Development and Qualification of Analytical Methods to Support Low Concentration Drug Product in-use Studies.

The emergence of highly potent therapeutics with low expected clinical doses creates a challenge for analytical characterization of simulated drug product in-use samples. The low expected protein concentration (often µg/mL) and highly charged and sub-optimal sample matrices like 0.9% saline or 5% dextrose make ensuring dose solution stability and characterizing product quality changes difficult. Health authority expectations require analysis of low concentration in-use samples to be completed with suitable assays to ensure little to no changes are occurring during drug product dose preparation and administration, thus ensuring patient safety. However, characterization of these samples for protein concentration, size variants, charge variants and potency often necessitates additional analytical method development to improve sensitivity and compatibility with in-use samples. Here we report the development and qualification of reliable in-use methods to characterize simulated in-use samples to assist during drug product development.

No. 362-Ovulation Induction in Polycystic Ovary Syndrome.

OBJECTIVE: To review current non-pharmacologic and pharmacologic options for ovulation induction in women with polycystic ovary syndrome (PCOS). OPTIONS: This guideline reviews the evidence for the various options for ovulation induction in PCOS. OUTCOMES: Ovulation, pregnancy and live birth rates, risks, and side effects are the outcomes of interest. EVIDENCE: Published literature was retrieved through searches of Medline using appropriate controlled vocabulary and key words spanning from 2000 to 2016. Results were restricted to systematic reviews, randomized control trials/controlled clinical trials, and observational studies. Grey (unpublished) literature was identified through searching the websites of health technology assessment and of health technology assessment-related agencies, clinical practice guideline collections, clinical trial registries, and national and international medical specialty societies. VALUES: The evidence gathered was reviewed and evaluated by the Reproductive Endocrinology and Infertility Committee of the Society of Obstetricians and Gynaecologists of Canada. The quality of evidence was quantified using the Canadian Task Force on Preventive Health Care. BENEFITS, HARMS, AND COSTS: Benefits include weight reduction and improvements in ovulation, pregnancy, and live birth rates. Potential harms include medication side effects and multiple pregnancies. VALIDATION: These guidelines have been reviewed and approved by the Reproductive Endocrinology and Infertility Committee of the SOGC. CONCLUSION: First line management of infertility once a diagnosis of PCOS is made should include weight loss and exercise with goals to below class 2 obesity (BMI <35 kg/m2) as applicable. Subsequently, first line medical therapy for ovulation induction should include aromatase inhibitors (now considered both safe and effective) and selective estrogen receptor modulators as available. Insulin sensitizers should not be used as first line therapy but as adjuncts as appropriate. Referral to a reproductive endocrinologist should be considered if there is failure or resistance to these approaches to consider ovulation induction with gonadotropins or IVF as appropriate. SPONSOR: The Society of Obstetricians and Gynaecologists of Canada.

Cysteine modifiers suggest an allosteric inhibitory site on the CAL PDZ domain.

Protein-protein interactions have become attractive targets for both experimental and therapeutic interventions. The PSD-95/Dlg1/ZO-1 (PDZ) domain is found in a large family of eukaryotic scaffold proteins that plays important roles in intracellular trafficking and localization of many target proteins. Here, we seek inhibitors of the PDZ protein that facilitates post-endocytic degradation of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR): the CFTR-associated ligand (CAL). We develop and validate biochemical screens and identify methyl-3,4-dephostatin (MD) and its analog ethyl-3,4-dephostatin (ED) as CAL PDZ inhibitors. Depending on conditions, MD can bind either covalently or non-covalently. Crystallographic and NMR data confirm that MD attacks a pocket at a site distinct from the canonical peptide-binding groove, and suggests an allosteric connection between target residue Cys319 and the conserved Leu291 in the GLGI motif. MD and ED thus appear to represent the first examples of small-molecule allosteric regulation of PDZ:peptide affinity. Their mechanism of action may exploit the known conformational plasticity of the PDZ domains and suggests that allosteric modulation may represent a strategy for targeting of this family of protein-protein binding modules.

Discovery of novel, orally bioavailable, antileishmanial compounds using phenotypic screening.

