Pretreatment of rhesus monkeys with transdermal patches containing physostigmine and procyclidine: implications of the delivery system for the potential application against VX nerve agent intoxication in humans.

Physostigmine (Phs) is a reversible inhibitor of acetylcholinesterase (AChE) that penetrates the blood-brain barrier (BBB) and could be used to protect the central nervous system (CNS) against the effects of nerve agents. For prophylactic effectiveness, long, steady, and adequate inhibition of AChE activity by Phs is needed to broadly protect against the CNS effects of nerve agents. Here, we evaluated the efficacy of transdermal patches containing Phs and procyclidine (PC) as prophylactic agents. Patches (25 cm2) containing 4.4 mg Phs and 17.8 mg PC had a protective ratio of approximately 78.6-fold in rhesus monkeys challenged with VX nerve agent and given an antidote. Physiologically based pharmacokinetic model in conjunction with an indirect pharmacodynamic (PBPK/PD) was developed for Phs and scaled to rhesus monkeys. The model was able to reproduce the concentration profile and inhibitory effect on AChE of Phs in monkeys, as evidenced by correlation coefficients of 0.994 and 0.992 for 25 cm2 and 49 cm2 patches, respectively (i.e., kinetic data), and 0.989 and 0.968 for 25 cm2 and 49 cm2 patches, respectively (i.e., dynamic data). By extending the monkey PBPK/ PD model to humans, the effective human dose was predicted to be five applications of a 25 cm2 patch (i.e., 22 mg Phs), and two applications of a 49 cm2 patch (i.e., 17.4 mg Phs). Therefore, given that patch application of Phs in rhesus monkeys has a prolonged effect (namely, AChE inhibition of 19.6% for the 25 cm2 patch and 23.0% for the 49 cm2 patch) for up to 216 h, patch formulation of Phs may provide similar protection against nerve agent intoxication in humans.

Therapeutic Efficacy of ABN401, a Highly Potent and Selective MET Inhibitor, Based on Diagnostic Biomarker Test in MET-Addicted Cancer.

The receptor tyrosine kinase c-MET regulates processes essential for tissue remodeling and mammalian development. The dysregulation of c-MET signaling plays a role in tumorigenesis. The aberrant activation of c-MET, such as that caused by gene amplification or mutations, is associated with many cancers. c-MET is therefore an attractive therapeutic target, and inhibitors are being tested in clinical trials. However, inappropriate patient selection criteria, such as low amplification or expression level cut-off values, have led to the failure of clinical trials. To include patients who respond to MET inhibitors, the selection criteria must include MET oncogenic addiction. Here, the efficacy of ABN401, a MET inhibitor, was investigated using histopathologic and genetic analyses in MET-addicted cancer cell lines and xenograft models. ABN401 was highly selective for 571 kinases, and it inhibited c-MET activity and its downstream signaling pathway. We performed pharmacokinetic profiling of ABN401 and defined the dose and treatment duration of ABN401 required to inhibit c-MET phosphorylation in xenograft models. The results show that the efficacy of ABN401 is associated with MET status and they highlight the importance of determining the cut-off values. The results suggest that clinical trials need to establish the characteristics of each sample and their correlations with the efficacy of MET inhibitors.

New Preclinical Development of a c-Met Inhibitor and Its Combined Anti-Tumor Effect in c-Met-Amplified NSCLC.

c-Met is a receptor tyrosine kinase with no commercially available product despite being a pivotal target in cancer progression. Unlike other c-Met inhibitors that fail clinically, ABN401 is a newly synthesized c-Met inhibitor that is not potentially degraded by aldehyde oxidase (AO) in human liver cytosol. This study aimed to determine the physicochemical stability, pharmacokinetics in beagle dogs, and therapeutic effect of ABN401 in a c-Met-amplified non-small-cell lung cancer (NSCLC) patient-derived xenograft (PDX) model. ABN401 was found to be a weak basic compound, with pKa and log P values of 7.49 and 2.46, respectively. It is poorly water-soluble but soluble at acidic pH. The accelerated storage stability is dependent on temperature, but the purity remains at over 97% after 6 months. The bioavailability is approximately 30% in dogs and it is highly efficient in the PDX model, achieving around 90% tumor growth inhibition in combination with erlotinib. These observations indicate that the compound is acceptable for the next phase of trials.

Development of an Equine Antitoxin by Immunizing the Halla Horse with the Receptor-Binding Domain of Botulinum Neurotoxin Type A1.

Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are the most toxic substances known. However, the number of currently approved medical countermeasures for these toxins is very limited. Therefore, studies on therapeutic antitoxins are essential to prepare for toxin-related emergencies. Currently, more than 10,000 Halla horses, a crossbreed between the native Jeju and Thoroughbred horses, are being raised in Jeju Island of Korea. They can be used for equine antitoxin experiments and production of hyperimmune serum against BoNT/A1. Instead of the inactivated BoNT/A1 toxoid, Halla horse was immunized with the receptor-binding domain present in the C-terminus of heavy chain of BoNT/A1 (BoNT/A1-HCR) expressed in Escherichia coli. The anti-BoNT/A1-HCR antibody titer increased rapidly by week 4, and this level was maintained for several weeks after boosting immunization. Notably, 20 µL of the week 24 BoNT/A1-HCR(-immunized) equine serum showed an in vitro neutralizing activity of over 8 international unit (IU) of a reference equine antitoxin. Furthermore, 20 µL of equine serum and 100 µg of purified equine F(ab')2 showed 100% neutralization of 10,000 LD50 in vivo. The results of this study shall contribute towards optimizing antitoxin production for BoNT/A1, which is essential for emergency preparedness and response.

Early gonadotropin-releasing hormone antagonist protocol in women with polycystic ovary syndrome: A preliminary randomized trial.

OBJECTIVE: To prospectively evaluate the efficacy and safety of a fixed early gonadotropin-releasing hormone (GnRH) antagonist protocol compared to a conventional midfollicular GnRH antagonist protocol and a long GnRH agonist protocol for in vitro fertilization (IVF) in patients with polycystic ovary syndrome (PCOS). METHODS: Randomized patients in all three groups (early antagonist, n=14; conventional antagonist, n=11; long agonist, n=11) received 21 days of oral contraceptive pill treatment prior to stimulation. The GnRH antagonist was initiated on the 1st day of stimulation in the early antagonist group and on the 6th day in the conventional antagonist group. The GnRH agonist was initiated on the 18th day of the preceding cycle. The primary endpoint was the number of oocytes retrieved, and the secondary endpoints included the rate of moderate-to-severe ovarian hyperstimulation syndrome (OHSS) and the clinical pregnancy rate. RESULTS: The median total number of oocytes was similar among the three groups (early, 16; conventional, 12; agonist, 19; p=0.111). The early GnRH antagonist protocol showed statistically non-significant associations with a higher clinical pregnancy rate (early, 50.0%; conventional, 11.1%; agonist, 22.2%; p=0.180) and lower incidence of moderate-to-severe OHSS (early, 7.7%; conventional, 18.2%; agonist, 27.3%; p=0.463), especially among subjects at high risk for OHSS (early, 12.5%; conventional, 40.0%; agonist, 50.0%; p=0.324). CONCLUSION: In PCOS patients undergoing IVF, early administration of a GnRH antagonist may possibly lead to benefits due to a reduced incidence of moderate-to-severe OHSS in high-risk subjects with a better clinical pregnancy rate per embryo transfer. Further studies with more subjects are required.

Effects of Estradiol on the Paracrine Regulator Expression of In Vitro Maturated Murine Ovarian Follicles.

The preservation of female germ cells is important in the individuals with ovarian dysfunction and failure. For this purpose, ovarian follicle in vitro maturation (OFIVM) is an important technology for the retrieval of mature oocytes. In the in vivo follicular development, paracrine factors such as angiotensin (AT) and anti-Müllerian hormone (AMH) play important roles. We attempted to add estrogen during the OFIVM and to assess their expression on the follicular cells. The ovaries and pre-antral follicles were collected from 13-day C57BL/6 mice and cultured in vitro with estradiol (E2) treatment for up to two weeks. In the whole ovaries, the expression of AT II was decreased and the expression of AMH was similar between control and E2-treated ovaries after in vitro culture. Although there was no difference in the survival, ovulation, maturation and fertilization rates between control and E2-treated groups, the expression of AT II in the follicular cells was down-regulated after E2 treatment at mRNA level, and AMH showed similar expression. In conclusion, adding E2 in OFIVM may regulate paracrine factors and their receptors that are related to follicular development. Further investigations are necessary to elucidate the roles of various sex hormones in the regulation of AT and AMH expression during the OFIVM.

Novel helical assembly in arginine methyltransferase 8.

Protein arginine methyltransferase 8 (PRMT8) is unique among PRMTs, as it is specifically expressed in brain and localized to the plasma membrane via N-terminal myristoylation. Here, we describe the crystal structure of human PRMT8 (hPRMT8) at 3.0-Å resolution. The crystal structure of hPRMT8 exhibited a novel helical assembly. Biochemical, biophysical and mutagenesis experiments demonstrated that hPRMT8 forms an octamer in solution. This octameric structure is necessary for proper localization to the plasma membrane and efficient methyltransferase activity. The helical assembly might be a relevant quaternary form for hPRMT1, which is the predominant PRMT in mammalian cells and most closely related to hPRMT8.

