ and MIPBPZ, were prepared using methacrylic acid (MAA) as a functional monomer and ethylene glycol dimethacrylate as a crosslinker by multi-step swelling and polymerization. The retention and molecular-recognition properties of the obtained MIPs were evaluated using LC in hydrophilic interaction chromatography (HILIC) and reversed-phase modes. In computational approaches, intermolecular interaction modes and energies between PZ derivatives and MAAs were evaluated at the HF/6-311G(d,p) level. The interaction energies of PZ, MPZ, CPZ and BPZ with 4 equivalents of MAAs were calculated. The results indicated that the interaction of the aliphatic amine moiety of a PZ derivative with MAA gave almost similar interaction energies at the HF/6-311G(d,p) level, and that the interaction of the sulfur atom of a phenothiazine scaffold with MAA was also the case. The third interaction of the aromatic amine of a PZ derivative with MAA was in the order of MPZ > PZ > CPZ > BPZ presumably due to the change of basicity by the electron-donating or electron-withdrawing effect of a subsituent. Furthermore, the fourth attractive modes of CPZ and BPZ were suggested to be the interaction of their halogen atoms with MAA through both halogen bonding and hydrogen bonding, while PZ and MPZ were suggested to have the weak C-H ⋅⋅⋅ π interaction with MAA. In HILIC mode, the interaction energies at the HF method had good correlation with the retention factor of a PZ derivative on each MIP, indicating that in addition to the shape recognition, the attractive electrostatic interactions would be more responsible for its retention rather than the dispersion energies. Furthermore, in addition to the shape recognition, ionic and hydrophobic interactions, and halogen bonding and hydrogen bonding (the last interaction seems to be weak) seem to work for the retention and molecular-recognition of PZ derivatives on the MIPs in reversed-phase mode.

Evaluation of molecularly imprinted polymers for chlorpromazine and bromopromazine prepared by multi-step swelling and polymerization method-The application for the determination of chlorpromazine and its metabolites in rat plasma by column-switching LC.

Monodisperse molecularly imprinted polymers (MIPs) for chlorpromazine (CPZ) and bromopromazine (BPZ), MIPCPZ and MIPBPZ, were prepared using methacrylic acid as a functional monomer and ethylene glycol dimethacrylate as a crosslinker by multi-step swelling and polymerization. The retention and molecular-recognition properties of MIPCPZ and MIPBPZ were evaluated using a mixture of potassium phosphate buffer and acetonitrile or a mixture of water and acetonitrile including ammonium formate as a mobile phase in reversed-phase LC. On MIPBPZ, CPZ, BPZ and imipramine (IMP) gave the maximal retention factors at a mobile-phase pH 8, while the maximal imprinting factors were obtained at a mobile-phase pH 7. Each MIP recognized a template molecule the most, while CPZ metabolites, desmethyl CPZ (DM-CPZ), CPZ sulfoxide (CPZ-SO) and 7-hydroxy CPZ (7-OH-CPZ), were moderately recognized on MIPCPZ and MIPBPZ. Furthermore, both MIPs gave the similar retention and molecular-recognition for CPZ and its metabolites. For avoiding the template-leakage problems, MIPBPZ was used as the pretreatment column for the determination of CPZ and its metabolites in rat plasma in column-switching LC with UV detection. In addition to DM-CPZ and CPZ-SO, didesmethyl CPZ (DDM-CPZ) and CPZ N-oxide (CPZ-NO) were speculated as the metabolite in rat plasma after administration of CPZ using LC-ESI-TOF-MS, while 7-OH-CPZ was not detected. The column-switching LC method was validated and applied for the determination of CPZ and its metabolites, DM-CPZ, DDM-CPZ, CPZ-SO and CPZ-NO, in rat plasma after intravenous and oral administration of CPZ using IMP as an internal standard.

Preparation and Evaluation of Molecularly Imprinted Polymers for Promazine and Chlorpromazine by Multi-step Swelling and Polymerization: the Application for the Determination of Promazine in Rat Serum by Column-switching LC.