Leishmaniasis is a parasitic infection that afflicts approximately 12 million people worldwide. There are several limitations to the approved drug therapies for leishmaniasis, including moderate to severe toxicity, growing drug resistance, and the need for extended dosing. Moreover, miltefosine is currently the only orally available drug therapy for this infection. We addressed the pressing need for new therapies by pursuing a two-step phenotypic screen to discover novel, potent, and orally bioavailable antileishmanials. First, we conducted a high-throughput screen (HTS) of roughly 600,000 small molecules for growth inhibition against the promastigote form of the parasite life cycle using the nucleic acid binding dye SYBR Green I. This screen identified approximately 2,700 compounds that inhibited growth by over 65% at a single point concentration of 10 µM. We next used this 2700 compound focused library to identify compounds that were highly potent against the disease-causing intra-macrophage amastigote form and exhibited limited toxicity toward the host macrophages. This two-step screening strategy uncovered nine unique chemical scaffolds within our collection, including two previously described antileishmanials. We further profiled two of the novel compounds for in vitro absorption, distribution, metabolism, excretion, and in vivo pharmacokinetics. Both compounds proved orally bioavailable, affording plasma exposures above the half-maximal effective concentration (EC50) concentration for at least 12 hours. Both compounds were efficacious when administered orally in a murine model of cutaneous leishmaniasis. One of the two compounds exerted potent activity against trypanosomes, which are kinetoplastid parasites related to Leishmania species. Therefore, this compound could help control multiple parasitic diseases. The promising pharmacokinetic profile and significant in vivo efficacy observed from our HTS hits highlight the utility of our two-step phenotypic screening strategy and strongly suggest that medicinal chemistry optimization of these newly identified scaffolds will lead to promising candidates for an orally available anti-parasitic drug.

The Impact of Standardized Acuity Assessment and a Fast-Track on Length of Stay in Obstetric Triage: A Quality Improvement Study.

To prospectively assess the impact of a standardized 5-category Obstetrical Triage Acuity Scale (OTAS) and a fast-track for lower-acuity patients on patient flow. Length of stay (LOS) data of women presenting to obstetric triage were abstracted from the electronic medical record prior to (July 1, 2011, to March 30, 2012) and following OTAS implementation (April 1 to December 31, 2012). Following computerized simulation modeling, a fast-track for lower acuity women was implemented (January 1, 2013, to February 28, 2014). Prior to OTAS implementation (8085 visits), the median LOS was 105 (interquartile range [IQR] = 52-178) minutes. Following OTAS implementation (8131 visits), the median LOS decreased to 101 (IQR = 49-175) minutes (P = .04). The LOS did not correlate well with acuity. Simulation modeling predicted that a fast-track for OTAS 4 and 5 patients would reduce the LOS. The LOS for lower-acuity patients in the fast-track decreased to 73 (IQR = 40-140) minutes (P = .005). In addition, the overall LOS (12 576 visits) decreased to 98 (IQR = 47-172) minutes (6.9% reduction; P < .001). Standardized assessment of acuity and a fast-track for lower acuity pregnant women decreased the overall LOS and the LOS of lower-acuity patients.

Diversity-oriented natural product platform identifies plant constituents targeting Plasmodium falciparum.

BACKGROUND: A diverse library of pre-fractionated plant extracts, generated by an automated high-throughput system, was tested using an in vitro anti-malarial screening platform to identify known or new natural products for lead development. The platform identifies hits on the basis of in vitro growth inhibition of Plasmodium falciparum and counter-screens for cytotoxicity to human foreskin fibroblast or embryonic kidney cell lines. The physical library was supplemented by early-stage collection of analytical data for each fraction to aid rapid identification of the active components within each screening hit. RESULTS: A total of 16,177 fractions from 1300 plants were screened, identifying several P. falciparum inhibitory fractions from 35 plants. Although individual fractions were screened for bioactivity to ensure adequate signal in the analytical characterizations, fractions containing less than 2.0 mg of dry weight were combined to produce combined fractions (COMBIs). Fractions of active COMBIs had EC50 values of 0.21-50.28 and 0.08-20.04 µg/mL against chloroquine-sensitive and -resistant strains, respectively. In Berberis thunbergii, eight known alkaloids were dereplicated quickly from its COMBIs, but berberine was the most-active constituent against P. falciparum. The triterpenoids α-betulinic acid and ß-betulinic acid of Eugenia rigida were also isolated as hits. Validation of the anti-malarial discovery platform was confirmed by these scaled isolations from B. thunbergii and E. rigida. CONCLUSIONS: These results demonstrate the value of curating and exploring a library of natural products for small molecule drug discovery. Attention given to the diversity of plant species represented in the library, focus on practical analytical data collection, and the use of counter-screens all facilitate the identification of anti-malarial compounds for lead development or new tools for chemical biology.

