Crystallization of a Metastable Solvate and Impact of the Isolation Method on the Material Properties of the Anhydrous Product

We report the crystallization of a metastable small-molecule solvate and the effect of the isolation method on the physical and material properties of the resulting anhydrous material. The anhydrous crystalline products obtained from two different isolation routes using either a temperature-driven form change or a solvent-wash-mediated form change were analyzed by a suite of material-sparing characterization methods probing both physical form and material properties such as particle size distribution and powder flow behavior. The temperature-driven desolvation method was found to be time-consuming and undesirable. A relatively rapid desolvation approach was obtained using an ethyl acetate wash-mediated process. However, this method leads to powder with a broader particle size distribution, poorer flowability, higher interparticulate friction, and lower bulk density compared with the powder obtained by the temperature-driven desolvation process. The direct impact of the method of isolation on the material properties of the drug substance highlights the importance of not only understanding the crystallization process and form landscape but also the ability to implement systematic characterization to identify key powder properties of drug candidates early in the drug development process

Crystal Engineering of Isostructural Quaternary Multicomponent Crystal Forms of Olanzapine

Pharmaceutical cocrystals have gained increased attention at least in part because of their potential for enhancing physicochemical and biopharmaceutical properties of existing drugs. As a result, design, screening, and large-scale preparation of pharmaceutical cocrystals have been emphasized in recent research. The design of pharmaceutical cocrystals has focused primarily on determining the empirical guidelines regarding the hierarchy of supramolecular synthons. However, this approach is typically less predictive when considering drugs that are complex in nature, such as those having a multiplicity of functional groups and/or numerous degrees of conformational flexibility. In this manuscript, we report a crystal engineering design strategy to facilitate the synthesis of multicomponent crystal forms of the atypical antipsychotic drug olanzapine, marketed as a drug product under the trade name Zyprexa. Comprehensive analysis and data mining of existing crystal structures of olanzapine were followed by grouping into categories according to the crystal packing exhibited and systematically using this information to crystal engineer new compositions. This approach afforded isostructural, quaternary multicomponent crystal forms of olanzapine composed of a stoichiometric ratio of four molecular components: olanzapine; a cocrystal former; water; solvent (isopropylacetate). To our knowledge this study is unprecedented in that the observed quaternary structures can be classified as solvates, hydrates, or cocrystals

Crystal Engineering of Isostructural Quaternary Multicomponent Crystal Forms of Olanzapine

Pharmaceutical cocrystals have gained increased attention at least in part because of their potential for enhancing physicochemical and biopharmaceutical properties of existing drugs. As a result, design, screening, and large-scale preparation of pharmaceutical cocrystals have been emphasized in recent research. The design of pharmaceutical cocrystals has focused primarily on determining the empirical guidelines regarding the hierarchy of supramolecular synthons. However, this approach is typically less predictive when considering drugs that are complex in nature, such as those having a multiplicity of functional groups and/or numerous degrees of conformational flexibility. In this manuscript, we report a crystal engineering design strategy to facilitate the synthesis of multicomponent crystal forms of the atypical antipsychotic drug olanzapine, marketed as a drug product under the trade name Zyprexa. Comprehensive analysis and data mining of existing crystal structures of olanzapine were followed by grouping into categories according to the crystal packing exhibited and systematically using this information to crystal engineer new compositions. This approach afforded isostructural, quaternary multicomponent crystal forms of olanzapine composed of a stoichiometric ratio of four molecular components: olanzapine; a cocrystal former; water; solvent (isopropylacetate). To our knowledge this study is unprecedented in that the observed quaternary structures can be classified as solvates, hydrates, or cocrystals

Baseline movement and tactile sensitivity of Nav1.7 KOs are not different from WT/HET.

<p>Overall locomotion was evaluated in Nav1.7 WT/HET and KO animals by scoring both total rearing behavior and basic movement using beam breaks in an automated open-field box. <b>A.</b> There was no statistically significant difference in rearing behavior between WT/HET (11187±492, n = 20) and KO (11036±781, n = 17) littermates (pairwise t-test, p = 0.2438). <b>B.</b> There was no statistically significant difference in basic movement between WT/HET (935±85, n = 20) and KO (835±75, n = 17) littermates (mean ± S.E.M.; pairwise t-test, p = 0.528). <b>C.</b> Tactile sensitivity, as assayed by measuring threshold of paw withdrawal to von Frey fibers of increasing force, was not significantly different between WT/HET (1.225 g ±0.05 g, n = 11) and KO littermates (1.18 g ±0.08 g, n = 7) (mean ± S.E.M.; Wilcoxon Two-Sample Exact Test, p = 0.6434). Dashed line represents the level at which the animal’s paw was physically lifted by the von Frey monofilament and is included to show that the measured responses are due to true behavioral tactile response.</p

Normal anatomy of Nav1.7 KOs.