The expression profile of angiotensin system on thawed murine ovaries.

Freezing and thawing is one of the most widely used tissue engineering techniques for the preservation of ovaries. Many cells and tissues demonstrate changes in functional gene expression after thawing. Several studies have reported the important roles of angiotensin (AT) system during the ovarian follicular growth. AT system consists of ATII, and ATII receptors type I (ATII-RI) and type II (ATII-RII). However, little is known whether frozen-thawed ovaries show any alteration of AT system member gene expression when treated with survival-enhancing factors. We aimed to investigate whether mass freezing and thawing with or without the use of Rho-associated kinase (ROCK) inhibitors up- or down-regulate the expression of ATII, ATII-RI, and ATII-RII genes on frozen-thawed ovarian tissues. Significant changes in the expression of ATII, ATII-RI, and ATII-RII genes were observed on thawed ovaries when compared to fresh control. The treatment with ROCK inhibitors did not significantly alter their expression. In conclusion, freezing and thawing of ovarian tissue may affect the mRNA expression levels of intra-ovarian AT system genes, and modulation of ROCK inhibitor activity may not regulate AT system on the frozenthawed ovarian tissue.

PRMT8 as a phospholipase regulates Purkinje cell dendritic arborization and motor coordination.

The development of vertebrate neurons requires a change in membrane phosphatidylcholine (PC) metabolism. Although PC hydrolysis is essential for enhanced axonal outgrowth mediated by phospholipase D (PLD), less is known about the determinants of PC metabolism on dendritic arborization. We show that protein arginine methyltransferase 8 (PRMT8) acts as a phospholipase that directly hydrolyzes PC, generating choline and phosphatidic acid. We found that PRMT8 knockout mice (prmt8 (-/-)) displayed abnormal motor behaviors, including hindlimb clasping and hyperactivity. Moreover, prmt8 (-/-) mice and TALEN-induced zebrafish prmt8 mutants and morphants showed abnormal phenotypes, including the development of dendritic trees in Purkinje cells and altered cerebellar structure. Choline and acetylcholine levels were significantly decreased, whereas PC levels were increased, in the cerebellum of prmt8 (-/-) mice. Our findings suggest that PRMT8 acts both as an arginine methyltransferase and as a PC-hydrolyzing PLD that is essential for proper neurological functions.

Detection of choline and phosphatidic acid (PA) catalyzed by phospholipase D (PLD) using MALDI-QIT-TOF/MS with 9-aminoacridine matrix.

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC), the most abundant phospholipids of plasma membrane, resulting in the production of choline and phosphatidic acid (PA). Choline is a precursor of the neurotransmitter acetylcholine, whereas PA functions as an intracellular lipid mediator of diverse biological functions. For assessing PLD activity in vitro, PLD-derived choline has been often analyzed with radioactive or non-radioactive methods. In this study, we have developed a new method for detecting choline and PA with MALDI-QIT-TOF/MS by using 9-aminoacridine as a matrix. The standard calibration curves showed that choline and PA could be detected with linearity over the range from 0.05 and 1 pmol, respectively. Importantly, this method enables the concomitant detection of choline and PA as a reaction product of PC hydrolysis by PLD2 proteins. Thus, our simple and direct method would be useful to characterize the enzymatic properties of PLD, thereby providing insight into mechanisms of PLD activation.

Effects of insulin-sensitizing agents and insulin resistance in women with polycystic ovary syndrome.

OBJECTIVE: The aim of this study was to investigate the effect of insulin sensitizing agents on hormonal and metabolic parameters as well as menstrual patterns in women with polycystic ovary syndrome (PCOS). METHODS: One hundred and twenty-three patients with PCOS were included. Metformin was administered to patients at 1,500 mg or 1,700 mg daily for 3 months. If the patients had no improvement of the menstrual cycle or metformin-related adverse effects developed, the patients changed medication to a daily dose of either 15 mg pioglitazone or up to 45 mg. Then resumption of a regular menstrual cycle or recovery of ovulation was evaluated. Hormonal and metabolic profiles were compared between the response and non-response group to insulin sensitizing agents. RESULTS: One hundred and five patients with PCOS were treated with metformin for 3 months. Forty-eight patients (45.7%) showed improvement of menstrual cycle regularity after 3 months of metformin use, whereas 57 patients (54.3%) had no change. The mean free testosterone measured after 3 months of treatment was significantly lower in metformin responders than in non-responders. The other parameters did not differ between the groups. Of the 23 patients who used pioglitazone for 3 to 6 months, 19 patients (82.6%) showed improvement in their menstrual cycles. CONCLUSION: Metformin treatment seems to be effective for the improvement of menstrual cyclicity irrespective of insulin resistance in women with PCOS. When metformin related adverse effect occurred, pioglitazone would be effective for aiding the resumption of the menstrual cycle.