Acuity Assessment in Obstetrical Triage.

OBJECTIVE: A five-category Obstetrical Triage Acuity Scale (OTAS) was developed with a comprehensive set of obstetrical determinants. The purposes of this study were: (1) to compare the inter-rater reliability (IRR) in tertiary and community hospital settings and measure the intra-rater reliability (ITR) of OTAS; (2) to establish the validity of OTAS; and (3) to present the first revision of OTAS from the National Obstetrical Triage Working Group. METHODS: To assess IRR, obstetrical triage nurses were randomly selected from London Health Sciences Centre (LHSC) (n = 8), Stratford General Hospital (n = 11), and Chatham General Hospital (n= 7) to assign acuity levels to clinical scenarios based on actual patient visits. At LHSC, a group of nurses were retested at nine months to measure ITR. To assess validity, OTAS acuity level was correlated with measures of resource utilization. RESULTS: OTAS has significant and comparable IRR in a tertiary care hospital and in two community hospitals. Repeat assessment in a cohort of nurses demonstrated significant ITR. Acuity level correlated significantly with performance of routine and second order laboratory investigations, point of care ultrasound, nursing work load, and health care provider attendance. A National Obstetrical Triage Working Group was formed and guided the first revision. Four acuity modifiers were added based on hemodynamics, respiratory distress, cervical dilatation, and fetal well-being. CONCLUSION: OTAS is the first obstetrical triage scale with established reliability and validity. OTAS enables standardized assessments of acuity within and across institutions. Further, it facilitates assessment of patient care and flow based on acuity.

Development of a phenotypic high-content assay to identify pharmacoperone drugs for the treatment of primary hyperoxaluria type 1 by high-throughput screening.

Primary hyperoxaluria is a severe disease for which the best current therapy is dialysis or organ transplantation. These are risky, inconvenient, and costly procedures. In some patients, pyridoxine treatment can delay the need for these surgical procedures. The underlying cause of particular forms of this disease is the misrouting of a specific enzyme, alanine:glyoxylate aminotransferase (AGT), to the mitochondria instead of the peroxisomes. Pharmacoperones are small molecules that can rescue misfolded proteins and redirect them to their correct location, thereby restoring their function and potentially curing disease. In the present study, we miniaturized a cell-based assay to identify pharmacoperone drugs present in large chemical libraries to selectively correct AGT misrouting. This assay employs AGT-170, a mutant form of AGT that predominantly resides in the mitochondria, which we monitor for its relocation to the peroxisomes through automated image acquisition and analysis. Over the course of a pilot screen of 1,280 test compounds, we achieved an average Z'-factor of 0.72±0.02, demonstrating the suitability of this assay for HTS.

UPLC-MS-ELSD-PDA as a powerful dereplication tool to facilitate compound identification from small-molecule natural product libraries.

The generation of natural product libraries containing column fractions, each with only a few small molecules, using a high-throughput, automated fractionation system, has made it possible to implement an improved dereplication strategy for selection and prioritization of leads in a natural product discovery program. Analysis of databased UPLC-MS-ELSD-PDA information of three leads from a biological screen employing the ependymoma cell line EphB2-EPD generated details on the possible structures of active compounds present. The procedure allows the rapid identification of known compounds and guides the isolation of unknown compounds of interest. Three previously known flavanone-type compounds, homoeriodictyol (1), hesperetin (2), and sterubin (3), were identified in a selected fraction derived from the leaves of Eriodictyon angustifolium. The lignan compound deoxypodophyllotoxin (8) was confirmed to be an active constituent in two lead fractions derived from the bark and leaves of Thuja occidentalis. In addition, two new but inactive labdane-type diterpenoids with an uncommon triol side chain were also identified as coexisting with deoxypodophyllotoxin in a lead fraction from the bark of T. occidentalis. Both diterpenoids were isolated in acetylated form, and their structures were determined as 14S,15-diacetoxy-13R-hydroxylabd-8(17)-en-19-oic acid (9) and 14R,15-diacetoxy-13S-hydroxylabd-8(17)-en-19-oic acid (10), respectively, by spectroscopic data interpretation and X-ray crystallography. This work demonstrates that a UPLC-MS-ELSD-PDA database produced during fractionation may be used as a powerful dereplication tool to facilitate compound identification from chromatographically tractable small-molecule natural product libraries.

Positive perception of pharmacogenetic testing for psychotropic medications.

