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1.  Commentary: Current Perspectives on the Aggregation of Protein Drugs 
The AAPS Journal  2014;16(3):413-414.
doi:10.1208/s12248-014-9580-0
PMCID: PMC4012046  PMID: 24563118
aggregation; analytical methods; protein drugs; symposium
2.  Screening of Bioactive Peptides Using an Embryonic Stem Cell-Based Neurodifferentiation Assay 
The AAPS Journal  2014;16(3):400-412.
Differentiation of pluripotent stem cells, PSCs, towards neural lineages has attracted significant attention, given the potential use of such cells for in vitro studies and for regenerative medicine. The present experiments were designed to identify bioactive peptides which direct PSC differentiation towards neural cells. Fifteen peptides were designed based on NCAM, FGFR, and growth factors sequences. The effect of peptides was screened using a mouse embryonic stem cell line expressing luciferase dual reporter construct driven by promoters for neural tubulin and for elongation factor 1. Cell number was estimated by measuring total cellular DNA. We identified five peptides which enhanced activities of both promoters without relevant changes in cell number. We selected the two most potent peptides for further analysis: the NCAM-derived mimetic FGLL and the synthetic NCAM ligand, Plannexin. Both compounds induced phenotypic neuronal differentiation, as evidenced by increased neurite outgrowth. In summary, we used a simple, but sensitive screening approach to identify the neurogenic peptides. These peptides will not only provide new clues concerning pathways of neurogenesis, but they may also be interesting biotechnology tools for in vitro generation of neurons.
doi:10.1208/s12248-014-9578-7
PMCID: PMC4012039  PMID: 24557747
bioactive peptides; embryonic stem cells; neural differentiation
3.  Stability: Recommendation for Best Practices and Harmonization from the Global Bioanalysis Consortium Harmonization Team 
The AAPS Journal  2014;16(3):392-399.
This paper provides a comprehensive overview of stability-related aspects of quantitative bioanalysis and recommends science-based best practices, covering small and large molecules as well as chromatographic and ligand-binding assays. It addresses general aspects, such as the use of reference values, transferability and treatment of failing stability results, and also focuses on specific types of stability assessment: bench-top, freeze/thaw and long-term frozen stability, stock stability, extract stability, stability in whole blood, tissue and urine, and stability of endogenous analytes, in special matrix types and in incurred samples.
doi:10.1208/s12248-014-9573-z
PMCID: PMC4012051  PMID: 24550081
GBC; regulated bioanalysis; stability assessment
4.  Sequential Bioequivalence Approaches for Parallel Designs 
The AAPS Journal  2014;16(3):373-378.
Regulators in EU, USA and Canada allow the use of two-stage approaches for evaluation of bioequivalence. The purpose of this paper is to evaluate such designs for parallel groups using trial simulations. The methods developed by Diane Potvin and co-workers were adapted to parallel designs. Trials were simulated and evaluated on basis of either equal or unequal variances between treatment groups. Methods B and C of Potvin et al., when adapted for parallel designs, protected well against type I error rate inflation under all of the simulated scenarios. Performance characteristics of the new parallel design methods showed little dependence on the assumption of equality of the test and reference variances. This is the first paper to describe the performance of two-stage approaches for parallel designs used to evaluate bioequivalence. The results may prove useful to sponsors developing formulations where crossover designs for bioequivalence evaluation are undesirable.
Electronic supplementary material
The online version of this article (doi:10.1208/s12248-014-9571-1) contains supplementary material, which is available to authorized users.
doi:10.1208/s12248-014-9571-1
PMCID: PMC4012040  PMID: 24526610
bioequivalence; parallel; power; sequential designs; type I errors
5.  Determination of the Most Influential Sources of Variability in Tacrolimus Trough Blood Concentrations in Adult Liver Transplant Recipients: A Bottom-Up Approach 
The AAPS Journal  2014;16(3):379-391.
