A Static Headspace Gas Chromatography Method for Quantitation of Residual Solvents in Nanoformulations.

Various organic solvents are widely used in the manufacturing, processing, and purification of drug substances, drug products, formulations, excipients, etc. These solvents must be removed to the lowest amount permitted, as they do not possess any therapeutic advantages and may cause undesirable toxicities. Therefore, a rapid and sensitive analytical method for the quantitation of residual solvents is needed. The following chapter presents a static headspace gas chromatographic (HSGC) method for determining the concentration of common residual solvents in various nanoformulations. An efficient and sensitive HSGC method has been developed using PerkinElmer's headspace autosampler/gas chromatographic system with a flame ionization detector (FID) and validated according to the International Conference for Harmonization (ICH) guideline Q3C. The method validation indicates that the method is specific, linear, accurate, precise, and sensitive for the analyzed solvents. The method is suitable for the analysis of 13 residual solvents (methanol, ethanol, acetone, diethyl ether, 2-propanol, acetonitrile, 1-propanol, ethyl acetate, tetrahydrofuran, dichloromethane, chloroform, 1-butanol, and pyridine) and utilizes an Elite 624 Crossbond 6% cyanopropylphenyl, 94% dimethylpolysiloxanes column with helium as a carrier gas.

Chemosensitization and inhibition of pancreatic cancer stem cell proliferation by overexpression of microRNA-205.

Treatment of pancreatic cancer with gemcitabine (GEM) is limited due to its rapid plasma metabolism and development of chemoresistance. MicroRNA (miRNA) regulates cancer stem cell (CSC) maintenance and induces chemoresistance in cancer cells. In this study, we observed differential downregulation of miR-205 (miR-205-5p) in human pancreatic cancer tissues and cells. Compared to GEM-sensitive MIA PaCa-2 cells, miR-205 was highly downregulated in GEM-resistant MIA PaCa-2R cells. Lentivirus-mediated overexpression of miR-205 inhibits MIA PaCa-2R cell proliferation after GEM-treatment. Further investigation confirmed that miR-205 alone significantly reduces the proliferation of CSCs and tumor growth in mouse models. However, miR-205 in combination with GEM was more efficient in reducing the proliferation of CSCs and 3D spheroids. Moreover, miR-205 overexpressing MIA PaCa-2R cells induced orthotopic tumor growth was significantly inhibited after intravenous administration of GEM-conjugated methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate)-graft-gemcitabine-graft-dodecanol (mPEG-b-PCC-g-GEM-g-DC) (mPEG-b-PCC-g-GEM-g-DC) polymeric micelles. Also, a reduction in CSCs, EMT and chemoresistance markers was observed in miR-205 overexpressing MIA PaCa-2R cells. Immunohistochemical analysis of orthotopic tumors showed a decrease in drug resistance protein caveolin-1 and cell proliferation marker Ki-67 in combination treatment. Overall, our findings suggest that miR-205 resensitizes GEM-resistant pancreatic cancer cells to GEM and acts as a tumor suppressor miRNA.

Biodistribution of Self-Assembling Polymer-Gemcitabine Conjugate after Systemic Administration into Orthotopic Pancreatic Tumor Bearing Mice.

Therapeutic efficacy of gemcitabine (GEM) is severely limited due to its rapid metabolism by enzymatic deamination in vivo. We recently determined its therapeutic efficacy before (F-GEM) and after conjugation to poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate) (mPEG-b-PCC-g-GEM-g-DC, abbreviated as P-GEM) in subcutaneous and orthotopic pancreatic tumor bearing mice. In this study, pharmacokinetic (PK) parameters and biodistribution profiles of F-GEM and P-GEM were determined after intravenous injection into orthotopic pancreatic tumor bearing NSG mice. To assess the short-term toxicity, the levels of hematological, hepatic, and renal injury markers were measured after 24 h postadministration into these mice. P-GEM was distributed to all the major organs, with higher accumulation in the liver, spleen, and tumor compared to F-GEM. Area under the curve (AUC), elimination half-life (t1/2), and mean residence time (MRT) of P-GEM treated group were significantly higher compared to those of F-GEM treated group: 246,425 ± 1605 vs 83,591 ± 1844 ng/mL × h as AUC, 5.77 ± 2.02 vs 1.99 ± 0.09 h as t1/2, and 4.45 ± 0.15 vs 1.12 ± 0.13 h as MRT. Further, P-GEM exhibited negligible systemic toxicity as evidenced by almost similar alanine aminotransferase (ALT) and aspartate aminotransferase (AST) values for both P-GEM and F-GEM. These results suggest that P-GEM protects GEM from degradation and provides sustained drug release, resulting in enhanced GEM delivery to the tumor by more than 2.5-fold compared to F-GEM. Hence, P-GEM is a promising gemcitabine conjugated polymeric micelle for treating pancreatic cancer.

