Safety and Efficacy of Riluzole in Acute Spinal Cord Injury Study (RISCIS): A Multi-Center, Randomized, Placebo-Controlled, Double-Blinded Trial.

Riluzole is a sodium-glutamate antagonist that attenuates neurodegeneration in amyotrophic lateral sclerosis (ALS). It has shown favorable results in promoting recovery in pre-clinical models of traumatic spinal cord injury (tSCI) and in early phase clinical trials. This study aimed to evaluate the efficacy and safety of riluzole in acute cervical tSCI. An international, multi-center, prospective, randomized, double-blinded, placebo-controlled, adaptive, Phase III trial (NCT01597518) was undertaken. Patients with American Spinal Injury Association Impairment Scale (AIS) A-C, cervical (C4-C8) tSCI, and <12 h from injury were randomized to receive either riluzole, at an oral dose of 100 mg twice per day (BID) for the first 24 h followed by 50 mg BID for the following 13 days, or placebo. The primary efficacy end-point was change in Upper Extremity Motor (UEM) scores at 180 days. The primary efficacy analyses were conducted on an intention to treat (ITT) and completed cases (CC) basis. The study was powered at a planned enrolment of 351 patients. The trial began in October 2013 and was halted by the sponsor on May 2020 (and terminated in April 2021) in the face of the global COVID-19 pandemic. One hundred ninety-three patients (54.9% of the pre-planned enrolment) were randomized with a follow-up rate of 82.7% at 180 days. At 180 days, in the CC population the riluzole-treated patients compared with placebo had a mean gain of 1.76 UEM scores (95% confidence interval: -2.54-6.06) and 2.86 total motor scores (CI: -6.79-12.52). No drug-related serious adverse events were associated with the use of riluzole. Additional pre-planned sensitivity analyses revealed that in the AIS C population, riluzole was associated with significant improvement in total motor scores (estimate: standard error [SE] 8.0; CI 1.5-14.4) and upper extremity motor scores (SE 13.8; CI 3.1-24.5) at 6 months. AIS B patients had higher reported independence, measured by the Spinal Cord Independence Measure score (45.3 vs. 27.3; d: 18.0 CI: -1.7-38.0) and change in mental health scores, measured by the Short Form 36 mental health domain (2.01 vs. -11.58; d: 13.2 CI: 1.2-24.8) at 180 days. AIS A patients who received riluzole had a higher average gain in neurological levels at 6 months compared with placebo (mean 0.50 levels gained vs. 0.12 in placebo; d: 0.38, CI: -0.2-0.9). The primary analysis did not achieve the predetermined end-point of efficacy for riluzole, likely related to insufficient power. However, on pre-planned secondary analyses, all subgroups of cervical SCI subjects (AIS grades A, B and C) treated with riluzole showed significant gains in functional recovery. The results of this trial may warrant further investigation to extend these findings. Moreover, guideline development groups may wish to assess the possible clinical relevance of the secondary outcome analyses, in light of the fact that SCI is an uncommon orphan disorder without an accepted neuroprotective treatment.

Stem Cell Theory of Cancer: Implications for Drug Resistance and Chemosensitivity in Cancer Care.

When it concerns cancer care and cancer therapy, drug resistance is more than an obstacle to successful treatment; it is a major cause of frustration in our attempts to optimize drug development versus therapy development. Importantly, overcoming the challenges of drug resistance may provide invaluable clues about the origin and nature of cancer. From this perspective, we discuss how chemoresistance and chemosensitivity in cancer therapy could be directly linked to the stem cell origin of cancer. A stem cell theory of cancer stipulates that both normal stem cells and cancer stem cells are similarly endowed with robust efflux pumps, potent antiapoptotic mechanisms, redundant DNA repair systems, and abundant antioxidation reserves. Cancer stem cells, like their normal stem cell counterparts, are equipped with the same drug resistance phenotypes (e.g., ABC transporters, anti-apoptotic pathways, and DNA repair mechanisms). Drug resistance, like other cancer hallmarks (e.g., tumor heterogeneity and cancer dormancy), could be intrinsically ingrained and innately embedded within malignancy. We elaborate that cellular context and the microenvironment may attenuate the effects of cancer treatments. We examine the role of circadian rhythms and the value of chronotherapy to maximize efficacy and minimize toxicity. We propose that a stem cell theory of drug resistance and drug sensitivity will ultimately empower us to enhance drug development and enable us to improve therapy development in patient care.