Tacrolimus, an immunosuppressant drug, presents a narrow therapeutic window and a large pharmacokinetic variability with poor correlation between drug dosing regimen and blood concentration. The objective was to identify predictive factors influencing tacrolimus trough concentrations (C0) using a bottom-up approach. A physiologically based pharmacokinetic (PBPK) model of tacrolimus was proposed, taking into account the body weight, the proportion of fat (Pfat), hematocrit, lipid fraction of organs, typical intrinsic clearance (CLityp), CYP3A5 genotype of liver donor, plasma unbound fraction of tacrolimus (fup), and concomitant drugs (CYP3A4 inhibitors). For the evaluation of the PBPK model, mean C0 and concentrations 2 h after oral dose of tacrolimus were compared with those from 66 liver transplant recipients included in a multicentric pharmacokinetic study and were found very close. Tacrolimus concentration profiles were simulated in a virtual population defined by a set of covariate values similar to those from the real population. The sensitivity of tacrolimus C0 with respect to each covariate has been tested to identify the most influential ones. With the range of covariate values tested, the impact of each covariate on tacrolimus C0 may be ranked as follows: fup, CLityp, bioavailability, body weight, hematocrit, CYP3A5 polymorphism, Pfat, and CYP3A4 inhibitory drug–drug interactions. Values for initial dosing regimen of tacrolimus in order to reach a C0 of 10 ng/ml at day 5 (assuming a constant dosing schedule) as a function of CYP3A5 donor genotype and patient’s hematocrit and body weight are proposed.
Electronic supplementary material
The online version of this article (doi:10.1208/s12248-014-9577-8) contains supplementary material, which is available to authorized users.
doi:10.1208/s12248-014-9577-8
PMCID: PMC4012056  PMID: 24526611
bottom-up approach; liver transplantation; tacrolimus; therapeutic drug monitoring
6.  Novel Lansoprazole-Loaded Nanoparticles for the Treatment of Gastric Acid Secretion-Related Ulcers: In Vitro and In Vivo Pharmacokinetic Pharmacodynamic Evaluation 
The AAPS Journal  2014;16(3):361-372.
The objective of this study is to combine nanoparticle design and enteric coating technique to sustain the delivery of an acid-labile drug, lansoprazole (LPZ), in the treatment of acid reflux disorders. Lansoprazole-loaded Eudragit® RS100 nanoparticles (ERSNP-LPZ) as well as poly(lactic-co-glycolic acid) (PLGA) nanoparticles (PLGANP-LPZ) were prepared using a solvent evaporation/extraction method. The effects of nanoparticle charge and permeation enhancers on lansoprazole uptake was assessed in Caco-2 cells. The confocal microscopic images revealed the successful localization of nanoparticles in the cytoplasm of Caco-2 cells. The cellular uptake of positively charged Eudragit nanoparticles was significantly higher than that of negatively charged PLGA nanoparticles, which were enhanced by sodium caprate via the transcellular pathway. Both types of nanoparticles exhibited sustained drug release behavior in vitro. The oral administration of enteric-coated capsules filled with nanoparticles sustained and prolonged the LPZ concentration up to 24 h in ulcer-induced Wistar rats, and 92.4% and 89.2% of gastric ulcers healed after a 7-day treatment with either EC-ERSNP1010-Na caprate or EC-PLGANP1005-Na caprate, respectively.
doi:10.1208/s12248-014-9564-0
PMCID: PMC4012042  PMID: 24519468
Caco-2 cell; lansoprazole; nanoparticles; pharmacodynamic study; pharmacokinetic study
7.  Recommendations and Best Practices for Reference Standards and Reagents Used in Bioanalytical Method Validation 
The AAPS Journal  2014;16(2):352-356.
The continued globalization of pharmaceutics has increased the demand for companies to know and understand the regulations that exist across the globe. One hurdle facing pharmaceutical and biotechnology companies developing new drug candidates is interpreting the current regulatory guidance documents and industry publications associated with bioanalytical method validation (BMV) from each of the different agencies throughout the world. The objective of this commentary is to provide our opinions on the best practices for reference standards and key reagents, such as metabolites and internal standards used in the support of regulated bioanalysis based on a review of current regulatory guidance documents and industry white papers for BMV.
doi:10.1208/s12248-014-9566-y
PMCID: PMC3933579  PMID: 24500277
bioanalytical method validation; internal standards; metabolites; reference standards; stock solutions
8.  New Frontiers—Accelerator Mass Spectrometry (AMS): Recommendation for Best Practices and Harmonization from Global Bioanalysis Consortium Harmonization Team 
The AAPS Journal  2014;16(2):357-359.