Synthesis, Magnetic Properties, Map Images, and Water Proton Relaxivities of D-Glucuronic Acid Coated Ln2O3 Nanoparticles (Ln = Ho and Er).

T2 MRI contrast agents cannot be synthesized by using molecules but nanoparticles because appreciable magnetic moments at room temperature are needed. Recently, some of lanthanide (Ln) oxide nanoparticles have shown decent magnetic moments at room temperature and even at ultrasmall particle diameters. In this study, we explored D-glucuronic acid coated Ln2O3 nanoparticles (Ln = Ho and Er) with ultrasmall particle diameters. They showed decent magnetic moments at room temperature and as a result, appreciable transverse water proton relaxivities (r2s) at 1.5 tesla MR field. Clear dose-dependent contrast enhancements in R2 map images were observed in both samples. These results showed that D-glucuronic acid coated Ln2O3 nanoparticles (Ln = Ho and Er) would be potential T2 MRI contrast agents at high MR fields.

Impact of CYP2C19 polymorphism on the pharmacokinetics of nelfinavir in patients with pancreatic cancer.

AIM: This study evaluated the influence of CYP2C19 polymorphisms on the pharmacokinetics of nelfinavir and its metabolite M8 in patients with pancreatic cancer. METHODS: Nelfinavir was administered orally to patients for over 10 days. The plasma concentrations of nelfinavir and M8 were measured by HPLC. The genotypes of CYP2C19*1, CYP2C19*2 and CYP2C19*3 were determined by the polymerase chain reaction-restriction fragment length polymorphism method. RESULTS: Pharmacokinetic profiles of nelfinavir and M8 were characterized by wide interindividual variability. The mean Cmax of nelfinavir in CYP2C19*1/*1 patients was 3.89 ± 0.40 (n = 3) and 5.12 ± 0.41 (n = 30) µg ml(-1) , while that of CYP2C19*1/*2 patients was 3.60 (n = 1) and 6.14 ± 0.31 (n = 5) µg ml(-1) at the doses of 625 and 1250 mg nelfinavir twice daily, respectively. For the M8 metabolite, the mean Cmax of CYP2C19*1/*1 patients was 1.06 ± 0.06 (n = 3) and 1.58 ± 0.27 (n = 30) µg ml(-1) , while those of CYP2C19*1/*2 patients were 1.01 (n = 1) and 1.23 ± 0.15 (n = 5) µg ml(-1) at the doses of 625 and 1250 mg nelfinavir twice daily, respectively. The area under the plasma concentration-time curve (AUC(0,12 h)) values of nelfinavir for CYP2C19*1/*1 patients were 28.90 ± 1.27 and 38.90 ± 4.99 µg ml(-1) ·h and for CYP2C19*1/*2 patients, AUC(0,12 h) was 28.20 (n = 1) and 40.22 ± 3.17 (n = 5) µg ml(-1) ·h at the doses of 625 and 1250 mg nelfinavir twice daily, respectively. The Cmax of nelfinavir was significantly higher (P <0.05) in CYP2C19*1/*2 patients but there was no statistical difference in AUC(0,12 h). CONCLUSION: CYP2C19*1/*2 genotype modestly affected the pharmacokinetic profiles of nelfinavir and M8 in patients with locally advanced pancreatic cancer.

Surface coated Eu(OH)3 nanorods: a facile synthesis, characterization, MR relaxivities and in vitro cytotoxicity.

The water-soluble and biocompatible D-glucuronic acid coated Eu(OH)3 nanorods (average thickness x average length = 9.0 x 118.3 nm) have been prepared in one-pot synthesis. The D-glucuronic acid coated Eu(OH)3 nanorods showed a strong fluorescence at approximately 600 nm with a narrow emission band width. A cytotoxicity test by using DU145 cells showed that D-glucuronic acid coated Eu(OH)3 nanorods are not toxic up to 100 microM, making them a promising candidate for biomedical applications such as fluorescent imaging. The minimum Eu concentration needed for a conventional confocal imaging was estimated to be approximately 0.1 mM. Therefore, D-glucuronic acid coated Eu(OH)3 nanorods can be applied to fluorescent imaging. However, a very tiny magnetization of approximately 1.2 emu/g at room temperature and at an applied field of 5 tesla was observed. As a result, very small r1 and r2 water proton relaxivities were estimated, implying that surface coated Eu(OH)3 nanorods are not sufficient for MRI contrast agents.

Paramagnetic nanoparticle T1 and T2 MRI contrast agents.