Pharmacokinetic Basis for Using Saliva Matrine Concentrations as a Clinical Compliance Monitoring in Antitumor B Chemoprevention Trials in Humans.

This study reports the first clinical evidence of significantly high secretion of matrine in a multi-component botanical (Antitumor B, ATB) into human saliva from the systemic circulation. This is of high clinical significance as matrine can be used as a monitoring tool during longitudinal clinical studies to overcome the key limitation of poor patient compliance often reported in cancer chemoprevention trials. Both matrine and dictamine were detected in the saliva and plasma samples but only matrine was quantifiable after the oral administration of ATB tablets (2400 mg) in 8 healthy volunteers. A significantly high saliva/plasma ratios for Cmax (6.5 ± 2.0) and AUC0-24 (4.8 ± 2.0) of matrine suggested an active secretion in saliva probably due to entero-salivary recycling as evident from the long half-lives (t1/2 plasma = 10.0 ± 2.8 h, t1/2 saliva = 13.4 ± 6.9 h). The correlation between saliva and plasma levels of matrine was established using a population compartmental pharmacokinetic co-model. Moreover, a species-relevant PBPK model was developed to adequately describe the pharmacokinetic profiles of matrine in mouse, rat, and human. In conclusion, matrine saliva concentrations can be used as an excellent marker compound for mechanistic studies of active secretion of drugs from plasma to saliva as well as monitor the patient's compliance to the treatment regimen in upcoming clinical trials of ATB.

Development & validation of LC-MS/MS assay for 5-amino-1-methyl quinolinium in rat plasma: Application to pharmacokinetic and oral bioavailability studies.

5-Amino-1-methyl quinolinium (5-AMQ) is a potent Nicotinamide N-methyl transferase (NNMT) inhibitor. NNMT is an enzyme that catalyzes the N-methylation of the endogenous substrate nicotinamide, as well as exogenous xenobiotics. NNMT is fundamental to cellular metabolism; NNMT is overexpressed in select tissues (e.g., adipose tissue, skeletal muscle, etc.) in pathophysiological conditions, making it a clinically relevant target for drug development in several chronic diseases including obesity and diabetes. The objective of this study was to develop and validate a simple, sensitive, and reproducible liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of 5-AMQ in rat plasma and urine samples. 5-AMQ was extracted from plasma and urine by protein precipitation. Chromatographic separation was achieved using an ACE® Excel™ C18 column (2 µm, 50 × 2.1 mm) with a binary gradient solvent system comprising of water (A) and acetonitrile (B) containing 0.1 % formic acid as the mobile phase. Analysis was performed using an API 4000 QTRAP hybrid triple quadruple mass spectrometer and multiple reaction monitoring (MRM) in positive mode at m/z transitions of 159.100 → 90.00 and 162.200 → 117.200 for 5-AMQ and the internal standard, respectively. The standard curves of 5-AMQ in rat urine and plasma samples were linear in the concentration range of 10-2500 ng/mL. The intra-day and inter-day precisions and accuracies for 5-AMQ at four concentration levels in rat plasma and urine samples were found to be within the 15 % FDA acceptance range. Similarly, the accuracy and precision of 5-AMQ quantification in samples diluted up to 20-fold using blank plasma were within the 15 % acceptable range. Furthermore, the extraction recoveries and matrix effects at three concentration levels of rat plasma samples ranged from 99.5 %-110.6 % and -6.1 %-14.1 %, respectively. 5-AMQ was stable in rat plasma samples subjected to standard storage, preparation, and handling conditions, with less than 15 % variation noted at two concentration levels. The validated, sensitive, and reproducible LC-MS/MS method for 5-AMQ in rat plasma and urine samples was effectively applied to a pharmacokinetic study in rats with IV and oral administration of 5-AMQ. 5-AMQ displayed substantial plasma exposures via IV and oral route, with a mean maximum plasma concentration of 2252 ng/mL after oral administration, mean area under the curve (AUC0-∞) of 3708 h.ng/mL and 14431 h.ng/mL for the IV and oral groups, respectively, mean terminal elimination half-life of 3.80 ± 1.10 h and 6.90 ± 1.20 h respectively after intravenous and oral dose, and a good oral bioavailability (F % = 38.4).

Simultaneous quantification of vincristine and its major M1 metabolite from dried blood spot samples of Kenyan pediatric cancer patients by UPLC-MS/MS.