Molecularly imprinted polymers (MIPs) for promazine (PZ) and chlorpromazine (CPZ), MIPPZ and MIPCPZ, were prepared by multi-step swelling and polymerization using methacrylic acid as a functional monomer and ethylene glycol dimethacrylate as a crosslinker. The retention and molecular-recognition properties of MIPPZ and MIPCPZ were evaluated using a mixture of potassium phosphate buffer and acetonitrile, or a mixture of ammonium formate and acetonitrile as the mobile phase in LC. PZ and CPZ gave the maximal retentions on MIPPZ and MIPCPZ at an apparent pH 8.2 using a mixture of potassium phosphate buffer and acetonitrile as the mobile phase. The retentions of PZ and CPZ decreased with an increase of acetonitrile contents from 70 to 90 vol% using a mixture of ammonium formate and acetonitrile as the mobile phase. The template molecules (PZ and CPZ, respectively) were recognized the most on the respective MIPs, and the imprinting factor of PZ was higher on MIPCPZ than on MIPPZ. These results indicate that in addition to shape recognition, ionic and hydrophobic interactions seem to work for the retention and molecular-recognition of PZ and CPZ on the MIPs. MIPCPZ was successfully utilized for the selective extraction of PZ in rat-serum samples in column-switching LC with fluorescence detection.

[Questionnaire Survey to Pharmacists after a Workshop on the Chemical Structural Formula of Drugs].

Recently, it has been reported that only a small number of sixth-year students who had undergone a long-term pre-clinical training in the fifth year found organic chemistry useful. To explain this, we hypothesized that pharmacists are unable to utilize the knowledge of organic chemistry to solve clinical problems. With the aim of addressing this problem, we conducted a workshop consisting of a series of lectures and exercises on structural similarity, solubility, absorption, and metabolism of drugs based on a chemical structural formula. Then, we administered a questionnaire survey to 253 participants who had participated in our workshop. The questionnaire comprised 17 questions, and free descriptions were analyzed using text mining. Results showed that, although about 45% of the participants confirmed the chemical structural formula described in the medical package insert, and about 22% of the participants had the opportunity to check the metabolites described in the drug interview form, more than 90% of the participants were interested in the workshop contents. Thus, pharmacists may want to learn how the process of utilizing the chemical structural formula can be applied to their clinical practice.

Effects of Reinforcement Method of Dissection Physiology Education on the Achievement in Pharmacology.

The Pharmaceutical Education Support Center was established in the Department of Pharmacy at the School of Pharmacy and Pharmaceutical Science of Mukogawa Women's University in 2014. We started teaching first and second years students according to proficiency from the 2014 academic year. Students were divided into two classes: the regular class (high proficiency class) and the basic class (low proficiency class), based on achievement in several basic subjects related to the study of pharmacy. The staffs in the Pharmaceutical Education Support Center reinforce what is taught to students in the basic class. In this reinforcement method of education, the class size is small, consisting of about 15 students, a quiz to review the previous lesson is given at the beginning of each lecture, and an additional five lectures are conducted, compared to the high proficiency class, which receives 15 lectures. In this study, we evaluated the effects of the reinforcement method of physiology education on achievement in pharmacology that was not conducted in the proficiency-dependent teaching method. The students in the basic class in physiology education were chosen based on achievement levels in anatomy. Achievement levels of pharmacology students in the basic class of physiology improved compared with those of students who had the same achievement levels in physiology but were not taught according to proficiency-dependent teaching in the 2013 academic year. These results suggest that the reinforcement method for education in basic subjects in pharmacy, such as physiology, can improve achievement in more advanced subjects, such as pharmacology.

A prescription survey about combined use of acetylcholinesterase inhibitors and anticholinergic medicines in the dementia outpatient using electronic medication history data from community pharmacies.

PURPOSE: We investigated prescriptions regarding the combined use of donepezil hydrochloride (DPZ) and anticholinergics for elderly outpatients in Japan to determine the impact that combination therapy has on decreasing their cognitive functions. METHODS: Using electronic medication records from 142 community pharmacies, outpatients older than 40 years of age taking DPZ, with or without other prescription medicines, were assessed over 6 years, beginning in 2007. We estimated the number of medicines administered along with DPZ, the number of anticholinergics administered along with DPZ, and the medicines' anticholinergic cognitive burden (ACB) scale cumulative score based on data from the top four pharmacies that filled the highest number of prescriptions for DPZ for outpatients with dementia in 2010. Data were gathered from records of 431 patients; only three patients were younger than 60 years. RESULTS: There was a 1.94-fold increase in the number of prescriptions including DPZ over 6 years. The proportion of patients to whom other medicines were administered along with DPZ was 65.6% (n=283) and the proportion of those taking at least one anticholinergic agent was 24.1% (n=104). The mean number of medicines among subjects taking at least one anticholinergic was 5.7, and the mean cumulative ACB score for anticholinergics contained in these medicines was 2.6. Among 104 patients to whom the anticholinergics were administered along with DPZ, two outpatients taking urologic medicines such as oxybutynin hydrochloride or tolterodine tartrate were found. CONCLUSION: Our findings suggest that it is necessary to pay attention to a decline in cognitive function when prescribing multiple medicines, especially to elderly patients who have already been prescribed DPZ.

