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A sensitive and robust liquid chromatography – tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously quantify the concentration levels of selective cyclo-oxygenase- 2 inhibitor, etoricoxib, and glutamate antagonist, riluzole, in rat plasma and brain tissue. Labeled internal standards (IS) etoricoxib D4 and [13C,15N2] riluzole were used for the quantifications of etoricoxib and riluzole, respectively. Analytes riluzole and etoricoxib, as well as their IS's, were separated from rat plasma and brain tissue homogenate by means of liquid – liquid extraction using organic solvent of ethyl acetate. A Waters Acquity UPLC BEH C18 (1.7 µm) column was used for chromatographic separation of analytes on a SCIEX ExionLC AD using a binary solvent system. In conjunction, a SCIEX QTRAP 5500 mass spectrometer was used to quantify etoricoxib at m/z of 359 → 244 (IS of m/z 363 → 248) and riluzole at m/z 235 → 166 (IS of m/z 238 → 169) mass transitions using positive mode electrospray ionization (ESI) with multiple reaction monitoring (MRM). The bioanalytical method was validated over a linear concentration range of 0.25 (LLOQ at average signal to noise >10) – 600 ng/ml in plasma and 0.625 – 1500 ng/g in brain tissue for both analytes. The methods intra/inter-day accuracy and precision are well within the FDA validation recommendations range for chromatographic assays for both riluzole and etoricoxib in rat plasma. A partial validation was conducted within rat brain biomatrix, yielding an acceptable intra-day accuracy and precision for both riluzole and etoricoxib. For riluzole and etoricoxib the average matrix effect and recovery from rat plasma and brain tissue were negligible. This bioanalytical method was effectively applied to perform a pharmacokinetic study of each compound administered concurrently in healthy male Long-Evan (L.E.) rats. This method was developed as a pre-requisite for the pre-clinical pharmacokinetic and pharmacodynamic evaluations of co-treatment with riluzole and etoricoxib in rats with a controlled cortical impact (CCI) induced traumatic brain injury.

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.

Background and aim: We have synthesized a novel lactone-stabilized camptothecin (CPT) analog named CZ48 and demonstrated its potent anticancer effects via bioconversion to the active CPT in earlier studies. Herein, we aimed to develop, optimize and characterize CZ48 nanosuspensions, for a sustained delivery of this drug in humans with an intravenous (i.v.) administration. Methods and materials: A three-factor, five-level central composite design (CCD) was employed to establish the impacts of the critical influencing factors (concentrations (wt%) of CZ48, polysorbate 80 (Tween-80), and Pluronic® F-108 (F-108)) on the responses (particle size and zeta potential). Based on the quantitative influencing factor-response relationships, two optimized CZ48 nanosuspensions of 197.22 ± 7.12 nm (NS-S) and 589.35 ± 23.27 nm (NS-L) were developed with the zeta potential values of -26.5 mV and -27.9 mV, respectively. Results: CZ48 released from the nanosuspensions in a sustained manner in contrast to the rapid release from cosolvent in both PBS and human plasma. Moreover, NS-S exhibited more favored pharmacokinetic properties than NS-L, with a 31-fold prolonged elimination half-life of CPT, and a 2.4-fold enhanced CPT exposure over cosolvent. In efficacy study, NS-S exhibited significant tumor suppression and an improved survival rate with a higher tolerable dose, compared to CZ48 cosolvent. Conclusion: We have successfully developed CZ48 nanosuspensions with significantly favorable pharmacokinetics and improved efficacy using CCD approach. The formulation offers potential merits as a preferred candidate for clinical trials with the prolonged CPT exposure, which is known to correlate with the clinical efficacy.

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).

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.

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.

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 hr. 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 x 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 to 10.11% and 5.78 to 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-hour 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.

In the present study, a sensitive and robust LC–MS/MS method has been developed and validated for the quantification of riluzole in human plasma and cerebrospinal fluid (CSF) in clinical samples from patients with spinal cord injury (SCI). Riluzole and its labeled internal standard (IS) were isolated from plasma and CSF by liquid–liquid extraction using ethyl acetate. Riluzole ( m / z 235 → 166) and IS ( m / z 238 → 169) were detected by electrospray ionization (ESI) using multiple reaction monitoring (MRM) in a positive mode. The assay was linear in the concentration range of 0.5 (LLOQ, signal/noise ratio > 10)–800 ng/ml in plasma, and 1.0 (LLOQ)–800 ng/ml in CSF samples. The intra- and inter-day accuracy in plasma were 94.2–110.0% and 97.8–102.0%, respectively, and those in CSF were 87.6–105.1% and 91.9–98.8%, respectively. The intra- and inter-day precision were 2.2–7.2% and 4.0–9.1%, respectively, in plasma, and 1.4–14.1% and 2.6–11.5%, respectively in CSF. Matrix effect was negligible from both matrices with signal percentages of 97.6–100.6% in plasma and 99.4–106.4% in CSF. The recoveries were >75% in plasma, >84% in CSF with low protein (53.9 mg/dl), and >68% in CSF with high protein (348.2 mg/dl). This method was successfully applied to quantify riluzole concentrations in plasma and CSF from patients with SCI.

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 one-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 months post-surgery. Conversely, simvastatin and its metabolite concentrations increased up to 6 months post-surgery, then declined to pre-operative levels by one-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.

Purpose Irinotecan is a potent and widely used anticancer drug for the treatment of metastatic colorectal cancer and elevated concentrations of its active metabolite, SN-38, causes severe diarrhea,...