Vincristine (VCR) is an integral part of chemotherapy regimens in the US and in developing countries. There is a paucity of information about its disposition and optimal therapeutic dosing. VCR is preferentially metabolized to its major M1 metabolite by the polymorphic CYP3A5 enzyme, which may be clinically significant as CYP3A5 expression varies across populations. Thus, it is important to monitor both VCR and M1 and characterize their dispositions. A previously developed HPLC-MS/MS method for VCR quantification was not sensitive enough to quantify the M1 metabolite beyond 1 h post VCR dose (not published). Establishing a highly sensitive assay is a pre-requisite to simultaneously quantify and monitor VCR and M1, which will enable characterization of drug exposure and dispositions of both analytes in a pediatric cancer population. The addition of formic acid during the extraction process enhanced M1 extraction from DBS samples. A sensitive, accurate, and precise UPLC-MS/MS assay method for the simultaneous quantification of VCR and M1 from human dried blood spots (DBS) was developed and validated. Chromatographic separation was performed on Inertsil ODS-3 C18 column (5 µm, 3.0 × 150 mm). A gradient elution of mobile phase A (methanol-0.2 % formic acid in water, 20:80 v/v) and mobile phase B (methanol-0.2 % formic acid in water, 80:20 v/v) was used with a flow rate of 0.4 mL/min and a total run time of 5 min. The analytes were ionized by electrospray ionization in the positive ion mode. The linearity range for both analytes in DBS were 0.6-100 ng/mL for VCR and 0.4-100 ng/mL for M1. The intra- and inter-day accuracies for VCR and M1 were 93.10-117.17 % and 95.88-111.21 %, respectively. While their intra- and inter-day precisions were 1.05-10.11 % and 5.78-8.91 %, respectively. The extraction recovery of VCR from DBS paper was 35.3-39.4 % and 10.4-13.4 % for M1, with no carryover observed for both analytes. This is the first analytical method to report the simultaneous quantification of VCR and M1 from human DBS. For the first time, concentrations of M1 from DBS patient samples were obtained beyond 1 h post VCR dose. The developed method was successfully employed to monitor both compounds and perform pharmacokinetic analysis in a population of Kenyan pediatric cancer patients.

Pharmacokinetic Model Analysis of Supralingual, Oral and Intravenous Deliveries of Mycophenolic Acid.

Mycophenolic acid (MPA) is commonly used for organ rejection prophylaxis via oral administration in the clinic. Recent studies have shown that MPA also has anticancer activities. To explore new therapeutic options for oral precancerous/cancerous lesions, MPA was designed to release topically on the dorsal tongue surface via a mucoadhesive patch. The objective of this study was to establish the pharmacokinetic (PK) and tongue tissue distribution of mucoadhesive MPA patch formulation after supralingual administration in rats and also compare the PK differences between oral, intravenous, and supralingual administration of MPA. Blood samples were collected from Sprague Dawley rats before and after a single intravenous bolus injection, a single oral dose, or a mucoadhesive patch administration on the dorsal tongue surface for 4 h, all with a dose of 0.5 mg/kg of MPA. Plots of MPA plasma concentration versus time were obtained. As multiple peaks were found in all three curves, the enterohepatic recycling (EHR) model in the Phoenix software was adapted to describe their PK parameters with an individual PK analysis method. The mean half-lives of intravenous and oral administrations were 10.5 h and 7.4 h, respectively. The estimated bioavailability after oral and supralingual administration was 72.4% and 7.6%, respectively. There was a 0.5 h lag-time presented after supralingual administration. The results suggest that the systemic plasma MPA concentrations were much lower in rats receiving supralingual administration compared to those receiving doses from the other two routes, and the amount of MPA accumulated in the tongue after patch application showed a sustained drug release pattern. Studies on the dynamic of drug retention in the tongue after supralingual administration showed that ~3.8% of the dose was accumulated inside of tongue right after the patch removal, ~0.11% of the dose remained after 20 h, and ~20.6% of MPA was not released from the patches 4 h after application. The data demonstrate that supralingual application of an MPA patch can deliver a high amount of drug at the site of administration with little systemic circulation exposure, hence lowering the potential gastrointestinal side effects associated with oral administration. Thus, supralingual administration is a potential alternative route for treating oral lesions.

Longitudinal Impact of Acute Spinal Cord Injury on Clinical Pharmacokinetics of Riluzole, a Potential Neuroprotective Agent.