There is no doubt that magnetic resonance imaging contrast agents (MRI CAs) can play a vital role in diagnosing diseases. Therefore, demand for new MRI CAs with an enhanced sensitivity and advanced functionalities is very high. Here, paramagnetic nanoparticles (NPs) are reviewed as new potential candidates for either T(1) or T(2) MRI CAs or both. These include surface coated lanthanide (Ln) oxide NPs (Ln = Gd, Dy, and Ho) and manganese oxide NPs. Surface coating materials should be biocompatible and hydrophilic. Compared to conventional large NPs, these surface coated paramagnetic NPs can be made ultrasmall with core particle diameter ranging from 1 to 3 nm, but their magnetic properties are still sufficient for MRI CAs. At this particle diameter, they can be easily excreted from the body through the renal system, which is prerequisite for in vivo applications. Mixed lanthanide oxide NPs into which a fluorescent Ln material is incorporated will be valuable as multiple imaging agents for both MRI-fluorescent imaging (FI) and MRI-cellular imaging (CL). These paramagnetic NPs can be further functionalized towards target-specific imaging, multiplex imaging, and drug delivery.

Paramagnetic dysprosium oxide nanoparticles and dysprosium hydroxide nanorods as T2 MRI contrast agents.

We report here paramagnetic dysprosium nanomaterial-based T(2) MRI contrast agents. A large r(2) and a negligible r(1) is an ideal condition for T(2) MR imaging. At this condition, protons are strongly and nearly exclusively induced for T(2) MR imaging. The dysprosium nanomaterials fairly satisfy this because they are found to possess a decent r(2) but a negligible r(1) arising from L + S state 4f-electrons in Dy(III) ion ((6)H(15/2)). Their r(2) will also further increase with increasing applied field because of unsaturated magnetization at room temperature. Therefore, MR imaging and various physical properties of the synthesized d-glucuronic acid coated ultrasmall dysprosium oxide nanoparticles (d(avg) = 3.2 nm) and dysprosium hydroxide nanorods (20 × 300 nm) are investigated. These include hydrodynamic diameters, magnetic properties, MR relaxivities, cytotoxicities, and 3 tesla in vivo T(2) MR images. Here, MR imaging properties of dysprosium hydroxide nanorods have not been reported so far. These two samples show r(2)s of 65.04 and 181.57 s(-1)mM(-1), respectively, with negligible r(1)s at 1.5 tesla and at room temperature, no in vitro cytotoxicity up to 100 µM Dy, and clear negative contrast enhancements in 3 tesla in vivo T(2) MR images of a mouse liver, which will be even more improved at higher MR fields. Therefore, d-glucuronic acid coated ultrasmall dysprosium oxide nanoparticles with renal excretion can be a potential candidate as a sensitive T(2) MRI contrast agent at MR field greater than 3 tesla.

A facile synthesis, in vitro and in vivo MR studies of d-glucuronic acid-coated ultrasmall Ln2O3 (Ln = Eu, Gd, Dy, Ho, and Er) nanoparticles as a new potential MRI contrast agent.

A facile one-pot synthesis of d-glucuronic acid-coated ultrasmall Ln(2)O(3) (Ln = Eu, Gd, Dy, Ho, and Er) nanoparticles is presented. Their water proton relaxivities were studied to address their possibility as a new potential MRI contrast agent. We focused on the d-glucuronic acid-coated ultrasmall Dy(2)O(3) nanoparticle because it showed the highest r(2) relaxivity among studied nanoparticles. Its performance as a T(2) MRI contrast agent was for the first time proved in vivo through its 3 T T(2) MR images of a mouse, showing that it can be further exploited for the rational design of a new T(2) MRI contrast agent at high MR fields.

Water-soluble MnO nanocolloid for a molecular T1 MR imaging: a facile one-pot synthesis, in vivo T1 MR images, and account for relaxivities.

A facile one-pot synthesis of a water-soluble MnO nanocolloid (i.e., D-glucuronic acid-coated MnO nanoparticle) is presented. The MnO nanoparticle in the MnO nanocolloid was coated with a biocompatible and hydrophilic D-glucuronic acid, and its particle diameter was nearly monodisperse and ranged from 2 to 3 nm. The average hydrodynamic diameter of the MnO nanocolloid was estimated to be 5 nm. The MnO nanoparticle was nearly paramagnetic down to T=3 K. The MnO nanocolloid showed a high longitudinal water proton relaxivity of r1=7.02 s(-1) mM(-1) with the r2/r1 ratio of 6.83 due to five unpaired S-state electrons of Mn(II) ion (S=5/2) as well as a high surface to volume ratio of the MnO nanoparticle. High contrast in vivo T1 MR images were obtained for various organs, showing the capability of the MnO nanocolloid as a sensitive T1 MRI contrast agent. The suggested three key-parameters which control the r1 and r2 relaxivities of nanocolloids (i.e., the S value of a metal ion, the spin structure, and the surface to volume ratio of a nanoparticle) successfully accounted for the observed r1 and r2 relaxivities of the MnO nanocolloid.