The aim of this study was to develop optimal micelles loaded with calcipotriol (Cal) and paclitaxel (PTX) for the treatment of pancreatic ductal adenocarcinoma (PDAC) using a Design of Experiment (DOE) approach. The central composite design (CCD), a type of DOE was used to tune the size and drug release properties of the drug-loaded micelles. This approach yielded optimal Cal and PTX co-loaded micelles (M-Cal/PTX) with size of 40-100 nm, a polydispersity index (PDI) of 0.25 and a zeta potential (ζ) of − 6.2 ± 0.8 mV. When evaluated in vitro, drug release from the micelles showed a biphasic pattern. The initial release, defined as the cumulative 2-hr drug release was less than 25% in all relevant media. This phase was followed by a gradual release with less than 80% of drugs released after 5 days. In vivo, the micelles prolonged the apparent biological half-life of Cal by more than 3 times and a marginal increase for PTX in an orthotopic mouse model of PDAC. The micelle-encapsulated drugs showed extended tumor accumulation when compared to non-encapsulated Cal and PTX at equivalent dose levels. Future studies on the antitumor activity of this novel dual drug payload delivery system are warranted.

The aim of this study was to develop a reliable UPLC-MS/MS assay for accurate quantification of mycophenolic acid (MPA) and its glucuronide conjugates in human plasma. Plasma proteins were precipitated with acetonitrile and the chromatographic separation was achieved on a C18 column with a gradient elution. The detection was performed by a triple quadrupole mass spectrometer in the positive electrospray ionization and multiple reaction monitoring mode. Linearity of the assay was demonstrated over the range of 20-10,000 ng/mL for MPA and MPA glucuronide (MPAG), and 2-1000 ng/mL for acyl MPA glucuronide in human plasma. The assay was precise and accurate with coefficient of variation and bias

Purpose: Pharmacokinetic (PK) dispositions of vincristine (VCR) and its M1 metabolite were characterized through PK co-modeling for Kenyan pediatric cancer patients with acute lymphoblastic leukemia or nephroblastoma. VCR dosing strategies are largely empirical regardless of its extensive use in pediatric oncology. Little information is known about its disposition and optimal therapeutic dosing. Renbarger and associates reported that CYP3A5 enzyme metabolizes VCR to M1 more efficiently than CYP3A4 enzyme. This may be clinically significant as CYP3A5 expression varies among Kenyans (90%), African Americans (AA, 70%) and Caucasians (10-20%). Therefore, it is essential to characterize the disposition of M1 in humans to provide an insight into the inter-ethnic variability in VCR metabolism and clearance, which will be helpful for future dosing regimen optimization. Methods: Pharmacokinetic Study: Dried blood spot (DBS) samples were collected, via finger stick at various time points depending on the feasibility, from 25 Kenyan pediatric cancer patients (13 males/12 females, 1-14 years, BSA of 0.36 - 1.3 m2) after an IV dose of VCR (1.6 – 3.1 mg/m2). Concentrations of VCR and M1 from DBS were quantified using a validated LC-MS-MS assay. Pharmacokinetic Modeling: A population PK (pop PK) co-model was developed using Phoenix® NLMETM software to simultaneously capture and predict the PK of VCR and M1 concentrations. A correlation between the observed and predicted concentrations of VCR and M1 PK was established by pop PK fitting. Model discrimination was performed on data by visual inspection, the goodness of fit plots and AIC values. Results: The best fit pop PK co-model for VCR and its M1 metabolite was established. PK parameter estimates were derived for both analytes. Volumes of distribution for VCR and M1 were 0.12 L (33.55 %CV) and 249.07 L (33.42 %CV), respectively. The elimination rate constants for VCR and M1 were 5.6 hr-1 (22.81 %CV) and 0.00015 hr-1 (66.85 %CV), respectively. The conversion rate constant of VCR to M1 was 9.66 hr-1 (32.49 %CV). Finally, clearance values were 0.67 L/hr for VCR and 0.04 L/hr for M1. In conclusion, VCR and its M1 metabolite exhibit distinct PK characteristics, in that M1 distribution is substantially larger than that of VCR (249 vs 0.12 L), and clearance is slower than that of VCR (0.04 vs 0.67 L/hr). The conversion kinetics of VCR to M1 is characterized for the first time, which may potentially offer a rationale for ethnic disparity in VCR therapy. In addition, a large inter-patient variability is documented even within the same ethnic Kenyan population. After model refinement and validation, our model could potentially serve as a tool for rational VCR dosing regimen modification for Kenyan/AA pediatric cancer patients. Citation Format: Lorita Agu, Lei Wu, Jamie Renbarger, Jodi Skiles, Andi Masters, Diana S-L. Chow. Population pharmacokinetics of vincristine and its metabolite in Kenyan pediatric cancer patients [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-117.