INTRODUCTION: Pharmacogenetics attempts to identify inter-individual genetic differences that are predictive of variable drug response and propensity to side effects, with the prospect of assisting physicians to select the most appropriate drug and dosage for treatment. However, many concerns regarding genetic tests exist. We sought to test the opinions of undergraduate science and medical students in southern Ontario universities toward pharmacogenetic testing. METHODS AND RESULTS: Questionnaires were completed by 910 undergraduate medicine and science students from 2005 to 2007. Despite students' concerns that the results of genetic tests may be used for other purposes without consent (71%) or lead to discrimination (78%), an overwhelming number of students were in favor of pharmacogenetic testing (90%). DISCUSSION: To our knowledge, this study is the first to survey a large sample for their attitude toward pharmacogenetic testing for psychotropic medications. Our results indicate that, although concerns remain and scientific advancements are required, respondents were in support of pharmacogenetic testing for medications used to treat schizophrenia. © 2014 The Authors. Human Psychopharmacology: Clinical and Experimental published by John Wiley & Sons, Ltd.

Transitioning pharmacoperones to therapeutic use: in vivo proof-of-principle and design of high throughput screens.

A pharmacoperone (from "pharmacological chaperone") is a small molecule that enters cells and serves as molecular scaffolding in order to cause otherwise-misfolded mutant proteins to fold and route correctly within the cell. Pharmacoperones have broad therapeutic applicability since a large number of diseases have their genesis in the misfolding of proteins and resultant misrouting within the cell. Misrouting may result in loss-of-function and, potentially, the accumulation of defective mutants in cellular compartments. Most known pharmacoperones were initially derived from receptor antagonist screens and, for this reason, present a complex pharmacology, although these are highly target specific. In this summary, we describe efforts to produce high throughput screens that identify these molecules from chemical libraries as well as a mouse model which provides proof-of-principle for in vivo protein rescue using existing pharmacoperones.

Optimization of the electrophile of chloronitrobenzamide leads active against Trypanosoma brucei.

We previously reported the phenylchloronitrobenzamides (PCNBs), a novel class of compounds active against the species of trypanosomes that cause Human African Trypanosomiasis (HAT). Herein, we explored the potential to adjust the reactivity of the electrophilic chloronitrobenzamide core. These studies identified compound 7d that potently inhibited the growth of trypanosomes (EC50=120nM for Trypanosoma b. brucei, 18nM for Trypanosoma b. rhodesiense, and 38nM for Trypanosoma b. gambiense) without significant cytotoxicity against mammalian cell lines (EC50>25µM for HepG2, HEK293, Raji, and BJ cell lines) and also had good stability in microsomal models (t1/2>4h in both human and mouse). Overall these properties indicate the compound 7d and its analogs are worth further exploration as potential leads for HAT.

High-throughput screen for pharmacoperones of the vasopressin type 2 receptor.

Pharmacoperone drugs correct the folding of misfolded protein mutants and restore function (i.e., "rescue") by correcting the routing of (otherwise) misrouted mutants. Assays for pharmacoperones have not been applied to screen large libraries previously. Currently, most pharmacoperones possess intrinsic agonist or antagonist activities since these were identified using high-throughput screens aimed at discovering direct agonists or antagonists. Here we describe an ultra-high-throughput compatible no-wash assay system designed to specifically identify pharmacoperones of the vasopressin type 2 receptor (V2R). Development of such assays is important and novel since useful chemical structures with the ability to control cellular trafficking but lacking intrinsic agonist or antagonist properties have not likely been identified using existing screens. In the described assay, the level of functional human V2R (hV2R) (mutant) present in each test well is quantitated by stimulation with saturating levels of agonist followed by use of a luminescent-based cyclic adenosine monophosphate assay. This allows the assay to identify compounds that increase the trafficking of mutant hV2R[L(83)Q] in our model system.

Optimization of chloronitrobenzamides (CNBs) as therapeutic leads for human African trypanosomiasis (HAT).

We previously reported the discovery of the activity of chloronitrobenzamides (CNBs) against bloodstream forms of Trypanosoma brucei . Herein we disclose extensive structure-activity relationship and structure-property relationship studies aimed at identification of tractable early leads for clinical development. These studies revealed a promising lead compound, 17b, that exhibited nanomolar potency against T. brucei (EC50 = 27 nM for T. b. brucei, 7 nM for T. b. rhodesiense, and 2 nM for T. b. gambiense ) with excellent selectivity for parasite cells relative to mammalian cell lines (EC50 > 25 µM). In addition compound 17b displayed suitable physiochemical characteristics and microsomal stability (t1/2 > 4 h for human and mouse) to justify pursuing in vivo studies.