Riluzole, a benzothiazole sodium channel blocker that received US Food and Drug Administration approval to attenuate neurodegeneration in amyotrophic lateral sclerosis in 1995, was found to be safe and potentially efficacious in a spinal cord injury (SCI) population, as evident in a phase I clinical trial. The acute and progressive nature of traumatic SCI and the complexity of secondary injury processes can alter the pharmacokinetics of therapeutics. A 1-compartment with first-order elimination population pharmacokinetic model for riluzole incorporating time-dependent clearance and volume of distribution was developed from combined data of the phase 1 and the ongoing phase 2/3 trials. This change in therapeutic exposure may lead to a biased estimate of the exposure-response relationship when evaluating therapeutic effects. With the developed model, a rational, optimal dosing scheme can be designed with time-dependent modification that preserves the required therapeutic exposure of riluzole.

Sensitive and rapid UHPLC-MS/MS assay for simultaneous quantifications of calcipotriol and paclitaxel in rat whole blood and plasma samples.

Vitamin-D analogues have emerged as potential stroma-modulating agents for the treatment of pancreatic ductal adenocarcinoma (PDAC). One such agent, calcipotriol (Cal) has shown significant activity in in vitro and in vivo models of pancreatic ductal adenocarcinoma. Attempts in our lab have been focused on establishing the therapeutic merits of co-formulating this agent with the chemotherapeutic drug paclitaxel (PTX) in animal models. Accurate and reliable quantifications of these agents is critical to successful pharmacokinetic/pharmacodynamic (PK/PD) projections from animals into humans. Herein, we developed a LC-MS/MS assay for measuring Cal and PTX in whole blood and plasma. A liquid-liquid analyte extraction procedure, using a mixture of water-MeOH (50:50, v/v) and hexane-dichloromethane- isopropyl alcohol (150:15:5, v/v/v) was used. Chromatographic separation was carried out on Kinetex C18 column (1.7 µm, 100 × 2.10 mm) under an isocratic elution at a flow rate of 0.4 mL/min with a total runtime of 3.5 min. The mobile phase was composed of ammonium acetate (pH 6.51; 5 mM)-methanol (15:85, v/v). The analytes were ionized by positive electrospray ionization using API 5500-Qtrap triple quadrupole mass spectrometer (Applied Biosystem/AB SCIEX). The linearity of calibration curves for both analytes were established at 0.5 (LLOQ)-500 ng/mL with correlation coefficients exceeding 0.99. Spiked whole blood and plasma samples were used as surrogates for matrix validation. For both analytes, the intra-day and inter-day accuracies were 90.5-105 % and 96.6-106 %, respectively, while the corresponding precisions were 3.09-10.7 % and 5.20-12.9 %. No carryover was observed for the analytes which also remained acceptably stable in the surrogate matrices under relevant conditions. The assay is robust, reliable and sensitive in rat whole blood and plasma. The analytes extraction procedure performs acceptably well in both matrices with high recoveries and minimal matrix effects. Additionally, only 20 µL of rat whole blood or plasma is required and the total run time per sample is 3.5 min. PK studies enabled by the assay revealed that when co-administered, PTX AUC0→∞ and Cmax increased while those of Cal decreased. This finding alerts a potential drug-drug interaction and warrants further investigation in studies using this combination regimen.

Doxorubicin-loaded hollow gold nanospheres for dual photothermal ablation and chemoembolization therapy.

BACKGROUND: Doxorubicin-loaded hollow gold nanospheres (Dox@HAuNS) are a promising technology for simultaneous trans-arterial tumor-targeted chemotherapy delivery and thermal ablation. We evaluated the efficacy of intra-arterial delivery of Dox@HAuNS followed by photothermal ablation (PTA) in a rabbit model of liver cancer. Adult New Zealand white rabbits (N=25) were inoculated with VX2 tumors into the left lobe of the liver. The animals were then randomized to sham surgery (N=5), PTA only (N=3), Dox@HAuNS only (N=5), HAuNS + PTA (N=5), and Dox@HAuNS + PTA (N=7). Nanoparticles were delivered as an emulsion with Lipiodol (Guerbet, France) via a trans-arterial approach. Following nanoparticle delivery, PTA was performed using an 808nm fibered laser at 1.5W for 3 minutes. Thermography during PTA demonstrated a sustained elevation in tumoral temperature in both HAuNS + laser and Dox@HAuNS + laser treatment groups relative to animals that underwent laser treatment without prior nanoparticle delivery. RESULTS: There was a significant decrease in tumor volumes in all three treatment arms relative to control arms (P = 0.004). Concentrations of intratumoral doxorubicin were significantly greater in animals treated with laser compared to those that were not treated with laser (P< 0.01). CONCLUSIONS: Doxorubicin-loaded HAuNS is a promising therapeutic agent for dual ablation/chemoembolization treatment of liver cancer.