Introduction: The 5-year survival rate for Pancreatic Ductal Adenocarcinoma (PDAC) remains 7%. An emerging treatment strategy involves the combination of vitamin D receptor (VDR) ligands with chemotherapeutic agents to re-program the activated cancer-associated fibroblast (CAFs). Calcipotriol (CAP), a synthetic analogue of vitamin D can reprogram CAFs, but is toxic with side effects including cachexia and edema. Due to the abundance of the VDR in the body, diminished plasma exposure and elimination of calcipotriol together with enhanced accumulation in the tumor are desirable to reduce toxicity and increase efficacy. We propose to achieve this with a slow-releasing micellar nanoformulation with a well-controlled size to take advantage of the leaky tumor fenestrations and enhance delivery. Method: 8-week old male C57BL/6 mice were administered with either formulated or non-formulated single IV bolus dose of CAP and paclitaxel (PTX). Drugs from blood samples were extracted, and simultaneously quantified using validated LC-MS/MS assay. Pharmacokinetic modeling was performed with Phoenix® WinNonlin (8.0) using 2-compartment model. Results: Our co-formulation of CAP and PTX resulted in >2-fold decrease in plasma exposure (AUC) and 5-fold reduction of peak concentration (Cmax) of CAP. Similarly, PTX AUC and Cmax were halved in mice administered with the formulated drugs. The PTX total clearance (CL) was similar between both groups, but CAP CL was 5-fold lower in formulation-administered mice. Further, CAP elimination (k10) from central compartment and from central to peripheral compartments (k12) were 2 and >3-fold slower, respectively, in the formulation group. The reverse elimination (k21) are similar. Similarly, the k10 of PTX was almost halved, while the k12 was 44-fold slower in mice receiving formulated drugs. The reverse elimination from the peripheral to the central compartment (k21) for formulated PTX is almost 10 times slower than in the mice receiving non-formulated PTX. Conclusion: Our micellar co-formulation of CAP and PTX is able to reduce total clearance of these agents from the systemic circulation. Further, rate constants derived show that PTX elimination from the systemic circulation to tissues is very slow and we expect this to correlate with an extended duration of action of this drug. Finally, our formulation halved the systemic exposure and peak concentrations of CAP that should lead to a more favorable safety profile of this agent. Ongoing work will focus on studying the pharmacokinetics of co-formulated CAP and PTX in a larger cohort of animals and we eventually hope to demonstrate the therapeutic merits of our nanoformulation strategy in pancreatic tumor-bearing mice. Citation Format: Victor R Lincha, Jun Zhao, Chun Li, Diana S-L Chow. Pharmacokinetic analysis of co-formulation of calcipotriol and paclitaxel for pancreatic ductal adenocarcinoma shows reduced systemic exposures and eliminations: Implications for safety and efficacy [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr D052.

Objectives: The aim of this study was to develop a pharmacokinetic (PK) model that can determine the PK of vincristine (VCR) and its M1 metabolite using concentration data from Kenyan pediatric cancer patients. VCR is one of the most widely used anticancer agents in treating a variety of malignancies in pediatric oncology. Regardless of its extensive pediatric use, dosing strategies for VCR are largely empirical as there is little information about its disposition and optimal therapeutic dosing. Our collaborator, Dr. Jamie Renbarger and her group have reported that CYP3A5 enzyme generates the major VCR metabolite, M1, more efficiently than CYP3A4 enzyme. This finding maybe clinically significant because CYP3A5 expression varies with up to 90%, 70% and 10-20% of expressions in Kenyans, African Americans and Caucasians, respectively. Therefore, it is crucial to monitor M1 and characterize its disposition in humans to provide an insight of inter-ethnic variability in VCR metabolism and clearance. Methods: Kenyan pediatric cancer patients (8 males:8 females, age range: 1-13 years, weight range: 7.0 - 36.6 kg, body surface area: 0.36 - 1.24 m2) were intravenously administered with VCR (delivered dose, 0.4 - 2.5 mg). Blood samples were collected using human dried blood spot (DBS) collection paper via finger prick at various time points depending on the feasibility and duration of patient stay for care in the hospital. Using a developed LC-MS-MS assay, the concentrations of VCR and M1 were measured from patient DBS samples. A PK compartmental model was developed through co-modeling of VCR and M1 using Phoenix (version 8.0). The model was selected based on comparison of quality of fit plots and on the likelihood ratio test on the difference of criteria (-2LL). Results: The best fit structural model for VCR and its M1 metabolite was established. The model consists of a one-way metabolite formation transfer from VCR to M1 compartments. Good correlations were observed between observed and predicted values of VCR and M1 in the subjects used. The best-fit PK parameter estimates were derived from the PK compartmental model. In one subject, values estimated for PK parameters that only our study could derive include: conversion rate constant from VCR to M1 of 0.04 1/hr, M1 metabolite volume of distribution of 51.49 L, and M1 metabolite elimination rate constant of 0.44 1/hr. Conclusion: For the first time, a compartmental PK model that could determine the PK parameters of VCR and its M1 metabolite from DBS samples through co-modeling was developed. The model resulted in a good fit for the subjects used. The model will be validated after further adjustments and could potentially be used to rationally modify future VCR dosing regimen for Kenyan pediatric patients. Further, this modification could be possibly extrapolated for African American pediatric patients. Additionally, using our validated model, we will characterize and predict the population PK of VCR and its M1 metabolite among different ethnic groups. Citation Format: Lorita Agu, Jamie Renbarger, Diana S-L. Chow. Pharmacokinetic analysis of vincristine and its m1 metabolite in Kenyan pediatric cancer patients through co-modeling [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr C008.

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.

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 here demonstrate effective stromal modulation by a polymeric micelle-based nanoformulation to co-deliver 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 PD-1 checkpoint blockade significantly prolonged animal survival in an orthotopic murine PDAC model as well as a genetically engineered mouse model (GEMM) of PDAC. The superior anti-tumor efficacy was mediated by enhanced tumor infiltration of CD8(+) T cells without concomitant infiltration of suppressive regulatory T cells (Tregs) or myeloid-derived suppressor cells (MDSCs), and by the coordinated action of PTX and interferon-gamma (IFN-γ). Our results demonstrate that stroma-modulating nano-formulations are a promising approach to potentiate immune checkpoint blockade therapy of pancreatic cancer.