Successful birth with preimplantation genetic diagnosis using single-cell allele-specific PCR and sequencing in a woman with hypochondroplasia due to FGFR3 mutation (c.1620C>A, p.N540K).

Hypochondroplasia (HCH) is an autosomal dominant inherited skeletal dysplasia, usually caused by a heterozygous mutation in the fibroblast growth factor receptor 3 gene (FGFR3). A 27-year-old HCH woman with a history of two consecutive abortions of HCH-affected fetuses visited our clinic for preimplantation genetic diagnosis (PGD). We confirmed the mutation in the proband (FGFR3:c.1620C>A, p.N540K), and established a nested allele-specific PCR and sequence analysis for PGD using single lymphocyte cells. We performed this molecular genetic analysis to detect the presence of mutation among 20 blastomeres from 18 different embryos, and selected 9 embryos with the wild-type sequence (FGFR3:c.1620C). A successful pregnancy was achieved through a frozen-thawed cycle and resulted in the full-term birth of a normal neonate. To the best of our knowledge, this is the first report of a successful pregnancy and birth using single-cell allele-specific PCR and sequencing for PGD in an HCH patient.

Effects of urinary and recombinant gonadotropins on in vitro maturation outcomes of mouse preantral follicles.

Gonadotropins including follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play a crucial role in human-assisted reproduction techniques. Despite wide use of recombinant gonadotropins in clinical practice, the efficacy of urinary gonadotropins and the dosage of LH component have not yet been elucidated. This study was designed to investigate the difference of follicle culture outcomes according to various compositions of gonadotropins during in vitro culture of mouse preantral follicles. Ovaries were obtained from the 14-day-old C57BL/6 mice, and preantral follicles were isolated and cultured in culture media supplemented with human menopausal gonadotropin (hMG) 200 mIU/mL (group 1), recombinant FSH and LH (rFSH + rLH) 200 mIU/mL each (group 2), rFSH 200 mIU/mL + rLH 100 mIU/mL (group 3), or rFSH 200 mIU/mL + rLH 20 mIU/mL (group 4). Follicle survival rate was significantly lower in group 4. Antral follicles in lower doses of LH (groups 3, 4) showed a statistically significant larger diameter and tended to have a higher antral formation rate. However, follicles in group 1 tended to have a higher oocyte maturation rate. Estradiol concentration from conditioned media from 2:1 FSH/LH (group 3) was significantly higher than those from 1:1 FSH/LH (group 2) or 10:1 FSH/LH (group 4). Half dose of rLH to rFSH facilitated upregulation of growth differentiation factor 9 (Gdf9) expression in granulosa cells when compared to 1:1 FSH/LH or 10:1 FSH/LH. Conclusively, recombinant gonadotropins provided a comparable condition to hMG, and half dose of rLH to rFSH seems to be more suitable for follicular development during in vitro culture.

Vesicovaginal fistula following large-loop excision of the transformation zone in a chronic systemic glucocorticoid user.

A 42-year-old woman with a history of a large-loop excision of transformation zone for cervical intraepithelial neoplasia III four years previously visited our outpatient department for cervical intraepithelial neoplasia I that newly detected by colposcopy-directed punch biopsy. Her other medical conditions include systemic lupus erythematosus on daily prednisolone 15 mg during 19 years. After second large-loop excision of transformation zone, delayed vesicovaginal fistula was confirmed by indigo carmine leakage test, computed tomography scan, and cystoscopy. Fistula was primarily repaired with indwelling double-J catheters with anchoring omentum on suture site.

The use of tenaculum during intrauterine insemination may not affect the pregnancy outcome.

PURPOSE: The association between tenaculum application to the cervix just before embryo transfer and lower pregnancy rate has been reported. However, studies on the use of tenaculum in intrauterine insemination (IUI) cycles are not available. This study attempted to investigate whether the use of tenaculum affects the clinical outcomes of IUI. MATERIALS AND METHODS: Two hundred and thirty three CC/hMG/ IUI cycles of one hundred and forty three couples were recruited at Seoul National University Hospital from October 2006 to December 2008. Mock insemination and IUI with or without tenaculum application to the cervix were also performed, and clinical pregnancy rate was compared. RESULTS: The incidence of difficult mock insemination at the beginning of cycle was higher in the tenaculum use group (p < 0.001). No significant statistical difference in the clinical pregnancy rate was observed between the groups with or without tenaculum application (12.1% vs. 18.5%; p = 0.175), which was not influenced by the difficulty of mock insemination. CONCLUSION: The use of tenaculum during IUI may not affect the pregnancy outcome. Our results need to be confirmed by a prospective study in a larger population.