Pioglitazone prevents reactive hypoglycemia in impaired glucose tolerance.

A 42-year-old woman with hypoglycemic symptoms that occurred several hours after a meal visited our hospital. The hypoglycemic symptoms appeared when she was 37 years old, and her plasma glucose level had been assessed as less than 60 mg/dL when she experienced the symptoms. One year before, she had been diagnosed with reactive hypoglycemia by 75 g-oral glucose tolerance test (OGTT), which showed a normal glucose tolerance (NGT) pattern, and had begun taking an alpha-glucosidase inhibitor and nutritional treatment. A 75 g-OGTT on admission showed hypoglycemia at 240 min after glucose loading, excessive insulin secretion and an impaired glucose tolerance (IGT) pattern. A euglycemic-hyperinsulinemic clamp study demonstrated decreased insulin sensitivity. Therefore, we suspected that she had reactive hypoglycemia associated with insulin resistance and treated her with 15 mg/day pioglitazone. Her hypoglycemic symptoms completely disappeared after treatment with pioglitazone; insulin sensitivity in a euglycemic-hyperinsulinemic clamp study improved. Another 75 g-OGTT revealed that the excessive insulin secretion and hypoglycemia at 240 min after glucose loading had disappeared, and glucose tolerance was normalized from an IGT pattern to an NGT pattern. Thus, we believe that pioglitazone is effective for reactive hypoglycemia and aggravated glycemic metabolism associated with insulin resistance.

Expression of type 1 corticotropin-releasing hormone (CRH) receptor mRNA in the hypothalamic paraventricular nucleus following restraint stress in CRH-deficient mice.

Previous studies have demonstrated that various types of stress increase type 1 corticotropin-releasing hormone (CRH) receptor (currently abbreviated to CRF1 receptor) mRNA in the hypothalamic paraventricular nucleus (PVN) of rats, but not mice. This study investigated whether different sensitivities of glucocorticoid-mediated negative feedback effects can explain this species difference in stress-induced PVN CRF1 receptor mRNA expression. First, the CRF1 receptor mRNA level in the PVN of CRH knockout (KO) mice during acute restraint stress was compared with that in wild-type (WT) mice. Consistent with previous findings, WT mice showed no induction of CRF1 receptor mRNA in the PVN following acute restraint, regardless of normal hypothalamic-pituitary-adrenocortical responses. In contrast, CRF1 receptor mRNA in the PVN of CRH KO mice was increased following 2 h of restraint. Since the response of tyrosine hydroxylase (TH) mRNA in the locus coeruleus (LC) to restraint was similar between CRH KO and WT mice, it is unlikely that enhanced noradrenergic input into the PVN was responsible for the CRF1 receptor mRNA induction in CRH KO mice. Second, to determine whether CRH KO per se or a low corticosterone response to stress is required to induce CRF1 receptor mRNA expression in the PVN in mice, the response of adrenalectomized WT mice was examined. Acute restraint increased the CRF1 receptor mRNA level in the PVN of adrenalectomized WT mice, similar to the case for CRH KO mice. TH mRNA in the LC showed similar increases in sham and adrenalectomized WT mice. These results indicate that PVN CRF1 receptor mRNA is much more sensitive to glucocorticoid-mediated negative feedback in mice than in rats, such that a normal increase in plasma corticosterone during stress can mask CRF1 receptor mRNA induction in the PVN of mice.

A case of anterior hypopituitarism showing recurrent pituitary mass associated with central diabetes insipidus.

We report a case of anterior hypopituitarism showing recurrent pituitary mass associated with central diabetes insipidus. A 76-year old woman was hospitalized with general fatigue and 5 kg body weight loss. Endocrinological examinations and pituitary provocative tests demonstrated hypopituitarism and central diabetes insipidus. T1-weighted image of magnetic resonance imaging (MRI) revealed an intrasellar cystic mass with ring enhancement suggesting pituitary abscess. MRI films subsequently obtained from another hospital and studied retrospectively showed intrasellar cystic mass with ring enhancement 4 years earlier, and a mass shape that was decreased after 2 years. Over the subsequent years, the patient has remained asymptomatic with hormone replacement therapy only. Cystic pituitary adenoma or Rathke's cleft cyst with repeated infection may be involved in the repeated change of pituitary mass shape although neither pituitary surgery nor a pituitary biopsy was performed because of the patient's age and condition. It is reported that apparent recurrence of Rathke's cleft cysts after initially successful surgery was higher than suggested by previous reports, and that long-term follow-up with pituitary imaging and neuroophthalmological assessment is essential. Careful evaluation by follow-up brain MRI is needed in the present case to prevent future recurrence of pituitary abscess.