Effects of inflammation on irinotecan pharmacokinetics and development of a best-fit PK model.

Irinotecan is a chemotherapeutic drug used in the treatment of advanced colorectal cancer and elevated blood concentrations of its active metabolite, SN-38 leads to increased gastrointestinal (GI) toxicity and diarrhea in patients. In this study, we investigated the effects of inflammation on the pharmacokinetics (PK) of irinotecan (CPT-11) and its active metabolite, SN-38. Mice were i.p.-injected with either saline or lipopolysaccharide (LPS) to induce inflammation. After 16 h, irinotecan was administered orally. Blood was collected from the tail vein of mice from 0 to 24 h after dosing. Concentrations of irinotecan, SN-38 and SN-38G were analyzed using LC-MS/MS. The AUC, Cmax, and tmax were derived using WinNonlin® 5.2. A PK model was developed using Phoenix NLME® to describe the PK of irinotecan and SN-38 during inflammation. Results indicated a significant increase in the blood concentrations of irinotecan and SN-38 in mice during inflammation. The AUC of irinotecan and SN-38 in LPS group were 2.6 and 2-folds, respectively, of those in control saline-treated mice. The Cmax of irinotecan and SN-38 in LPS treated mice were 2.4 and 2.3-folds of those in saline-treated mice. The PK model was successfully developed and validated. The best-fit plots of individual PK analysis showed a good correlation between observed and predicted concentrations of irinotecan and SN-38. Together, this study reveals that SN-38 concentrations are elevated during inflammation, which may increase the GI toxicity and diarrhea in patients who receive irinotecan; and the developed PK model can quantitatively describe the PK of irinotecan and SN-38 during inflammation.

Copper-64 Labeled PEGylated Exosomes for In Vivo Positron Emission Tomography and Enhanced Tumor Retention.

Exosomes have attracted tremendous attention due to their important role in physiology, pathology, and oncology, as well as promising potential in biomedical applications. Although great efforts have been dedicated to investigating their biological properties and applications as natural cancer drug-delivery systems, the systemic biodistribution of exosomes remains underexplored. In addition, exosome-based drug delivery is inevitably hindered by the robust liver clearance, leading to suboptimal tumor retention and therapeutic efficiency. In this study, we report one of the first examples using in vivo positron emission tomography (PET) for noninvasive monitoring of copper-64 (64Cu)-radiolabeled polyethylene glycol (PEG)-modified exosomes, achieving excellent imaging quality and quantitative measurement of blood residence and tumor retention. PEGylation not only endowed exosomes with a superior pharmacokinetic profile and great accumulation in the tumor versus traditionally reported native exosomes but also reduced premature hepatic sequestration and clearance of exosomes, findings that promise enhanced therapeutic delivery efficacy and safety in future studies. More importantly, this study provides important guidelines about surface engineering, radiochemistry, and molecular imaging in obtaining accurate and quantitative biodistribution information on exosomes, which may benefit future exploration in the realm of exosomes.

Correction to: Longitudinal Impacts of Gastric Bypass Surgery on Pharmacodynamics and Pharmacokinetics of Statins.

In the original article the authors failed to include the following footnote.

Longitudinal Impacts of Gastric Bypass Surgery on Pharmacodynamics and Pharmacokinetics of Statins.