The technique of accelerator mass spectrometry (AMS) is applicable to the analysis of a wide range of trace elemental isotopes. However, in the context of the pharmaceutical industry, it is invariably used to measure radiocarbon (14C). There are two broad modes of application: analysis of total 14C sometimes termed “direct AMS” and analysis of specific 14C-labelled analytes in a variety of matrices following some method of isolation. It is the latter application which is within the remit of the GBC team, and the team has made efforts to propose harmonized recommendations for the validation of AMS when used in a regulatory bioanalytical mode, i.e. the quantification of specific analyte(s) using liquid chromatography with off-line detection by AMS now known as “LC + AMS”. The GBC team has reached a position where they have agreed to many aspects, but also differ on some aspects of what constitutes a bioanalytical assay validation in support of clinical studies using this technology. The detail of most of this will be covered under separate publication(s), but for the purposes of this paper, we have outlined the points of consensus. The purpose of this article is not to provide a roadmap for validation of LC + AMS assays, but to highlight agreements amongst the industry representative experts and the practitioners, as well as identifying specific areas essential for establishing assay quality but where additional discussion is required to reach agreement.
doi:10.1208/s12248-014-9568-9
PMCID: PMC3933583  PMID: 24500278
accelerator mass spectrometry; bioanalysis; LC + AMS
9.  Ethinyl Estradiol and Other Human Pharmaceutical Estrogens in the Aquatic Environment: A Review of Recent Risk Assessment Data 
The AAPS Journal  2014;16(2):299-310.
Interest in pharmaceuticals in the environment has increased substantially in recent years. Several studies in particular have assessed human and ecological risks from human pharmaceutical estrogens, such as 17α-ethinyl estradiol (EE2). Regulatory action also has increased, with the USA and other countries developing rules to address estrogens and other pharmaceuticals in the environment. Accordingly, the Center for Drug Evaluation and Research at the US Food and Drug Administration has conducted a review and analysis of current data on the long-term ecological exposure and effects of EE2 and other estrogens. The results indicate that mean-flow long-term predicted environmental concentrations (PECs) of EE2 in approximately 99% or more of US surface water segments downstream of wastewater treatment plants are lower than a predicted no-effect concentration (PNEC) for aquatic chronic toxicity of 0.1 ng/L. Exceedances are expected to be primarily in localized, effluent-dominated water segments. The median mean-flow PEC is more than two orders of magnitude lower than this PNEC. Similar results exist for other pharmaceutical estrogens. Data also suggest that the contribution of EE2 more broadly to total estrogenic load in the environment from all sources (including other human pharmaceutical estrogens, endogenous estrogens, natural environmental estrogens, and industrial chemicals), while highly uncertain and variable, appears to be relatively low overall. Additional data and a more comprehensive approach for data collection and analysis for estrogenic substances in the environment, especially in effluent-dominated water segments in sensitive environments, would more fully characterize the risks.
doi:10.1208/s12248-014-9561-3
PMCID: PMC3933577  PMID: 24470211
aquatic ecology; environmental impact; estrogens; regulatory science; toxicity
10.  Novel Endogenous Glycan Therapy for Retinal Diseases: Safety, In Vitro Stability, Ocular Pharmacokinetic Modeling, and Biodistribution 
The AAPS Journal  2014;16(2):311-323.
Asialo, tri-antennary oligosaccharide (NA3 glycan) is an endogenous compound, which supports proper folding of outer segment membranes, promotes normal ultrastructure, and maintains protein expression patterns of photoreceptors and Müller cells in the absence of retinal pigment epithelium support. It is a potential new therapeutic for atrophic age-related macular degeneration (AMD) and other retinal degenerative disorders. Herein, we evaluate the safety, in vitro stability, ocular pharmacokinetics and biodistribution of NA3. NA3 was injected into the vitreous of New Zealand white rabbits at two concentrations viz. 1 nM (minimum effective concentration (MEC)) and 100 nM (100XMEC) at three time points. Safety was evaluated using routine clinical and laboratory tests. Ocular pharmacokinetics and biodistribution of [3H]NA3 were estimated using scintillation counting in various parts of the eye, multiple peripheral organs, and plasma. Pharmacokinetic parameters were estimated by non-compartmental modeling. A 2-aminobenzamide labeling and hydrophilic interaction liquid interaction chromatography were used to assess plasma and vitreous stability. NA3 was well tolerated by the eye. The concentration of NA3 in eye tissues was in the order: vitreous > retina > sclera/choroid > aqueous humor > cornea > lens. Area under the curve (0 to infinity) (AUC∞) was the highest in the vitreous thereby providing a positive concentration gradient for NA3 to reach the retina. Half-lives in critical eye tissues ranged between 40 and 60 h. NA3 concentrations were negligible in peripheral organs. Radioactivity from [3H]NA3 was excreted via urine and feces. NA3 was stable at 37°C in vitreous over a minimum of 6 days, while it degraded rapidly in plasma. Collectively, these results document that NA3 shows a good safety profile and favorable ocular pharmacokinetics.