<p>Postnatal day 4 Nav1.7 WT (A to E) and KO (F to J) neonates stained with hematoxylin and eosin (H&E). <b>Wild type:</b><b>A.</b> Sagittal section of the entire pup. <b>B</b> through <b>E</b> show magnifications of various regions of the central and peripheral nervous systems: <b>Knock Out:</b><b>F.</b> Sagittal section of the entire pup. <b>G</b> through <b>J</b> show magnifications of various regions of the central and peripheral nervous systems. (<b>B, G</b>) cortex; (<b>C, H</b>) hippocampus; (<b>D, I</b>) olfactory bulb; (<b>E, J</b>) dorsal root ganglia. (Scale bars: <b>A, F</b> = 5 mm; <b>B, G</b> = 100 µm; <b>C, H</b> and <b>D, I</b> = 400 µm; <b>E, J</b> = 300 µm).</p

Nav1.7 KOs show minimal pain behaviors upon injection of veratridine or grayanotoxin III.

<p><b>A.</b> hNav1.7 currents recorded from HEK 293 cells in control (left) and after addition of 30 µM veratridine (right). Currents were evoked by a family (traces overlaid) of depolarizing voltage pulses incremented by +10 mV from a holding voltage of −100 mV, with repolarization to −80 mV followed by return to the holding voltage of −100 mV. Inward currents were evoked by depolarizations to −50 mV and more positive. Note the prolonged opening following repolarization in veratridine. Scale bars, 500 pA and 10 ms. <b>B.</b> Nav1.7 currents recorded with 300 µM grayanotoxin III in the internal (pipette) solution. Currents shown at left are in response to a family (traces overlaid) of step depolarizations in +5 mV increments from −120 mV to −50 mV, followed by repolarization to −120 mV. Depolarizations to −95 mV and more positive evoked inward currents, and these currents showed little or no inactivation during the test pulse. Dotted line marks the zero current level (I = 0). Currents shown at right are records from the same cell, after switching the holding voltage to −80 mV. Note that the holding current increased despite the decreased driving force (compare current at −80 mV to the dashed line marking the holding current at −120 mV), presumably reflecting steady influx of sodium ions through grayanotoxin-modified Nav1.7. Step depolarizations from −80 mV to −40 mV at +10 mV increments evoked additional currents with slow inactivation and deactivation kinetics. Scale bars, 500 pA and 20 ms. <b>C.</b> Licking and lifting in male CD-1 mice in response to increasing intraplantar doses of veratridine. At doses of 1 µg and 10 µg, veratridine caused a statistically significant increase in paw licking and lifting behaviors compared to saline or vehicle (1% ethanol/99% PBS) controls. The licking and lifting caused by 1 µg veratridine was completely prevented by pre-dosing the animals with mexiletine (MEX; 30 mg/kg, i.p. or p.o.). A separate animal cohort was used for each dose. <b>D.</b> Licking and lifting in male CD-1 mice in response to increasing intraplantar doses of grayanotoxin III. At doses of 0.1 µg and 1 µg, grayanotoxin III caused a statistically significant increase in paw licking and lifting behaviors compared to saline or vehicle (1% ethanol/99% PBS) controls. A separate animal cohort was used for each dose. <b>E.</b> In a separate experiment, the licking and lifting induced by grayanotoxin III was prevented by pre-dosing the animals with mexiletine (MEX; 30 mg/kg, i.p. or p.o.). <b>F.</b> Total paw licking and lifting behavior time in Nav1.7 KO (n = 5) and WT/HET littermates (n = 7) mice in the 20 minutes following i.pl. injection of 1 µg veratridine. Responses from KO (44.4 sec ±26.6 sec) were smaller than from WT/HET (292.1 sec ±34.7 sec) (p = 0.0073) (mean ± S.E.M., homogeneous ANOVA model with Tukey-Kramer adjusted t-test). <b>G.</b> Total painful paw lifting and licking behavior time in Nav1.7 KO (n = 3) and WT/HET littermates (n = 7) in the 15 minutes following i.pl. injection of 0.1 µg grayanotoxin III. Responses from KO (7.33 sec ±3.5 sec) were smaller than from WT/HET (170.9 sec ±23.1 sec) (p = 0.0003) (mean ± S.E.M., heterogeneous ANOVA model with Welch’s test).</p

Genotyping and phenotyping of global Nav1.7 knockout animals.