PURPOSE: Undergoing Roux-en-Y gastric bypass (RYGB) is expected to affect orally administered drug absorption. Statins are commonly prescribed to patients with obesity for the prevention of atherosclerotic cardiovascular diseases by lowering cholesterol. This is the first longitudinal prospective study on impacts of RYGB on weight loss, pharmacodynamics, and pharmacokinetics of atorvastatin, rosuvastatin, and simvastatin, and their active metabolites, up to 1-year post-surgery. METHODS: Forty-six patients were recruited, five patients on atorvastatin, twelve on rosuvastatin, nine on simvastatin, and twenty on no statin. The concentrations of atorvastatin, rosuvastatin, and simvastatin with their active metabolites were monitored. RESULTS: Mean plasma concentrations of atorvastatin and metabolites and rosuvastatin normalized by the unit dose [(nM)/(mg/kg)] decreased by 3- to 6-month post-surgery. Conversely, simvastatin and its metabolite concentrations increased up to 6-month post-surgery, then declined to preoperative levels by 1-year post-surgery. The metabolisms of atorvastatin to hydroxyl-metabolites and simvastatin to simvastatin acid were decreased after RYGB. The weight loss and PD outcomes were comparable between statin and non-statin groups suggesting the key impacts were from RYGB. The discontinuation or reduction of dose of atorvastatin or rosuvastatin post-RYGB exhibited rebounds of LDL levels in some subjects, but the rebound was not apparent with patients on simvastatin pre-surgery. CONCLUSION: Discontinuations of statin dosing post-RYGB require LDL monitoring and reducing the dose to half seems to have better results. Patients on statin treatment post-RYGB should be followed-up closely based on our pharmacokinetic findings, to ensure therapeutic effects of the treatment with minimal adverse effects.

Development and validation of a rapid and sensitive UPLC-MS/MS assay for simultaneous quantification of paclitaxel and cyclopamine in mouse whole blood and tissue samples.

The prominent stromal compartment surrounds pancreatic ductal adenocarcinoma and protects the tumor cells from chemo- or radiotherapy. We hypothesized that our nano formulation carrying cyclopamine (CPA, stroma modulator) and paclitaxel (PTX, antitumor agent) could increase the permeation of PTX through the stromal compartment and improve the intratumoral delivery of PTX. In the present study a sensitive, reliable UPLC-MS/MS method was developed and validated to quantify PTX and CPA simultaneously in mouse whole blood, pancreas, liver and spleen samples. Docetaxel was used as the internal standard. The method demonstrated a linear range of 0.5-2000 ng/mL for whole blood and tissue homogenates for both PTX and CPA. The accuracy and precision of the assay were all within ±15%. Matrix effects for both analytes were within 15%. Recoveries from whole blood, liver, spleen and pancreas homogenates were 92.7-105.2% for PTX and 72.8-99.7% for CPA. The stability was within ±15% in all test biomatrices. The validated method met the acceptance criteria according to US Food and Drug Administration regulatory guidelines. The method was successfully applied to support a pharmacokinetic and biodistribution study for PTX and CPA in mice biomatrices.

Simultaneous LC-MS/MS analysis of simvastatin, atorvastatin, rosuvastatin and their active metabolites for plasma samples of obese patients underwent gastric bypass surgery.

Statins, HMG-CoA reductase inhibitors, are considered the first line treatment of hyperlipidemia to reduce the risk of atherosclerotic cardiovascular diseases. The prevalence of hyperlipidemia and the risk of atherosclerotic cardiovascular diseases are higher in obese patients. Published methods for the quantification of statins and their active metabolites did not test for matrix effect of or validate the method in hyperlipidemic plasma. A sensitive, specific, accurate, and reliable LC-MS/MS method for the simultaneous quantification of simvastatin (SMV), active metabolite of simvastatin acid (SMV-A), atorvastatin (ATV), active metabolites of 2-hydroxy atorvastatin (2-OH-ATV), 4-hydroxy atorvastatin (4-OH-ATV), and rosuvastatin (RSV) was developed and validated in plasma with low (52-103 mg/dl, <300 mg/dl) and high (352-403 mg/dl, >300 mg/dl) levels of triglyceride. The column used in this method was ACQUITY UPLC BEH C18 column (2.1 × 100 mm I.D., 1.7 µm). A gradient elution of mobile phase A (10 mM ammonium formate and 0.04% formic acid in water) and mobile phase B (acetonitrile) was used with a flow rate of 0.4 ml/min and run time of 5 min. The transitions of m/z 436.3 → 285.2 for SMV, m/z 437.2 → 303.2 for SMV-A, m/z 559.2 → 440.3 for ATV, m/z 575.4 → 440.3 for 2-OH-ATV and 4-OH-ATV, m/z 482.3 → 258.1 for RSV, and m/z 412.3 → 224.2 for fluvastatin (internal standard, IS) were determined by Selected Reaction Monitoring (SRM) method to detect transitions ions in the positive ion mode. The assay has a linear range of 0.25 (LLOQ) -100 ng/ml for all six analytes. Accuracy (87-114%), precision (3-13%), matrix effect (92-110%), and extraction recovery (88-100%) of the assay were within the 15% acceptable limit of FDA Guidelines in variations for plasma with both low and high triglyceride levels. The method was used successfully for the quantification of SMV, ATV, RSV, and their active metabolites in human plasma samples collected for an ongoing clinical pharmacokinetic and pharmacodynamic study on patients prior to and post gastric bypass surgery (GBS).