Electronic supplementary material
The online version of this article (doi:10.1208/s12248-014-9563-1) contains supplementary material, which is available to authorized users.
doi:10.1208/s12248-014-9563-1
PMCID: PMC3933590  PMID: 24470212
age-related macular degeneration (AMD); NA3 glycan; pharmacokinetics; safety
11.  Synthesis, Spectral Characterization, and In Vitro Cellular Activities of Metapristone, a Potential Cancer Metastatic Chemopreventive Agent Derived from Mifepristone (RU486) 
The AAPS Journal  2014;16(2):289-298.
Mifepristone (RU486) is marketed and used widely by women as an abortifacient, and experimentally for psychotic depression and anticancer treatments. After administration, metapristone is found to be the most predominant metabolite of mifepristone. We hypothesized that adhesion of circulating tumor cells (CTCs) to vascular endothelial bed is a crucial starting point in metastatic cascade, and that metapristone can serve as a cancer metastatic chemopreventive agent that can interrupt adhesion and invasion of CTCs to the intima of microvasculature. In the present study, we modified the synthesis procedure to produce grams of metapristone, fully characterized its spectral properties and in vitro cellular activities, including its cytostatic effects, cell cycle arrest, mitochondrial membrane potential, and apoptosis on human colorectal cancer HT-29 cells. Metapristone concentration dependently interrupted adhesion of HT-29 cells to endothelial cells. Metapristone may potentially be a useful agent to interrupt metastatic initiation.
doi:10.1208/s12248-013-9559-2
PMCID: PMC3933578  PMID: 24442753
cancer metastasis chemoprevention; in vitro cellular activities; metapristone; mifepristone; spectral properties
12.  Combined Inhaled Salbutamol and Mannitol Therapy for Mucus Hyper-secretion in Pulmonary Diseases 
The AAPS Journal  2014;16(2):269-280.
This study focuses on the co-engineering of salbutamol sulphate (SS), a common bronchodilator, and mannitol (MA), a mucolytic, as a potential combination therapy for mucus hypersecretion. This combination was chosen to have a synergic effect on the airways: the SS will act on the β2-receptor for relaxation of smooth muscle and enhancement of ciliary beat frequency, whilst mannitol will improve the fluidity of mucus, consequently enhancing its clearance from the lung. A series of co-spray-dried samples, containing therapeutically relevant doses of SS and MA, were prepared. The physico-chemical characteristics of the formulations were evaluated in terms of size distribution, morphology, thermal and moisture response and aerosol performance. Additionally, the formulations were evaluated for their effects on cell viability and transport across air interface Calu-3 bronchial epithelial cells, contractibility effects on bronchial smooth muscle cells and cilia beat activity using ciliated nasal epithelial cells in vitro. The formulations demonstrated size distributions and aerosol performance suitable for inhalation therapy. Transport studies revealed that the MA component of the formulation enhanced penetration of SS across the complex mucus layer and the lung epithelia cells. Furthermore, the formulation in the ratios of SS 10−6 and MA 10−3 M gave a significant increase in cilia beat frequency whilst simultaneously preventing smooth muscle contraction associated with mannitol administration. These studies have established that co-spray dried combination formulations of MA and SS can be successfully prepared with limited toxicity, good aerosol performance and the ability to increase ciliary beat frequency for improving the mucociliary clearance in patients suffering from hyper-secretory diseases, whilst simultaneously acting on the underlying smooth muscle.
Electronic supplementary material
The online version of this article (doi:10.1208/s12248-014-9560-4) contains supplementary material, which is available to authorized users.
doi:10.1208/s12248-014-9560-4
PMCID: PMC3933573  PMID: 24431080
cilia responce; epithelia transport; lung delivery; mannitol; salbutamol; smooth muscle responce
13.  Dietary Flavonoids Modulate CYP2C to Improve Drug Oral Bioavailability and Their Qualitative/Quantitative Structure–Activity Relationship 
The AAPS Journal  2014;16(2):258-268.
