[Conducting Assured Nonclinical Studies in the Pharmaceutical Industry: Good Laboratory Practice (GLP) Study, GLP Inspection, and Standards for Assurance].

Many nonclinical and clinical studies are conducted to submit new drug applications for chemical entities. Nonclinical studies cover pharmacology, pharmacokinetic, and toxicology aspects and provide pharmacologic evidence as well as kinetic and toxicologic profiles of the compounds. The risks and benefits of compounds are evaluated based on these nonclinical studies, especially before initiation of the first human trials. Therefore, using adequate procedures, highly controlled, quality nonclinical data should be obtained. This section shares and discusses items required of good laboratory practice (GLP)-compliant organizations and management systems in GLP facilities in the pharmaceutical industry as well as those required for GLP inspections by the Japanese Pharmaceuticals and Medical Devices Agency. In addition, it explains standard operating procedures for conducting quality, non-GLP, pharmacology, and pharmacokinetic studies.

Combination of circulating microRNAs as indicators of specific targets of retinal toxicity in rats.

Circulating miR-96-5p, -124-3p, and 183-5p have been reported as safety biomarkers for retinal toxicity. In the present research, 5 serum microRNAs (miRNAs), which are highly specific to and abundant in the retina, including the 3 miRNAs previously mentioned, were assessed in 3 different models of retinal toxicity. Distinct types of retinal lesions were induced in rats by a single dose of N-methyl-N-nitrosourea (MNU: 10, 30, and 50 mg/kg, i.p.), N-methyl-d-aspartate (NMDA: 200 nmol/eye, intravitreal injection), or sodium iodate (NaIO3: 30 mg/kg, i.v.). Time-course change of serum miRNAs was evaluated by RT-PCR for up to 1 week after administration. Ophthalmologic and histologic examinations and electroretinogram recording were also performed. MNU at 50 mg/kg induced photoreceptor cell death, with elevation in serum miR-96-5p, -124-3p, and -183-5p levels. NMDA induced retinal ganglion and inner nuclear layer cell death, with elevation in serum miR-124-3p. In both models, serum miRNA elevations occurred in parallel with the onset of neuroretinal cell death and retinal dysfunction. NaIO3 induced retinal pigment epithelial cell death without changes in neuroretinal cell or serum miRNAs. In the present research, circulating miR-124-3p was elevated in a case of retinal ganglion and inner nuclear layer cell death as well as photoreceptor cell death. Our data suggest that different patterns of circulating miRNA elevations correspond to death of a specific neuroretinal cell. A miRNA panel consisting of miR-96-5p, -124-3p, and -183-5p may be used as a biomarker to detect neuroretinal cell death and identify the specific target cell.

Brunner's gland lesions in rats induced by a vascular endothelial growth factor receptor inhibitor.

Vascular endothelial growth factor (VEGF) receptor tyrosine kinase (RTK) inhibitors are reported to cause reversible mucosal hyperplasia (adenosis) in the duodenum of rats; however, the pathogenesis is not fully elucidated. Using lenvatinib, a VEGF RTK inhibitor, we characterized the histologic time course of this duodenal change in rats. At 4 weeks, there was degeneration and necrosis of Brunner's gland epithelium accompanied by neutrophil infiltration around the affected glands. At 13 weeks, the inflammation was more extensive, and Brunner's gland epithelium was attenuated and flattened and was accompanied by reactive hyperplasia of duodenal epithelium. At 26 weeks, the changes became more severe and chronic and characterized by marked cystic dilation, which extended to the external muscular layer. These dilated glands exhibited morphological characteristics of duodenal crypt epithelium, suggestive of replacement of disappeared Brunner's glands by regenerative duodenal crypt epithelial cells. Similar changes were not present in similar time course studies in dog and monkey studies, suggesting that this is a rodent- or species-specific change. Based on the temporal progression of Brunner's gland lesion, we identify degeneration and necrosis of the Brunner's glands as the primary change leading to inflammation, cystic dilatation, and regeneration with cells that are morphologically suggestive of duodenal crypt epithelium.

E2012-induced cataract and its predictive biomarkers.