Integrin αvß3-Targeted [64Cu]CuS Nanoparticles for PET/CT Imaging and Photothermal Ablation Therapy.

Copper sulfide (CuS) nanoparticles have been considered one of the most clinical relevant nanosystems because of their straightforward chemistry, small particle size, low toxicity, and intrinsic theranostic characteristics. In our previous studies, radioactive [64Cu]CuS nanoparticles were successfully developed to be used as efficient radiotracers for positron emission tomography and for photothermal ablation therapy of cancer cells using near-infrared laser irradiation. However, the major challenge of CuS nanoparticles as a theranostic platform is the lack of a means for effective targeted delivery to the tumor site. To overcome this challenge, we designed and synthesized angiogenesis-targeting [64Cu]CuS nanoparticles, which are coupled with cyclic RGDfK peptide [c(RGDfK)] through polyethylene glycol (PEG) linkers using click chemistry. In assessing their tumor-targeting efficacy, we found that the tumor uptakes of [64Cu]CuS-PEG-c(RGDfK) nanoparticles at 24 h after intravenous injection were significantly greater (8.6% ± 1.4% injected dose/gram of tissue) than those of nontargeted [64Cu]CuS-PEG nanoparticles (4.3% ± 1.2% injected dose/gram of tissue, p < 0.05). Irradiation of tumors in mice administered [64Cu]CuS-PEG-c(RGDfK) nanoparticles induced 98.7% necrotic areas. In contrast, irradiation of tumors in mice administered nontargeted CuS-PEG nanoparticles induced 59% necrotic areas ( p < 0.05). The angiogenesis-targeting [64Cu]CuS nanoparticles may serve as a promising platform for image-guided ablation therapy with high efficacy and minimal side effects in future clinical translation of this novel class of multifunctional nanomaterials.

Rational design and development of a stable liquid formulation of riluzole and its pharmacokinetic evaluation after oral and IV administrations in rats.

Enterally administered riluzole is currently being investigated in a Phase II/III clinical trial for the treatment of acute spinal cord injury (SCI). Many SCI patients suffer from severe motor dysfunction and exhibit swallowing difficulties and cannot swallow riluzole tablets. The purpose of the present study was to develop a liquid solution formulation of riluzole, which can be administered more easily to this patient population with the capability to adjust the dose if needed. Riluzole was solubilized using water miscible organic solvents, namely, polyethylene glycol 400, propylene glycol and glycerin. A Central Composite Design (CCD) approach was used to develop an optimum co-solvent composition that can solubilize the entire 50 mg dose of riluzole in 5 ml. A three-factor five-level design was employed to investigate the effects of composition of co-solvents on riluzole solubility. The selected optimum formulation consists of 15% v/v PEG 400, 20% v/v propylene glycol and 10% v/v glycerin, with riluzole concentration of 10 mg/ml. The optimum composition was assessed for stability at different temperatures. Satisfactory stability was obtained at room temperature and 4 °C (t90 of 17 and 35 months, respectively). The optimum formulation of riluzole was suitable for both oral and intravenous administrations. Single dose pharmacokinetic studies of the optimum formulation by oral and IV routes were evaluated in rats, using commercially available Rilutek® tablets as a reference. The co-solvent formulation was well tolerated both orally and intravenously. In comparison to the commercial tablet, the co-solvent formulation had a faster rate of absorption and more sustained plasma levels with a significantly longer elimination half-life. Higher concentrations of riluzole in brain and spinal cord were achieved from co-solvent formulation as compared to tablet. The riluzole solution formulation is stable and offers advantages of ease of administration, consistent dosing, rapid onset and longer duration of action, better availability at site of action which can be extremely beneficial for the therapy in SCI patients.