This study aims to improve the drug oral bioavailability by co-administration with flavonoid inhibitors of the CYP2C isozyme and to establish qualitative and quantitative (QSAR) structure–activity relationships (SAR) between flavonoids and CYP2C. A total of 40 naturally occurring flavonoids were screened in vitro for CYP2C inhibition. Enzyme activity was determined by measuring conversion of tolbutamide to 4-hydroxytolbutamide by rat liver microsomes. The percent inhibition and IC50 of each flavonoid were calculated and used to develop SAR and QSAR. The most effective flavonoid was orally co-administered in vivo with a cholesterol-reducing drug, fluvastatin, which is normally metabolized by CYP2C. The most potent CYP2C inhibitor identified in vitro was tamarixetin (IC50 = 1.4 μM). This flavonoid enhanced the oral bioavailability of fluvastatin in vivo, producing a >2-fold increase in the area under the concentration–time curve and in the peak plasma concentration. SAR analysis indicated that the presence of a 2,3-double bond in the C ring, hydroxylation at positions 5, 6, and 7, and glycosylation had important effects on flavonoid–CYP2C interactions. These findings should prove useful for predicting the inhibition of CYP2C activity by other untested flavonoid-like compounds. In the present study, tamarixetin significantly inhibited CYP2C activity in vitro and in vivo. Thus, the use of tamarixetin could improve the therapeutic efficacy of drugs with low bioavailability.
doi:10.1208/s12248-013-9549-4
PMCID: PMC3933575  PMID: 24431079
bioavailability; CYP2C; flavonoid; structure–activity relationship; tamarixetin
14.  Headway and Hurdles in the Clinical Development of Dietary Phytochemicals for Cancer Therapy and Prevention: Lessons Learned from Vitamin A Derivatives 
The AAPS Journal  2014;16(2):281-288.
Accumulating epidemiologic and preclinical evidence support the pharmacologic use of a variety of dietary chemicals for the prevention and treatment of cancer. However, it will be challenging to translate these findings into routine clinical practice since phytochemicals have pleiotropic biological activities that have to be balanced for optimal efficacy without unacceptable and potentially unanticipated toxicities. Correctly matching patient populations and settings with optimal, natural product-based phytochemical therapies will require a greater understanding of the specific mechanisms underlying the efficacy, toxicity, and resistance of each agent in a variety of normal, premalignant, and malignant settings. This, in turn, necessitates continued commitment from the basic research community to guide carefully designed and informed clinical trials. The most developed class of anticancer phytochemicals consists of the derivatives of vitamin A called retinoids. Unlike other natural product chemicals currently under study, the retinoids have been extensively tested in humans. Over 30 years of clinical investigation has resulted in several disappointments, but there were some spectacular successes where certain retinoid-based protocols are now FDA-approved standard of care therapies to treat specific malignancies. Furthermore, retinoids are one of the most evaluated pharmacologic agents in the ultra-challenging setting of interventional cancer prevention. This review will summarize the development of retinoids in cancer therapy and prevention with an emphasis on currently proposed mechanisms mediating their efficacy, toxicity, and resistance.
doi:10.1208/s12248-014-9562-2
PMCID: PMC3933572  PMID: 24431081
chemoprevention; clinical trials; differentiation therapy; retinoids
15.  Antitumor Activity of Gemcitabine Can Be Potentiated in Pancreatic Cancer through Modulation of TLR4/NF-κB signaling by 6-Shogaol 
The AAPS Journal  2014;16(2):246-257.
Advanced pancreatic cancer still has a poor prognosis, even with the approval of several drugs, such as gemcitabine. Therefore, developing effective and safe antitumor agents is urgently needed. 6-Shogaol, a phenol extracted from ginger, has been linked to suppression of proliferation and survival of cancer with different mechanisms. In the present study, we investigated whether 6-shogaol could suppress pancreatic cancer progress and potentiate pancreatic cancer to gemcitabine treatment in vitro and in vivo. We found that 6-shogaol prevented the activation of toll like receptor 4 (TLR4)/NF-κB signaling. The modulation of NF-κB signaling by 6-shogaol was ascertained by electrophoretic mobility shift assay and western blot analysis. The suppression of NF-κB signaling and key cell survival regulators including COX-2, cyclinD1, survivin, cIAP-1, XIAP, Bcl-2, and MMP-9 brought the anti-proliferation effects in pancreatic cancer cells and sensitized them to gemcitabine treatment. Furthermore, in a pancreatic cancer xenograft model, we found a decreased proliferation index (Ki-67) and increased apoptosis by TUNEL staining in 6-shogaol treated tumors. It was also shown that 6-shogaol combined with gemcitabine treatment was more effective than drug alone, consistent with the downregulation of NF-κB activity along with its target genes COX-2, cyclinD1, survivin, cIAP-1, and XIAP. Overall, our results suggest that 6-shogaol can inhibit the growth of human pancreatic tumors and sensitize them to gemcitabine by suppressing of TLR4/NF-κB-mediated inflammatory pathways linked to tumorigenesis.