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).

The determination of dopamine facilitates better understanding of the complex brain disorders in the central nervous system and the regulation of endocrine system, cardiovascular functions and renal functions in the periphery. The purpose of this study was to develop a highly sensitive and reliable assay for the quantification of dopamine in human neonate plasma. Dopamine was extracted from human plasma by strong cation exchange (SCX) solid phase extraction (SPE), and subsequently derivatized with propionic anhydride. The derivatized analyte was separated by a Waters Acquity UPLC BEH C18 column using gradient elution at 0.4 ml/min with mobile phases A (0.2% formic acid in water [v/v]) and B (MeOH-ACN [v/v, 30:70]). Analysis was performed under positive electrospray ionization tandem mass spectrometer (ESI-MS/MS) in the multiple reaction monitoring (MRM) mode. The stable and relatively non-polar nature of the derivatized analyte enables reliable quantification of dopamine in the range of 10-1000 pg/ml using 200 μl of plasma sample. The method was validated with intra-day and inter-day precision less than 7%, and the intra-day and inter-day accuracy of 91.9-101.9% and 92.3-102.6%, respectively. The validated assay was applied to quantify dopamine levels in two preterm neonate plasma samples. In conclusion, a sensitive and selective LC-MS/MS method has been developed and validated, and successfully used for the determination of plasma dopamine levels in preterm neonates.

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.

Background/aim Phosphaplatin platinum (IV) (RRD4) complex has exceptional antitumor properties. The aim of this study was to investigate the effects and the mechanism of action of free and liposome-encapsulated RRD4 in breast cancer. Materials and methods Liposome-encapsulated RRD4 prepared by thin-film dehydration: hydration and free RRD4 were tested in vivo and in vitro against 4T1 breast cancer cells. Cell proliferation, migration and viability were determined. Tissue and cell production and expression of pigment epithelium-derived factor (PEDF) were assessed by ELISA and western blot. 4T1 cells treated with PEDF siRNA were evaluated for viability and apoptosis. Results RRD4 inhibited tumor growth and prevented distant metastasis. Liposome formulation enhanced this therapeutic benefit without increasing toxicity and prolonged RRD4 retention in tumor tissues. In vitro, RRD4 induced 4T1 apoptosis through up-regulation of FAS, BAX, and PUMA, and down-regulation of BCL2. RRD4 facilitates a FAS-intrinsic signaling mechanism. PEDF up-regulation represents another antitumor mechanism associated with this phosphaplatin compound. Discussion Free RRD4 or formulated into liposomes, are excellent candidates for adjuvant therapy against breast tumor growth and metastasis.

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.

OBJECTIVES A rapid, sensitive and robust ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the quantification of four major polymyxin B components (polymyxin B1, polymyxin B2, polymyxin B3 and isoleucine-polymyxin B1) in serum and epithelial lining fluid (ELF) samples. METHODS A Waters Acquity UPLC HSS C18 column was used with 0.1% formic acid in water/acetonitrile as mobile phases. Analysis was performed in a positive ionization mode with multiple-reactions monitoring scan type. Five percent trichloroacetic acid was used to precipitate proteins in biological samples and to increase the sensitivity of detection. RESULTS Our results showed a linear concentration range of 0.0065-3.2 mg/L for all the major polymyxin B components in both serum and ELF, respectively; the interday variation was

Purpose The stability of polymyxin B sulfate in infusion bags containing 0.9% sodium chloride injection stored at 4 and 25 °C was studied. Methods Seven manufacturing batches of polymyxin B from different sources were tested. The products were reconstituted in sterile water for injection, diluted in infusion bags containing 0.9% sodium chloride injection, and stored at room temperature (25 °C) or under refrigeration (4 °C). Samples were withdrawn at the same time on days 0, 1, 2, 3, 5, and 7. A modified microbiological assay was used to determine the concentrations, as indicated by zones of inhibition, of polymyxin B. Bordetella bronchiseptica served as the reference organism. Stability was defined as retention of >90% of the initial concentration. The decomposition kinetics of polymyxin B in 0.9% sodium chloride injection were evaluated by plotting the polymyxin B concentration remaining versus time. Results On average, the samples retained over 90% of their initial concentration for up to two days at both storage temperatures. All samples retained over 90% of their initial concentration at 24 hours. The decomposition kinetics of polymyxin B in infusion bags containing 0.9% sodium chloride injection exhibited pseudo-first-order kinetics, with rate constants of 0.024–0.075 day−1 at 25 °C and 0.022–0.043 day−1 at 4 °C ( p > 0.05). Conclusion Polymyxin B was stable for at least one day when stored at 4 or 25 °C in infusion bags containing 0.9% sodium chloride injection. Stability did not differ significantly between the two storage temperatures.

The lactone stability of camptothecins is critical for their anticancer activity. A stable liposomal 9-nitro-camptothecin formulation was developed to circumvent the drawbacks of low aqueous solubility and lactone instability and to provide sustained release of the agent in blood circulation. The potential merits of the formulation were demonstrated by its profoundly improved lactone stability in vivo, favorable pharmacokinetic and biodistribution characteristics in rats, and enhanced preclinical efficacy in tumor-bearing athymic mice.