<p><b>A.</b> Example of endogenous (top - 267 bp) and targeted (bottom - 389 bp) PCR products obtained from DNA isolated from Nav1.7 WT (lane 1), KO (lane 2) and HET (lane 3) animals, while the last lane serves as a negative control (no DNA). All PCRs were run using a 1 kb PLUS DNA ladder. <b>B.</b> Photos of postnatal day 2 littermates showing their capability in feeding naturally by the presence of milk spots in control (see arrow left) and KO (see arrow right) and slight size difference in young animals. <b>C.</b> Body weight (grams) comparison between six female littermates at nine weeks of age that represented all three genotype classes illustrating the absence of size difference in adult animals.</p

Nav1.7 KOs do not develop CFA-induced thermal hyperalgesia.

<p>Latency time to withdrawal from radiant heat stimulus for Nav1.7 KO (n = 4) and WT/HET littermates (n = 9) at baseline and 1, 3, and 7 days following CFA injection. WT/HET mice developed robust hyperalgesia in the affected paw 24 hours post-CFA injection that returned to baseline at day 7. KO mice did not develop hyperalgesia. Differences between KO and WT/HET were statistically significant at baseline (WT/HET = 11.5 sec ±0.8 sec; KO = 16 sec ±0.8 sec, p = 0.0001), day 1 (WT/HET = 4.0 sec ±0.5 sec, KO = 15.7 sec ±0.9 sec, p<0.0001) and day 3 (WT/HET = 9.6 sec ±1.0 sec, KO = 17.8 sec ±0.7 sec, p = 0.0028), but not at day 7 (WT/HET = 11.3 sec ±1.1 sec, KO = 15.3 sec ±1.0 sec, p = 0.5381), at which time control animals had recovered back to baseline (all values mean ± S.E.M, ANOVA).</p

Nav1.7 KOs display inability to smell and insensitivity to intra-dermal histamine.

<p><b>A.</b> Total time spent to find a scented food pellet buried in cage bedding for Nav1.7 KO (n = 19) and WT/HET littermates (n = 21). Only one KO found the pellet before the test was cut off at 15 minutes; for statistical purposes the other 18 KO animals were each assigned a time of 15 minutes (900 sec) for an average time of 838.4 sec ±40.2 sec. All WT/HET animals found the pellet, in average time 214.9 sec ±39.3 sec (p = 2.209 E-09) (mean ± S.E.M., Wilcoxon two-sample exact test). <b>B.</b> Total number of scratching bouts following i.d. injection of histamine for Nav1.7 KO (n = 8) and WT/HET littermates (n = 17). Nav1.7 KO animals showed a 98% reduction in scratching bouts (average 1.25±0.6) compared to WT/HET (60.9±13.7) (mean ± S.E.M., heterogeneous ANOVA analysis, p<0.0001).</p

TTX-sensitive currents and mechanically-evoked spiking of C-fibers are reduced in KO mice.

<p><b>A</b>. Representative current traces showing the effect of 200 nM TTX on a KO DRG neuron. Overlaid sweeps show the time course of selective TTX blockade of fast-inactivating currents. Sweeps shown are the three before and the three after TTX addition (inter-sweep interval 10 s). Scale bars, 200 pA and 2 ms. <b>B</b>. Average current densities from acutely dissociated Nav1.7 KO and age-matched WT adult DRG neurons. TTX-S currents: 10.1±3.06 pA/pF, n = 12 neurons from 2 KO animals; and WT/HET, 27.2±4.53 pA/pF, n = 16 neurons from 4 control animals (2 WT and 2 HET) (p = 0.0074, unpaired t-test, mean ± S.E.M.). TTX-R currents: 16.9±4.44 pA/pF from KO, 11.9±3.26 from WT/HET (p = 0.3638, unpaired t-test, mean ± S.E.M.). Cell capacitance averaged 50.2 pF ±5.7 pF from WT/HET and 65.4 pF ±8.6 pF from KO (p = 0.069, unpaired t-test, mean ± S.E.M.). <b>C</b>. Representative mechanically-evoked action potentials in saphenous nerve. Upper trace shows the stimulation protocol (mN); middle and lower traces show representative action potentials from a single C-fiber from a Nav1.7 HET and a Nav1.7 KO preparation respectively. <b>D.</b> Frequency of mechanically-evoked action potentials from C fibers was significantly reduced in Nav1.7 KO mice (filled squares, n = 9 fibers) at forces ≥150 mN when compared to those recorded in C fibers from Nav1.7 control animals (open circles, n = 13 fibers) (150 mN: 8.71±0.86 Hz in control; 3.20±0.76 Hz in KO (mean ± S.E.M., two way ANOVA; p<0.01); 200 mN: 8.1±1.11 in control, 4.14±1.14 Hz in KO (mean ± S.E.M., two way ANOVA, p<0.05)).</p