E2012, a gamma secretase modulator without affecting Notch processing, aimed at Alzheimer's disease by reduction of amyloid ß-42, induced cataract following repeated doses in the rat. Cataract appeared first at week 10-11 of treatment as a posterior subcapsular area with granular/punctate opaque or shiny dots along the suture line, characterized histologically as lenticular fiber degeneration, which eventually coalesced to form a triangular or circular opacity. It was associated with prolonged and sustained elevation of lenticular desmosterol (24-dehydrocholesterol), the final precursor of cholesterol, and decrease in lenticular cholesterol. In vitro studies to investigate the effect of E2012 on cholesterol metabolism demonstrated that E2012 inhibits 3ß-hydroxysterol Δ24-reductase (DHCR24) at the final step in the cholesterol biosynthesis. In vivo lenticular concentration of E2012 after 13-week repeated dose with cataract was well above those where inhibition was observed in vitro. There was no cataract formation at doses where desmosterol did not accumulate in the lens. The elevation of desmosterol and decreased cholesterol levels were also seen in the liver and plasma and preceded those in the lens. These results demonstrate that E2012 induces cataract in the rat by inhibiting DHCR24 at the final step of cholesterol synthesis with associated elevation in desmosterol within the lens, preceded by desmosterol changes that would serve as a predictive safety biomarker for lenticular opacity.

Elevated Bone Turnover in an Infantile Patient with Mucolipidosis II; No Association with Hyperparathyroidism.

This present report concerns an infantile patient with mucolipidosis II, who showed transient cortical bone hyperostosis followed by severe osteopenia. The diagnosis of mucolipidosis II was made based on the leakage of lysosomal enzymes in serum and conditioned media of the patient's skin fibroblasts, low activity of lysosomal enzymes of the fibroblasts and mutation of c.2086_2089insC (p.L697fs) and c.3565C>T (p.R1189X) in the GNPTAB gene. Bone X-ray analysis demonstrated a periosteal reaction and elevated bone resorption at the age of 2 mo. Bone markers, including alkaline phosphatase, osteocalcin and urine deoxypyridinoline, also indicated a high turnover of bone metabolism; however, no apparent rickets-like changes and no increased levels of PTH were observed. Elevated bone resorption is possibly associated with the leakage of lysosomal enzyme from osteoclasts into bone matrices. Bone formation gradually reduced, and increased bone resorption persisted. This led to severe osteopenia at the age of 6 mo. Characteristic bone findings may contribute to early diagnosis of mucolipidosis II, but their pathogenesis remains to be clarified.

Galantamine-induced amyloid-{beta} clearance mediated via stimulation of microglial nicotinic acetylcholine receptors.

Reduction of brain amyloid-ß (Aß) has been proposed as a therapeutic target for Alzheimer disease (AD), and microglial Aß phagocytosis is noted as an Aß clearance system in brains. Galantamine is an acetylcholinesterase inhibitor approved for symptomatic treatment of AD. Galantamine also acts as an allosterically potentiating ligand (APL) for nicotinic acetylcholine receptors (nAChRs). APL-binding site is located close to but distinct from that for acetylcholine on nAChRs, and FK1 antibody specifically binds to the APL-binding site without interfering with the acetylcholine-binding site. We found that in human AD brain, microglia accumulated on Aß deposits and expressed α7 nAChRs including the APL-binding site recognized with FK1 antibody. Treatment of rat microglia with galantamine significantly enhanced microglial Aß phagocytosis, and acetylcholine competitive antagonists as well as FK1 antibody inhibited the enhancement. Thus, the galantamine-enhanced microglial Aß phagocytosis required the combined actions of an acetylcholine competitive agonist and the APL for nAChRs. Indeed, depletion of choline, an acetylcholine-competitive α7 nAChR agonist, from the culture medium impeded the enhancement. Similarly, Ca(2+) depletion or inhibition of the calmodulin-dependent pathways for the actin reorganization abolished the enhancement. These results suggest that galantamine sensitizes microglial α7 nAChRs to choline and induces Ca(2+) influx into microglia. The Ca(2+)-induced intracellular signaling cascades may then stimulate Aß phagocytosis through the actin reorganization. We further demonstrated that galantamine treatment facilitated Aß clearance in brains of rodent AD models. In conclusion, we propose a further advantage of galantamine in clinical AD treatment and microglial nAChRs as a new therapeutic target.

A case of primary hyperparathyroidism in childhood found by a chance hematuria.

A 10-yr-old boy visited Minoh City Hospital complaining of gross hematuria. Laboratory investigations revealed hypercalcemia, hypophosphatemia, and elevated serum levels of parathyroid hormone. A stone was found in the right ureter with drip infusion pyelography. A parathyroid adenoma was successfully diagnosed with computed tomography, ultrasonography, and methoxy-2-isobutyl isonitrile (MIBI) scintigraphy. Multiple endocrine neoplasia was ruled out by normal results of endocrine laboratory examinations. Extracorporeal shock wave lithotripsy was performed to treat the urolithiasis, and the parathyroid adenoma was surgically removed. Primary hyperparathyroidism is rare in childhood; however, this case suggests that gross hematuria is an important sign of hyperparathyroidism.