Stromal Modulation Reverses Primary Resistance to Immune Checkpoint Blockade in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most difficult cancers to treat. It is refractory to most existing therapies, including immunotherapies, due to the presence of an excessive desmoplastic stroma, which restricts penetration of drugs and cytotoxic CD8+ T cells. Stromal modulation has shown promising results in the enhancement of immune checkpoint blockade treatment in PDAC. We demonstrate here effective stromal modulation by a polymeric micelle-based nanoformulation to codeliver a sonic hedgehog inhibitor (cyclopamine, abbreviated as CPA) and a cytotoxic chemotherapy drug (paclitaxel, abbreviated as PTX). The formulation, M-CPA/PTX, modulated the PDAC stroma by increasing the intratumoral vasculature density, which then promoted the tumor infiltration by cytotoxic CD8+ T cells without depletion of tumor-restraining α-smooth muscle action-positive fibroblasts and type I collage in the stroma. The combination of M-CPA/PTX and the PD-1 checkpoint blockade significantly prolonged animal survival in an orthotopic murine PDAC model as well as a genetically engineered mouse model of PDAC. The superior antitumor efficacy was mediated by enhanced tumor infiltration of CD8+ T cells without concomitant infiltration of suppressive regulatory T cells or myeloid-derived suppressor cells and by the coordinated action of PTX and interferon-gamma. Our results demonstrate that stroma-modulating nanoformulations are a promising approach to potentiate immune checkpoint blockade therapy of pancreatic cancer.

Ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for simultaneous quantifications of CZ48, lactone-stabilized camptothecin, and camptothecin and their pharmacokinetic and biliary evaluations in rats.

CZ48, a prodrug of camptothecin (CPT), has a broad spectrum of antitumor activity against various types of human tumors without severe toxicity in preclinical human tumor-xenografted mouse models, which facilitates further preclinical and clinical pharmacokinetic (PK) evaluations of CZ48. In this study, a UHPLC-MS/MS method was developed and validated to simultaneously quantify CZ48 and CPT in rat plasma and bile. Detection was performed using the API 3200 Q Trap triple quadrupole mass spectrometer in a positive ion mode. Chromatographic separation was achieved on Waters ACQUITY UPLC BEH Shield RP18 column with a gradient elution at a flow rate of 0.45 ml/min, using mobile phases of 0.1% acetic acid in water (A) and 0.1% acetic acid in acetonitrile (B). The method was linear at the concentration ranges of 0.98 (LLOQ) -1000 ng/ml of CZ48 and CPT in rat plasma and 3.9 (LLOQ) -1000 ng/ml in bile. Intra- and inter-day accuracy and precision values did not deviate by more than 6.57% and 10.15% for CZ48 and CPT, respectively, in plasma, and 12.09% and 13.48% in bile. Extraction recoveries of CZ48 were 90.18-95.42% from plasma and 86.51 -91.66% from bile. The recoveries of CPT were 91.56-97.06% from plasma and 84.89-89.15% from bile. No significant matrix effects were observed in plasma and bile within 14.00% and 16.19%, respectively. CZ48 and CPT in plasma were stable after extraction process and different storage conditions, including bench-top, processed sample in autosampler, three cycles of freeze and thaw, and long-term (3 month) stability at -80 °C. The application of the validated method was demonstrated by a PK study after an intravenous dose of CZ48 in rats.

Simultaneous inhibition of hedgehog signaling and tumor proliferation remodels stroma and enhances pancreatic cancer therapy.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers. It has an excessive desmoplastic stroma that can limit the intratumoral delivery of chemotherapy drugs, and protect tumor cells against radiotherapy. Therefore, both stromal and tumor compartments need to be addressed in order to effectively treat PDAC. We hereby co-deliver a sonic hedgehog inhibitor, cyclopamine (CPA), and a cytotoxic chemotherapy drug paclitaxel (PTX) with a polymeric micelle formulation (M-CPA/PTX). CPA can deplete the stroma-producing cancer-associated fibroblasts (CAFs), while PTX can inhibit tumor proliferation. Here we show that in clinically relevant PDAC models, M-CPA effectively modulates stroma by increasing microvessel density, alleviating hypoxia, reducing matrix stiffness while maintaining the tumor-restraining function of extracellular matrix. M-CPA/PTX also significantly extends animal survival by suppressing tumor growth and lowering the percentages of poorly to moderately differentiated tumor phenotypes. Our study suggests that using multifunctional nanoparticles to simultaneously target stromal and tumor compartments is a promising strategy for PDAC therapy.