Arginine vasopressin is an endogenous neuropeptide secreted in response to situations such as hyperosmolality, hypotension and hypovolemia. The purpose of this study was to develop a reliable assay using small volumes of plasma and urine samples to quantify vasopressin levels in preterm infants. Weak cation solid-phase extraction was used to extract vasopressin from 200μl human plasma and urine samples. Separation was achieved on a Waters Acquity UPLC BEH C18 column by gradient elution at 0.55ml/min, with a mobile phase composed of methanol and 0.02% aqueous acetic acid solution. Analysis was performed under a hybrid triple quadrupole linear ion trap mass spectrometer, operated in multiple reaction monitoring mode using positive ionization. The linear response range was 1.0-40pg/ml for vasopressin, with the lower limit of quantification (LLOQ) of 1.0pg/ml in human plasma and urine. Recoveries at concentrations of 3, 10 and 32pg/ml were all greater than 70%, and matrix effects were within 15%. The method was validated with intra-day and inter-day precision of less than 8% for human plasma and urine. The intra-day and inter-day accuracy for human plasma were 91.9-100.6% and 92.3-104.8%, respectively. The intra-day and inter-day accuracy for human urine were 89.2-95.9% and 89.3-91.3%, respectively. The validated method was successfully applied to analyze two preterm neonate plasma samples and one child urine sample. In conclusion, the developed and validated method was sensitive and reliable, and was successfully used to quantify endogenous vasopressin levels in neonate plasma and child urine.

Two cremophor-free microemulsions, lecithin:butanol:myvacet oil:water (LBMW) and capmul:myvacet oil:water (CMW) for paclitaxel (PAC) were developed for intravenous (i.v.) administration. Six surfactants and four oils were screened with various combinations for maximal water incorporation and PAC solubility. Microemulsion regions were subsequently determined in ternary phase diagrams. Cytotoxicity in an MDA-M231 human breast cancer cell line and hemolytic potential were assessed in these systems compared to Taxol (cremophor EL:ethanol, 1:1, 6 mgPAC/ml). The maximal water incorporation into the lecithin:butanol surfactant blend was greater than that incorporated into capmul when combined with the oils screened. PAC solubility in myvacet oil was increased 1389-fold over its aqueous solubility. LBMW had a larger microemulsion region (46.5% of total ternary phase diagram) than that seen with CMW (18.6%). The droplet size of the dispersed phase was 111.5 (4.18)nm for LBMW and 110.3 (8.09)nm for CMW. Cytotoxicity of PAC was in decreasing order of: Taxol>LBMW>CMW. The IC50 values for LBMW and CMW ranged from 4.5 to 5.7 and >10 microM, respectively, as compared to that of Taxol (1.3 to 1.8 microM). Eighty-three percent, 68%, and 63% of red blood cells remain unlysed at a formulation volume to blood ratio of 0.035 in LBMW, CMW and Taxol. Promising microemulsions, LBMW and CMW were developed that can incorporate approximately 12 mg/g of PAC, substantially higher than its aqueous solubility (10.8 microg/ml) and that in the Taxol vehicle (6 mg/ml). PAC retained its cytotoxicity in the LBMW and CMW and was less likely to cause hemolysis compared to Taxol. This higher drug loading results in a smaller vehicle volume in required doses of these formulations and potentially less vehicle-related side effects are anticipated.

Lazaroid U-74389G (LAZ) has potential radio-protective and anti-proliferative activities in treatment of glioblastoma, but it exhibits poor brain penetration and high hepatic clearance. To increase the brain exposure of LAZ, we formulated LAZ in co-solvent system and as nanostructured lipid carriers (NLC). The rationale behind this approach is that LAZ formulations due to their high drug payload for administration at higher doses and prolonged circulation results in greater systemic exposure compared to unformulated LAZ solution. The purpose of this study was to comparatively evaluate the brain exposures of unformulated LAZ solution in citrate buffer (pH 3), LAZ co-solvent formulation in mixture of propylene glycol and citrate buffer (pH 3), and LAZ NLC, prepared using ultra-sonication and optimized using Central Composite Design (CCD). Statistical analysis was carried out using Student's t-test. Male Sprague Dawley rats were randomized into 3 groups and were administered unformulated LAZ solution at 5 mg/kg dose and LAZ co-solvent and LAZ NLC at 15 mg/kg dose, intravenously through jugular vein cannula. The brain tissues were homogenized in normal saline and LAZ in the brain tissue was quantified using Absciex Qtrap 5500 LC/MS/MS with linear range of 1 (LLOQ) - 250 ng/mL. The concentrations of unformulated LAZ solution and the LAZ co-solvent solution were 2 mg/mL and 8.5 mg/mL, respectively. The optimized LAZ NLC had particle size of 150 ± 2.86 nm, zeta potential of (-6.79) ± 0.39 mV, encapsulation efficiency of 81.34 ± 6.06%. The LAZ NLC preparation is monodisperse with polydispersity index of 0.158 ± 0.01 (N = 3). The amount of LAZ in brain tissues after 4 hours is significantly higher in formulation groups (LAZ co-solvent 37.48 ± 7.06 ng/gm and LAZ NLC 27.49 ng/gm) than the unformulated groups (LAZ solution 6.84 ± 1.87 ng/gm). Upon dose normalization there was a significant increase in amount of LAZ in brain tissues (N = 3 each) after 4 hours in LAZ co-solvent group (2.72 ± 0.53 ng/gm/mg) in comparison to unformulated LAZ solution group (1.37 ± 0.37 ng/gm/mg), at p Developing LAZ formulations will offer potential merits of enabling administrations at higher doses, facilitating LAZ delivery to the brain at therapeutic levels and decreasing its systemic clearance. LAZ formulations are capable of incorporating LAZ at higher drug payload than unformulated solution. Upon dose normalization, the amount of LAZ in brain tissue at 4 hours is significantly higher (1.5-2 times) in LAZ formulations in comparison to LAZ unformulated group. This study helps to build a strong foundational background for use of LAZ as an alternative agent in treatment of glioblastoma utilizing its dual properties of lipid peroxidation inhibition and anti-proliferation. Citation Format: Prajakta Gadgil, Diana S-L. Chow, Pamela New, Jaymin Shah. Lazaroid formulations for brain delivery in treatment of glioblastoma multiforme. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4402. doi:10.1158/1538-7445.AM2015-4402