[Suspected case of pseudohypoparathyroidism type II].

We experienced a suspected case of pseudohypoparathyroidism type II. The patient came to our emergency room with no thermal convulsion. The Ellsworth-Howard test was applied to the patient to determine the type of PHP.

Extremely sensitive biomarker of acute organophosphorus insecticide exposure.

Egasyn-beta-glucuronidase complex is located at the luminal site of liver microsomal endoplasmic reticulum. When organophosphorus insecticides (OP) are incorporated into the liver microsomes, they become tightly bound to egasyn, a carboxylesterase isozyme, and subsequently, beta-glucuronidase (BG) is dissociated and released into blood. Consequently, the increase in plasma BG activity becomes a good biomarker of OP exposure. Thus, the single administration of EPN (O-ethyl O-p-nitrophenylphenylphosphonothioate), acephate and chlorpyrifos increased plasma BG activity in approximately 100-fold the control level in rats. The increase in plasma BG activity after OP exposure is a much more sensitive biomarker of acute OP exposure than acetylcholinesterase (AChE) inhibition.

[Clinical efficacy of shortened ACTH therapy --an individualized method for minimization of adverse effects--Part 1. The short-term outcome].

To minimize adverse effects and to get good efficacy of ACTH therapy against West syndrome, we tried a new 2-steps therapeutic protocol consisting of the shortened ACTH therapy and the additional ACTH therapy. In a prospective multi-institutional study, 20 patients with newly diagnosed West syndrome who had failed to respond to high-dose vitamin B6 and zonisamide were treated by this shortened ACTH therapy. Synthetic corticotropin (ACTH-Z 0.025 mg/kg/dose, max 0.25 mg) was administrated intramuscularly seven times on every other day for 14 days. At 1 month after discontinuing corticotropin, spasms and hypsarrhythmia disappeared in 10/20 (50%) and 13/17 (59%) patients respectively. Subsequently, 9 out of the 10 patients with persistent spasms received additional therapy for 1 or 2 weeks with daily intramuscular ACTH-Z, which was tapered off over a few weeks. Including the additional ACTH therapy, the disappearance of spasms and hypsarrhythmia were found in 13 patients (65%) and 13 patients (76%). Adverse effects during the shortened ACTH therapy were fewer than additional ACTH therapy but not statistically significant. Severe adverse effects were not observed in both ACTH therapy. In the 2-steps therapeutic protocol according to the response to ACTH, favorable results were obtained in seizure control, EEG findings and the degree of adverse effects.

A patient with membranoproliferative glomerulonephritis diagnosed by the third biopsy via endocapillary proliferative glomerulonephritis and focal membranoproliferative glomerulonephritis.

We present a girl with type I membranoproliferative glomerulonephritis (MPGN) diagnosed by the third renal biopsy. The first renal biopsy was performed at age 11.2 years after microscopic hematuria (which was revealed by school urinary screening) had persisted for 3 months, along with a low level of serum C3. Pathological examination of the biopsied specimen revealed endocapillary proliferative glomerulonephritis with multiple humps. The serum C3 level increased to within the normal range 2 months after the first renal biopsy, and the microscopic hematuria disappeared at age 12.3. However, microscopic hematuria, proteinuria, and the low serum complement level reappeared at age 12.8. Pathological examination of a further renal biopsy that was performed at age 13.2 revealed focal MPGN with humps. Prednisolone therapy was subsequently initiated. Fluvastatin was added to her treatment regime when she developed hypercholesterolemia at age 13.6 and was continued even after normal cholesterol levels were reestablished. Pathological examination of the third renal biopsy, which was performed at age 15.2, revealed type I MPGN with humps. Serum C3 normalized 6 months after the cessation of prednisolone at age 15.9. It is clinically important that patients with nontypical acute glomerulonephritis should be observed over a long period and repeated renal biopsies should be performed.

Successful resumption of trimethoprim-sulfamethoxazole after oral desensitisation in patients with chronic granulomatous disease.

Two patients with chronic granulomatous disease who had previously been intolerant to trimethoprim-sulfamethoxazole because of various adverse reactions completed a desensitisation protocol with a favourable clinical outcome.