Electronic supplementary material
The online version of this article (doi:10.1208/s12248-013-9558-3) contains supplementary material, which is available to authorized users.
doi:10.1208/s12248-013-9558-3
PMCID: PMC3933586  PMID: 24424498
chemosensitization; gemcitabine; pancreatic cancer; TLR4/NF-κB; 6-shogaol
16.  Recommendations from the Global Bioanalysis Consortium Team A8: Documentation 
The AAPS Journal  2014;16(2):240-245.
The A8 Global Harmonization Team focused on the documentation needed to support both small and large molecule bioanalysis. Current regulatory requirements were compiled and compared. The scope of the team’s discussions included the validation report, the bioanalytical report, study plans, raw data, and bioanalytical summaries for the common technical document (CTD). A common high-level table of contents for method validation and sample analysis reports is proposed. Suggestions have been made as to how the CTD can be standardized to improve usability and review. Additional comments have been made on reports of failure investigations, study plans, and raw data documentation. The recommendation is that no prescriptive guidelines are required in these areas but should be led by good scientific practices subject to particular circumstances.
doi:10.1208/s12248-013-9556-5
PMCID: PMC3933576  PMID: 24408787
A8; documentation; GBC; reports
17.  Large Molecule Run Acceptance: Recommendation for Best Practices and Harmonization from the Global Bioanalysis Consortium Harmonization Team 
The AAPS Journal  2014;16(2):221-225.
The L1 Global Harmonization Team provides recommendations specifically for run acceptance of ligand binding methods used in bioanalysis of macromolecules in support of pharmacokinetics. The team focused on standard curve calibrators and quality controls for use in both pre-study validation and in-study sample analysis, including their preparation and acceptance criteria. The team also considered standard curve editing and the concept of total error.
doi:10.1208/s12248-013-9553-8
PMCID: PMC3933574  PMID: 24395373
acceptance criteria; accuracy and precision (A&P); curve editing; quality controls; standard curve calibrators; total error
18.  Duel-Acting Subcutaneous Microemulsion Formulation for Improved Migraine Treatment with Zolmitriptan and Diclofenac: Formulation and In Vitro-In Vivo Characterization 
The AAPS Journal  2013;16(2):214-220.
Subcutaneous triptan provides immediate analgesia in migraine and cluster headache but is limited by high pain recurrence due to rapid drug elimination. A dual-acting subcutaneous formulation providing immediate release of a triptan and slow but sustained release of a nonsteroidal anti-inflammatory drug may provide a longer duration of relief. A microemulsion-based technology has various advantages over other technically complex dosage forms. Oil-in-water microemulsions of zolmitriptan and diclofenac acid using Labrafac Lipophile, Tween 80, Capryol 90 and water were prepared. One formulation was characterised in vitro and found to have uniformly dispersed nanosized globules. The formulation provided differential release of zolmitriptan and diclofenac acid both in vitro as well as in vivo that may be potentially beneficial to migraine patients.
doi:10.1208/s12248-013-9557-4
PMCID: PMC3933582  PMID: 24363199
diclofenac acid; dual-acting injection; migraine treatment; prolonged release microemulsion; subcutaneous injection; zolmitriptan
19.  Genetic Polymorphisms of Metabolic Enzymes and the Pharmacokinetics of Indapamide in Taiwanese Subjects 
The AAPS Journal  2013;16(2):206-213.
To understand the genetic makeup and impact on pharmacokinetics (PK) in the Taiwanese population, we analyzed the pharmacogenetic (PG) profile and demonstrated its effects on enzyme metabolism using indapamide as an example. A multiplex mass spectrometry method was used to examine the single nucleotide polymorphism (SNP) profile of eight major phases I and II metabolic enzymes in 1,038 Taiwanese subjects. A PG/PK study was conducted in 24 healthy subjects to investigate the possible effects of 28 SNPs on drug biotransformation. Among the genetic profile analyzed, eight SNPs from CYP2A6, CYP2C19, CYP2D6, CYP2E1, CYP3A5, and UGT2B7 showed higher variant frequencies than those previously reported in Caucasians or Africans. For instance, we observed 14.7% frequency of the SNP rs5031016 (I471T) from CYP2A6 in Taiwanese, whereas 0% variation was reported in Caucasians and Africans. The PG/PK study of indapamide demonstrated that the polymorphic SNPs CYP2C9 rs4918758 and CYP2C19 rs4244285 appeared to confer lowered enzyme activity, as indicated by increased Cmax (25% ∼ 64%), increased area under the plasma level-time curves (30∼76%), increased area under the time infinity (43% ∼ 80%), and lower apparent clearance values than PK for wild-type indapamide. Our results reinforce the biochemical support of CYP2C19 in indapamide metabolism and identify a possible new participating enzyme CYP2C9. The PG/PK approach contributed toward understanding the genetic makeup of different ethnic groups and associations of enzymes in drug metabolism. It could be used to identify two genetic markers that enable to differentiate subjects with varied PK outcomes of indapamide.