Purpose. The dissolution profiles of nonprescription extended-release niacin and inositol niacinate products were studied using the prescription extended-release niacin, Niaspan, as a reference. Methods. Seven nonprescription extended-release and 12 nonprescription inositol niacinate products were collected from community and online pharmacies in the United States. Extended-release Niaspan was used as a reference. Dissolution profiles were examined by the United States Pharmacopeia dissolution test, using a paddle method. Release samples were removed every 30 minutes for up to 240 minutes. Niacin was quantified by high-performance liquid chromatography. Results. Ten out of the 12 inositol niacinate products were capsules and 6 of the 7 extended-release formulations were tablets. During the initial 30-minute dissolution study of inositol niacinate products, free niacin was released to various degrees. One product achieved fast dissolution, with >30% cumulative release of niacin. The cumulative percentage of niacin released at 240 minutes of all inositol niacinate products was statistically different ( p < 0.0001). The majority of these products reached a plateau of releasing niacin in one to two hours, which was maintained until the end of the study. Six out of the seven extended-release niacin products had extended-release profiles. Five products showed a statistically higher dissolution rate ( p < 0.05) than that of Niaspan. Conclusion. Significant variations in dissolution profiles were noted among the 7 nonprescription extended-release and 12 nonprescription inositol niacinate products in vitro, and their dissolution rates were not comparable to that of the prescription extended-release niacin. Further studies are warranted to correlate such dissolution data with their in vivo efficacy.

Background: CZ48, a prodrug of camptothecin (CPT), shows promising antitumor activity as a topoisomerase I (Topo-I) inhibitor after its biotransformation to CPT in vivo. Studies establish that a prolonged exposure of Topo-I inhibitors with low concentrations is more efficacious in cancer treatment than a short term exposure with high concentrations, due to the S-phase cell cycle specific mechanism. However, poor solubility and the effective delivery challenges have been the obstacles for clinical applications of CZ48 to patients. Objectives: The research was to achieve the following objectives: (1) to prepare by wet media milling method and characterize CZ48 nanosuspensions (NSs); (2) to determine the pharmacokinetics (PK) of CZ48 and CPT from CZ48 NSs in Swiss nude mouse model; (3) to demonstrate the proof-of-concept efficacy of CZ48 NS in tumor-bearing athymic mouse model. Methods: CZ48 NSs of different sizes were formulated by media milling method. The in vitro release kinetics in plasma and phosphate buffered saline (PBS) solution (pH 7.4) were characterized, respectively, using the dialysis bag diffusion technique. The PK of CZ48 and CPT were evaluated in male Swiss nu-nu mice by intravenous (i.v.) doses of 5 and 25 mg/kg for cosolvent reference and NSs, respectively. The PK parameters of CZ48 and CPT were derived by WinNonlin 3.0. The proof-of-concept efficacy was performed in the subcutaneous tumor model of Non-small Cell Lung Cancer (NSCLC) H460 cell line in the nude mouse model. ANOVA with Tukey's post-hoc and Kaplan-Meier survival analysis were used for comparisons of PK modifications and efficacy of the CZ48 from NSs using the cosolvent as reference. Results: CZ48 NSs of particle sizes of 200 nm (NS-S) and 600 nm (NS-L) were successfully formulated and extensively characterized. The in vitro sustained release of CZ48 from NSs in plasma was achieved with 0.60 ± 0.12 %/h release rate and less than 40 % total release at 48 h, compared with 6.48 ± 0.29 %/h release rate and complete release by 28 h from cosolvent. NS-S and NS-L prolonged the blood circulation of CZ48 by 8.00, and 5.58 h, respectively, compare to 0.70 h from cosolvent. More importantly, NS-S yielded CPT with the longest half-life (12.51, 5.81 vs. 0.40 h) and largest AUC (19.78, 10.95 vs. 8.29 nM*h/(mg/kg)) than NS-L and cosolvent. CZ48-loaded NS-S exhibited significant tumor growth rate suppression compared to cosolvent (0.08 ± 0.03 vs. 0.18 ± 0.06/day) with a higher tolerable dose (25 vs. 5 mg/kg), and an improved survival rate (80% vs. 10%). Conclusion: This study is the first report to demonstrate the NS as a viable pharmaceutical carrier to deliver CZ48 in a sustained manner, achieving significant tumor growth suppression at a higher tolerable dose. The study provides a formulation strategy of great potentials to overcome the delivery barriers for other anticancer drugs. We anticipate that the CZ48 NS will be a lead candidate for the future clinical trials of CZ48. Citation Format: Diana S-L Chow, Dong Dong, Beppino C. Giovanella, Xin Liu, Cheng-Hui Hsiao, Yang Teng. Sustained delivery of CZ48, a lactone-stabilized camptothecin, by nanosuspensions. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5656. doi:10.1158/1538-7445.AM2013-5656

