A rapid solid form risk assessment workflow for ophthalmic drug candidates

This work introduces a material-sparing process that rapidly screens the solid form landscape for ophthalmic compound candidates. Crystalline form of compound candidates generated by a Form Risk Assessment (FRA) can be used to reduce their downstream development risk. This workflow evaluated nine model compounds with various molecular and polymorphic profiles by using less than 350 mg of drug substances. Kinetic solubility of the model compounds in a variety of solvents was screened to support the experimental design. The FRA workflow integrated several crystallization methods such as temperature-cycled slurrying (thermocycling), cooling, and evaporation. The FRA was also applied on ten ophthalmic compound candidates for verification. X-ray powder diffractometry (XRPD) was used for form identification. For the nine model compounds studied, multiple crystalline forms were generated. This demonstrates the potential of the FRA workflow to reveal polymorphic tendency. In addition, thermocycling process was found to be the most effective technique to capture the thermodynamically most stable form. Satisfactory results were observed with the discovery compounds intended for ophthalmic formulations. This work introduces a form risk assessment workflow by using sub-gram level of drug substances. The capability of this material-sparing workflow to discover polymorphs and capture the thermodynamically most stable forms within 2–3 weeks makes it suitable for discovery stage compounds, especially for ophthalmic candidates.</p

Phylogenetic relationships of the different CCE classes/clades.

The deduced amino acid sequences of 44 M. occidentalis CCE genes were aligned with those of selected CCEs from D. melanogaster (Dm), A. mellifera (Am) and T. urticae (Tu) (S5 Fig). The alignment was trimmed at both ends using criteria set previously [67]. The midpoint-rooted tree was generated using MrBayes. The M. occidentalis CCE genes are shown in red. Posterior probabilities are shown at the nodes. Details of the gene names for the CCEs from M. occidentalis and other arthropods are shown in S1 and S4 Tables, respectively.</p

Phylogenetic relationships of the different cytosolic GST classes.

The deduced amino acid sequences of 13 M. occidentalis cytosolic GST genes were aligned with those of selected GSTs from D. melanogaster (Dm), An. gambiae (Ag), A. mellifera (Am), T. urticae (Tu) and I. scapularis (Is) (S2 Fig). The midpoint-rooted tree was generated using MrBayes. The M. occidentalis GST genes are shown in red. Posterior probabilities are shown at the nodes. Details of the gene names for the GSTs from M. occidentalis and other arthropods are shown in S1 and S2 Tables, respectively.</p

The effects of oral delivery of <i>clathrin heavy chain</i> dsRNA on survival and egg production of <i>M. occidentalis</i> females. Egg hatch rates and gene knockdown efficiency were also evaluated.

<p>(<b>A</b>) and (<b>B</b>) The effects of <i>clathrin heavy chain</i> dsRNA delivery on survival and oviposition of females, respectively. N = 10 for each treatment group. One-way ANOVA results for days of survival and number of eggs are <i>F_2,29</i> = 20.55; <i>P</i><0.0001, <i>F_2,29</i> = 57.71; <i>P</i><0.0001, respectively. Bars labeled with different letters are significantly different (Tukey’s HSD test, <i>P</i><0.0004). (<b>C</b>) The effect of <i>clathrin heavy chain</i> dsRNA delivery on egg hatch rates. N = 10, 10, and 5 for TE buffer group, control dsRNA group, and <i>clathrin heavy chain</i> dsRNA group, respectively. (<b>D</b>) The effect of <i>clathrin heavy chain</i> dsRNA delivery on the mRNA levels of the <i>clathrin heavy chain</i> gene. N = 4 for each group. <i>Clathrin heavy chain</i> mRNA levels in control females were scaled to 1. Student’s <i>t</i> test result for the comparison of relative <i>clathrin heavy chain</i> mRNA levels between control dsRNA- and <i>clathrin heavy chain</i> dsRNA-treated mites is <i>P</i><0.0001.</p

The effects of <i>BTB1</i> and <i>BTB2</i> dsRNA deliveries on the mRNA levels of <i>BTB2</i> and <i>BTB1</i>, respectively.

<p><i>BTB1</i><b>(A)</b> and <i>BTB2</i> <b>(B)</b> dsRNA deliveries did not alter the mRNA levels of <i>BTB2</i> and <i>BTB1</i>, respectively, in either F10A or AO females. Student’s <i>t</i> test results for the relative <i>BTB2</i> (or <i>BTB1</i>) mRNA levels in F10A (or AO) mites that received control or <i>BTB1</i> (or <i>BTB2</i>) dsRNA are <i>P</i> > 0.1, 2-tailed <i>t</i> test. The <i>BTB2</i> (or <i>BTB1</i>) mRNA levels in control females were scaled to 1.</p

A comparison of cytosolic GST gene numbers in the genomes of nine arthropods.