doi:10.1208/s12248-013-9535-x
PMCID: PMC3933584  PMID: 24357089
indapamide; metabolic enzymes; pharmacogenetics; pharmacokinetics; SNPs
20.  Assay Formats: Recommendation for Best Practices and Harmonization from the Global Bioanalysis Consortium Harmonization Team 
The AAPS Journal  2013;16(2):194-205.
As part of the GBC (Global Bioanalysis Consortium), the L3 assay format team has focused on reviewing common platforms used to support ligand binding assays in the detection of biotherapeutics. The following review is an overview of discussions and presentations from around the globe with a group of experts from different companies to allow an international harmonization of common practices and suggestions for different platforms. Some of the major platforms include Gyrolab, Erenna, RIA, AlphaLISA, Delfia, Immuno-PCR, Luminex, BIAcore, and ELISAs. The review is meant to support bioanalysts in taking decisions between different platforms depending on the needs of the analyte with a number of recommendations to help integration of platforms into a GLP environment.
doi:10.1208/s12248-013-9552-9
PMCID: PMC3933581  PMID: 24343771
assay format; biotherapeutic; large molecule; platform; technology
21.  Hyaluronidase-Sensitive Nanoparticle Templates for Triggered Release of HIV/AIDS Microbicide In Vitro 
The AAPS Journal  2013;16(2):181-193.
This study was designed to test the hypothesis that a triggered release of a topical microbicide (tenofovir) from hyaluronic acid nanoparticles (HA-NPs) can be achieved under the influence of hyaluronidase (HAase) enzyme. A fractional factorial experimental design was used to examine the factors [molar concentrations of adipic acid dihydrazide (X1) and 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (X2), volume of acetone (X3) and reaction time (X4)] influencing the responses, Y1; particle mean diameter: PMD (nanometers: nm), Y2; polydispersity index: PDI and Y3; zeta (ζ) potential: (millivolts). The amide bond formation between HA and ADH after cross-linking was confirmed by FT-IR and 13C-NMR analyses. These NPs were also characterized for cytotoxicity on a human vaginal epithelial cell line and L. crispatus. When formulated with factors X1; 2.49 mM, X2; 9.96 mM, X3; 60 mL, X4; 6 h, HA-NPs exhibited a spherical shape with PMD, PDI, ζ potential, encapsulation efficiency, and drug loading of 70.6 ± 4.1 nm, 0.07 ± 0.02, −38.2 ± 2.8 mV, 51.8 ± 2.4% w/w and 26.1 ± 1.2% w/w, respectively, (n = 3). Unlike for HA based gel, HAase significantly triggered the drug release and HA degradation from the NPs after 24 h (∼90% w/w and 65% w/w, respectively); whereas, in its absence, these values were ∼39% w/w and 26% w/w, respectively. The NPs were non-cytotoxic to human vaginal VK2/E6E7, End1/E6E7 cells and Lactobacillus crispatus. These data highlight the potential of HAase-sensitive HA-NPs templates for the controlled and vaginal delivery of anti-HIV/AIDS microbicides.
Electronic supplementary material
The online version of this article (doi:10.1208/s12248-013-9546-7) contains supplementary material, which is available to authorized users.
doi:10.1208/s12248-013-9546-7
PMCID: PMC3933588  PMID: 24343770
experimental design; hyaluronic acid; hyaluronidase; microbicide; nanoparticles
22.  Use of Different Parameters and Equations for Calculation of IC50 Values in Efflux Assays: Potential Sources of Variability in IC50 Determination 
The AAPS Journal  2013;16(1):172-180.