The bifunctional alkylating agent busulfan (Bu) was solubilized in a cosolvent mixture of anhydrous dimethylacetamide (DMA), polyethylene glycol 400 (PEG400), and water at a ratio of 1:2:2 (v/v/v), to achieve a Bu concentration of 3 mg/ml, a preparation that would be suitable for parenteral administration in high-dose chemotherapy preceding bone marrow transplantation. The complete formulation was stable for more than 54 h at room temperature (RT, 22 degrees C). An accelerated stability study of Bu in anhydrous DMA or DMA/PEG400 (1:2) as stock solutions indicated shelf-lives of 191 and 180 days respectively, at RT, and 8.2 and 7.5 years, respectively, at 4 degrees C. Although the complete formulation with Bu was very hypertonic, hemolysis studies indicated that the formulation would be safe for intravenous (i.v.) administration, since it would be rapidly diluted to harmless tonicity levels in the blood. Cytotoxicity studies of the complete formulation in vitro proved that Bu retained its activity when dissolved in the complete vehicle. A preliminary pharmacokinetic study in a rodent model after the i.v. administration of Bu at a dose of 1 mg/kg body weight yielded high plasma concentrations of Bu for at least 5 h after injection.

Purpose: Lazaroid U-74389G (LAZ) is a 21-aminosteroid that has radioprotective effect against radiation-induced lipid peroxidation. In-vitro antiproliferative effect was reported against Glioblastoma primary cell lines. The aim of this study was to use a liposomal formulation in a proof-of-concept efficacy study to evaluate LAZ radioprotective and antiproliferative effects in Glioblastoma mouse model. Methods: LAZ PEGylated liposomes (Lipo G) were developed in our laboratory. Lipo G size was 80-90 nm with Zeta potential of -22mv. Glioblastoma cell line U87-Lexpressing firefly luciferase reporter gene (100,000 cells in 2 μL) was injected intracranially in each male SCID hairless outbred mouse. The mice were randomized among 4 treatment groups (n=8-9, each): brain model (M) without treatment (control), radiation of 2Gy once a week (M+R), Lipo G at 5 mg/kg dose IP twice a week (M+L) and radiation with Lipo G (M+R+L). The treatment lasted three weeks. The tumor size was monitored using non-invasive bioluminescence imaging (BLI), in each mouse, expressed as the relative BLI photon intensity on the second and third week to that of the first measurement. The mice were sacrificed at the end of the third week and the whole brain was harvested for immunohistochemistry examinations. Lipid peroxidation of brain tissues was quantified by measuring malondialdehyde (MDA) as a surrogate biomarker. The survival was evaluated using Kaplan Meier analysis at P = 0.05. Results: The relative BLI intensity was 4002.03±1737.67, 2034±737.72, 1387.36±684.53 and 2498.89±2521.32 % for M, M+R, M+L and M+R+L, respectively. The tumor size of the M+L group was reduced by 65% compared to the control group. For the radiation treated groups (M+R and M+R+L), there was no significant difference in the tumor size suppression compared to the control group. MDA brain concentration in M+L and M+R+L groups was significantly less than that in M+R group (8.27±0.78 and 10.37±3.30 µM/gm vs. 23.09± 3.79 µM/gm). The survival mean was 22.67, 25.33, 25.22 and 27.13 days for M, M+R, M+R+L and M+L groups, respectively. The mean survival duration of LAZ treated groups (M+L and M+R+L) was significantly longer than that of the control group (27.13 and 25.22 day vs. 22.67 day, respectively). Conclusion: LAZ liposomal formulations administered at 5 mg/kg dose delivered a therapeutic concentration of LAZ to the brain for the efficacy of suppressing Glioblastoma tumor growth (65% reduction). The liposomal LAZ also protected the brain tissue from radiation-induced lipid peroxidation by reducing MDA concentration (lipid peroxidation surrogate) to 50%. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5460. doi:10.1158/1538-7445.AM2011-5460

Purpose: To develop a sustained release nanosuspension formulation of diethylstilbestrol (DES) for subcutaneous delivery, in recurrent prostate cancer, to by-pass the first pass hepatic metabolism and potentially reduce the adverse effects of DES. Methods: Three nanosuspension formulations of DES were prepared by the wet milling technique with sizes of 160 (A), 300 (B) and 500 (C) nm, respectively. The formulations were characterized by particle size, zeta-potential and release kinetics. The release study was performed by adding each formulation, equivalent to 1mg of DES, to a dialysis bag (M.W cut off, 6000-8000). The bag was placed in 20 ml of PBS to simulate the subcutaneous conditions. The tubes were shaken at 100 rpm in a water bath at 37°C. The drug release was monitored for 14 days. DES concentrations were quantified by a validated HPLC assay. Results: Sizes of the nanosuspensions were 162.5±1.5 nm, 287.1±2.7 nm and 492.5±8.3 nm, and the polydispersity was 0.18±0.01, 0.15±0.01, 0.20±0.03 for formulations A, B and C, respectively. The time of milling required for A, B and C was 30, 8 and 6 hours respectively. The zeta potential was -18.11±0.65, -19.49±0.49, -18.08±0.45 mV for formulations A, B and C, respectively. The release profiles showed a biphasic pattern, with an initial rapid release in 4 days followed by a slower release and reaching plateau after 6 days. Formulations A and B showed a significantly higher release than the formulation C by using ANOVA and Tukey's Multiple Comparison Test (p Conclusion: Stable nanosuspension formulations were successfully formulated for DES with particle sizes of 162.5 nm, 287.1 nm and 492.5 nm. The particle size depends on the duration of milling. The 162.5 nm and 287.1 nm formulations achieved a much higher release than the 492.5 nm nanosuspension indicating that the particle size has a profound effect on the release of the drug. A sustained and extended release of DES was achieved over a period of 14 days in simulated subcutaneous conditions indicating that these formulations definitely warrant further preclinical evaluations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 376. doi:10.1158/1538-7445.AM2011-376