<p>Data are derived from Hayes et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160009#pone.0160009.ref073" target="_blank">73</a>], Oakeshott et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160009#pone.0160009.ref084" target="_blank">84</a>], Grbic et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160009#pone.0160009.ref058" target="_blank">58</a>], Reddy et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160009#pone.0160009.ref059" target="_blank">59</a>], and the current study.</p

The Glutathione-S-Transferase, Cytochrome P450 and Carboxyl/Cholinesterase Gene Superfamilies in Predatory Mite <i>Metaseiulus occidentalis</i>

<div><p>Pesticide-resistant populations of the predatory mite <i>Metaseiulus</i> (= <i>Typhlodromus</i> or <i>Galendromus) occidentalis</i> (Arthropoda: Chelicerata: Acari: Phytoseiidae) have been used in the biological control of pest mites such as phytophagous <i>Tetranychus urticae</i>. However, the pesticide resistance mechanisms in <i>M</i>. <i>occidentalis</i> remain largely unknown. In other arthropods, members of the glutathione-S-transferase (GST), cytochrome P450 (CYP) and carboxyl/cholinesterase (CCE) gene superfamilies are involved in the diverse biological pathways such as the metabolism of xenobiotics (e.g. pesticides) in addition to hormonal and chemosensory processes. In the current study, we report the identification and initial characterization of 123 genes in the GST, CYP and CCE superfamilies in the recently sequenced <i>M</i>. <i>occidentalis</i> genome. The gene count represents a reduction of 35% compared to <i>T</i>. <i>urticae</i>. The distribution of genes in the GST and CCE superfamilies in <i>M</i>. <i>occidentalis</i> differs significantly from those of insects and resembles that of <i>T</i>. <i>urticae</i>. Specifically, we report the presence of the Mu class GSTs, and the J’ and J” clade CCEs that, within the Arthropoda, appear unique to Acari. Interestingly, the majority of CCEs in the J’ and J” clades contain a catalytic triad, suggesting that they are catalytically active. They likely represent two Acari-specific CCE clades that may participate in detoxification of xenobiotics. The current study of genes in these superfamilies provides preliminary insights into the potential molecular components that may be involved in pesticide metabolism as well as hormonal/chemosensory processes in the agriculturally important <i>M</i>. <i>occidentalis</i>.</p></div

Clathrin Heavy Chain Is Important for Viability, Oviposition, Embryogenesis and, Possibly, Systemic RNAi Response in the Predatory Mite <i>Metaseiulus occidentalis</i>

<div><p>Clathrin heavy chain has been shown to be important for viability, embryogenesis, and RNA interference (RNAi) in arthropods such as <i>Drosophila melanogaster</i>. However, the functional roles of clathrin heavy chain in chelicerate arthropods, such as the predatory mite <i>Metaseiulus occidentalis,</i> remain unknown. We previously showed that dsRNA ingestion, followed by feeding on spider mites, induced systemic and robust RNAi in <i>M. occidentalis</i> females. In the current study, we performed a loss-of-function analysis of the <i>clathrin heavy chain</i> gene in <i>M. occidentalis</i> using RNAi. We showed that ingestion of <i>clathrin heavy chain</i> dsRNA by <i>M. occidentalis</i> females resulted in gene knockdown and reduced longevity. In addition, <i>clathrin heavy chain</i> dsRNA treatment almost completely abolished oviposition by <i>M. occidentalis</i> females and the few eggs produced did not hatch. Finally, we demonstrated that <i>clathrin heavy chain</i> gene knockdown in <i>M. occidentalis</i> females significantly reduced a subsequent RNAi response induced by ingestion of <i>cathepsin L</i> dsRNA. The last finding suggests that clathrin heavy chain may be involved in systemic RNAi responses mediated by orally delivered dsRNAs in <i>M. occidentalis</i>.</p></div

Phylogenetic relationships of the different CYP clans.

<p>The deduced amino acid sequences of 63 <i>M</i>. <i>occidentalis</i> CYP genes were aligned with those of selected CYPs from <i>D</i>. <i>melanogaster</i> (Dm), <i>A</i>. <i>mellifera</i> (Am) and <i>T</i>. <i>urticae</i> (Tu) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160009#pone.0160009.s003" target="_blank">S3 Fig</a>). The midpoint-rooted tree was generated using MrBayes. The <i>M</i>. <i>occidentalis</i> CYP genes are shown in colors. Posterior probabilities are shown at the nodes. Details of the gene names for the CYPs from <i>M</i>. <i>occidentalis</i> and other arthropods are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160009#pone.0160009.s005" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160009#pone.0160009.s007" target="_blank">S3</a> Tables, respectively.</p

A comparison of CCE gene numbers in the genomes of eight arthropods.

<p>Data are derived from Yu et al.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160009#pone.0160009.ref106" target="_blank">106</a>], Oakeshott et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160009#pone.0160009.ref084" target="_blank">84</a>]. Grbic et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160009#pone.0160009.ref058" target="_blank">58</a>]. and the current study.</p