Implementing an obstetric triage acuity scale: interrater reliability and patient flow analysis.

A 5-category Obstetric Triage Acuity Scale (OTAS) was developed with a comprehensive set of obstetrical determinants. The objectives of this study were as follows: (1) to test the interrater reliability of OTAS and (2) to determine the distribution of patient acuity and flow by OTAS level. To test the interrater reliability, 110 triage charts were used to generate vignettes and the consistency of the OTAS level assigned by 8 triage nurses was measured. OTAS performed with substantial (Kappa, 0.61 - 0.77, OTAS 1-4) and near perfect correlation (0.87, OTAS 5). To assess patient flow, the times to primary and secondary health care provider assessments and lengths of stay stratified by acuity were abstracted from the patient management system. Two-thirds of triage visits were low acuity (OTAS 4, 5). There was a decrease in length of stay (median [interquartile range], minutes) as acuity decreased from OTAS 1 (120.0 [156.0] minutes) to OTAS 3 (75.0 [120.8]). The major contributor to length of stay was time to secondary health care provider assessment and this did not change with acuity. The percentage of patients admitted to the antenatal or birthing unit decreased from 80% (OTAS 1) to 12% (OTAS 5). OTAS provides a reliable assessment of acuity and its implementation has allowed for triaging of obstetric patients based on acuity, and a more in-depth assessment of the patient flow. By standardizing assessment, OTAS allows for opportunities to improve performance and make comparisons of patient care and flow across organizations.

Therapeutic rescue of misfolded/mistrafficked mutants: automation-friendly high-throughput assays for identification of pharmacoperone drugs of GPCRs.

Mutations cause protein folding defects that result in cellular misrouting of otherwise functional proteins. Such mutations are responsible for a wide range of disease states, especially among G-protein coupled receptors. Drugs which serve as chemical templates and promote the proper folding of these proteins are valuable therapeutic molecules since they return functional proteins to the proper site of action. Small molecules have been identified that are able to function as pharmacological chaperones or "pharmacoperones" and stabilize the correct conformations of their target proteins with high specificity. Most of these are also agonists or antagonists of the proteins of interest, complicating potential therapeutic use. This is due, in part, to the fact that the majority of these were discovered during high-throughput screening campaigns using assays designed to detect agonists and antagonists, rather than compounds which improve the trafficking of misrouted mutants. The assays described in this report are designed specifically to identify compounds which result in the reactivation and correct trafficking of misfolded gonadotropin releasing hormone receptor and vasopressin type 2 receptor mutants, rather than those which act as agonists directly. The system reported is a generalizable approach amenable to use in automated (robotic) high-throughput screening efforts and can be used to identify compounds which affect protein conformation without necessarily acting as direct agonists or antagonists.

On the mechanism of action of SJ-172550 in inhibiting the interaction of MDM4 and p53.

SJ-172550 (1) was previously discovered in a biochemical high throughput screen for inhibitors of the interaction of MDMX and p53 and characterized as a reversible inhibitor (J. Biol. Chem. 2010; 285:10786). Further study of the biochemical mode of action of 1 has shown that it acts through a complicated mechanism in which the compound forms a covalent but reversible complex with MDMX and locks MDMX into a conformation that is unable to bind p53. The relative stability of this complex is influenced by many factors including the reducing potential of the media, the presence of aggregates, and other factors that influence the conformational stability of the protein. This complex mechanism of action hinders the further development of compound 1 as a selective MDMX inhibitor.

Chemical genetics of Plasmodium falciparum.

Malaria caused by Plasmodium falciparum is a disease that is responsible for 880,000 deaths per year worldwide. Vaccine development has proved difficult and resistance has emerged for most antimalarial drugs. To discover new antimalarial chemotypes, we have used a phenotypic forward chemical genetic approach to assay 309,474 chemicals. Here we disclose structures and biological activity of the entire library-many of which showed potent in vitro activity against drug-resistant P. falciparum strains-and detailed profiling of 172 representative candidates. A reverse chemical genetic study identified 19 new inhibitors of 4 validated drug targets and 15 novel binders among 61 malarial proteins. Phylochemogenetic profiling in several organisms revealed similarities between Toxoplasma gondii and mammalian cell lines and dissimilarities between P. falciparum and related protozoans. One exemplar compound displayed efficacy in a murine model. Our findings provide the scientific community with new starting points for malaria drug discovery.