Purpose Lazaroid U74389G (LAZ) is a 21-aminosteroid that has a radioprotective and antioxidant effects. It prevents the damage in the tissues after radiation by a selective action on vascular endothelium cells in normal tissues. The aim of this study is to develop liposomal formulations of LAZ with favorable pharmacokinetic (PK) profiles for potential therapeutic merits in an animal model. Methods Conventional (LipoB) and pegylated (LipoG) liposomes were prepared with various phosphatidylcholine: cholesterol ratios and lipid: LAZ ratio of 10:1. The liposomes were characterized with the size, charge and release profile. The PK study was performed in Sprague-Dawley rats. The rats were randomized into 3 groups (n=4 each) prior to the dosing of the formulations (LAZ solution, LipoB and LipoG) at a dose of 1 mg/kg by injection through the cannula at jugular veins. At pre-determined time points, blood samples were collected from the tail vein. The LAZ concentrations in plasma were quantified by a validated UPLC/MS/MS assay using Daidazine as the internal standard, with a lower limit of quantification (LLOQ) of 4 ng/ml for LAZ. The PK profiles were constructed by plotting the plasma concentration versus time and the PK parameters were derived using compartmental models of WinNonlin Ver 3.3 software. Results The liposomal preparations with the least amount of cholesterol had the lowest Encapsulation Efficiency (EE=26%). However, an increase of cholesterol ratio up to 20% increased the EE up to 58%. The size of the liposomes was in the range of 90-118 nm. In in-vivo study, the plasma profile of the solution fits 1-compartment model while the liposomal formulations fit 2-compartment model. The solution group yielded a higher Cmax and faster clearance (4.27 μg/ml and 546 ml/hr) than those of LipoB and LipoG (0.97 and 2.03 μg/ml and 380 and 216 ml/hr, respectively). The t1/2's of LAZ from LipoB and LipoG were 5 folds of that from the solution (0.64 and 0.52 hr vs 0.10hr). The solution showed a significantly smaller AUC compared to those of LipoB and LipoG (0.56 μg.hr/ml vs. 0.79 and 1.42 μg.hr/ml, respectively). The volume of distribution was small for the solution compared to LipoB and LipoG (73 ml vs. 1173 and 589 ml, respectively). The half-lives of distribution and elimination, t1/2 α and t1/2 β, were comparable between LipoB and LipoG (0.22 vs. 0.16 hr and 3.17 vs. 2.55 hr, respectively). Conclusion The liposomal formulations modified the pharmacokinetics of LAZ solution. The liposomal formulations resulted in a slower clearance and wider distribution of LAZ in-vivo compared to the solution. LAZ systemic exposures in rats after administration of LAZ liposomes were 1.4 −2.5 folds higher than that of solution. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2652.

A selective and stability-indicating high-performance liquid chromatographic assay with diazepam as the internal standard was developed for simultaneously analyzing physostigmine and tetrahydroaminoacridine in skin samples, permeation diffusates and dosage form. Baseline resolution was achieved with an octadecyl column for physostigmine, its two degradation products and tetrahydroaminoacridine. Peak homogeneity of physostigmine and tetrahydroaminoacridine was confirmed. The calibration curves for both drugs in skin samples were established at 1-50 micrograms per 200 mg skin. Those for diffusate samples were at 10-50 ng per 50 microliters for physostigmine and 30-150 ng per 50 microliters for tetrahydroaminoacridine. The assay was reproducible with within-day and between-day variations of 5-6 and 4-10%, respectively. Application of the assay for in vitro transdermal permeation study was demonstrated.

A new high-performance liquid chromatographic procedure for the analysis of phenprobamate, a skeletal muscle relaxant in biologic fluids was developed. The method used a C18 reverse phase column, a mobile phase of methanol/acetonitrile/water (33:15:52), and UV detection at 215 nm. The assay procedure was applied to the determination of phenprobamate binding to rat and human plasma proteins using the equilibrium dialysis method. In addition, the red blood cell/plasma partitioning was determined in the whole blood of rats and humans. Phenprobamate exhibited a moderate binding to plasma proteins of rat (74.3 +/- 2.2 per cent) and human (80.5 +/- 1.1 per cent). The protein binding was concentration-independent in the range of 10 to 80 micrograms ml-1. Phenprobamate binding to plasma proteins was also determined in the presence of 10 micrograms ml-1 acetaminophen. The protein binding of phenprobamate was not significantly altered by acetaminophen (74.4 +/- 0.6 per cent for rat plasma; 75.7 +/- 1.6 per cent for human plasma). The distribution ratios of phenprobamate between the red blood cells and plasma were greater than unity, 1.86 and 1.59 in rat and human, respectively, indicating a preferential partitioning of the drug in the red blood cells.