Drug interactions due to efflux transporters may result in one drug increasing or decreasing the systemic exposure of a second drug. The potential for in vivo drug interactions is estimated through in vitro cell assays. Variability in in vitro parameter determination (e.g., IC50 values) among laboratories may lead to different conclusions in in vivo interaction predictions. The objective of this study was to investigate variability in in vitro inhibition potency determination that may be due to calculation methods. In a Caco-2 cell assay, the absorptive and secretive permeability of digoxin was measured in the presence of spironolactone, itraconazole and vardenafil. From the permeability data, the efflux ratio and net secretory flux where calculated for each inhibitor. IC50 values were then calculated using a variety of equations and software programs. All three drugs decreased the secretory transport of digoxin in a concentration-dependent manner while increasing digoxin’s absorption to a lesser extent. The resulting IC50 values varied according to the parameter evaluated, whether percent inhibition or percent control was applied, and the computational IC50 equation. This study has shown that multiple methods used to quantitate the inhibition of drug efflux in a cell assay can result in different IC50 values. The variability in the results in this study points to a need to standardize any transporter assay and calculation methods within a laboratory and to validate the assay with a set of known inhibitors and non-inhibitors against a clinically relevant substrate.
Electronic supplementary material
The online version of this article (doi:10.1208/s12248-013-9554-7) contains supplementary material, which is available to authorized users.
doi:10.1208/s12248-013-9554-7
PMCID: PMC3889528  PMID: 24338112
Caco-2; drug interaction; IC50; inhibition; P-glycoprotein
23.  Automation Practices in Large Molecule Bioanalysis: Recommendations from Group L5 of the Global Bioanalytical Consortium 
The AAPS Journal  2013;16(1):164-171.
In recent years, the use of automated sample handling instrumentation has come to the forefront of bioanalytical analysis in order to ensure greater assay consistency and throughput. Since robotic systems are becoming part of everyday analytical procedures, the need for consistent guidance across the pharmaceutical industry has become increasingly important. Pre-existing regulations do not go into sufficient detail in regard to how to handle the use of robotic systems for use with analytical methods, especially large molecule bioanalysis. As a result, Global Bioanalytical Consortium (GBC) Group L5 has put forth specific recommendations for the validation, qualification, and use of robotic systems as part of large molecule bioanalytical analyses in the present white paper. The guidelines presented can be followed to ensure that there is a consistent, transparent methodology that will ensure that robotic systems can be effectively used and documented in a regulated bioanalytical laboratory setting. This will allow for consistent use of robotic sample handling instrumentation as part of large molecule bioanalysis across the globe.
doi:10.1208/s12248-013-9551-x
PMCID: PMC3889521  PMID: 24311307
automation; documentation; large molecule bioanalysis; robotic system
24.  Plant Phytochemicals as Epigenetic Modulators: Role in Cancer Chemoprevention 
The AAPS Journal  2013;16(1):151-163.
In recent years, “nutri-epigenetics,” which focuses on the influence of dietary agents on epigenetic mechanism(s), has emerged as an exciting novel area in epigenetics research. Targeting of aberrant epigenetic modifications has gained considerable attention in cancer chemoprevention research because, unlike genetic changes, epigenetic alterations are reversible and occur during early carcinogenesis. Aberrant epigenetic mechanisms, such as promoter DNA methylation, histone modifications, and miRNA-mediated post-transcriptional alterations, can silence critical tumor suppressor genes, such as transcription factors, cell cycle regulators, nuclear receptors, signal transducers, and apoptosis-inducing and DNA repair gene products, and ultimately contribute to carcinogenesis. In an effort to identify and develop anticancer agents which cause minimal harm to normal cells while effectively killing cancer cells, a number of naturally occurring phytochemicals in food and medicinal plants have been investigated. This review highlights the potential role of plant-derived phytochemicals in targeting epigenetic alterations that occur during carcinogenesis, by modulating the activity or expression of DNA methyltransferases, histone modifying enzymes, and miRNAs. We present in detail the epigenetic mode of action of various phytochemicals and discuss their potential as safe and clinically useful chemopreventive strategies.
doi:10.1208/s12248-013-9548-5
PMCID: PMC3889536  PMID: 24307610
cancer chemoprevention; dietary agents; DNA methylation; epigenetics; histone modification; microRNA
25.  Squarticles as a Lipid Nanocarrier for Delivering Diphencyprone and Minoxidil to Hair Follicles and Human Dermal Papilla Cells 
The AAPS Journal  2013;16(1):140-150.
Delivery of diphencyprone (DPCP) and minoxidil to hair follicles and related cells is important in the treatment of alopecia. Here we report the development of “squarticles,” nanoparticles formed from sebum-derived lipids such as squalene and fatty esters, for use in achieving targeted drug delivery to the follicles. Two different nanosystems, nanostructured lipid carriers (NLC) and nanoemulsions (NE), were prepared. The physicochemical properties of squarticles, including size, zeta potential, drug encapsulation efficiency, and drug release, were examined. Squarticles were compared to a free control solution with respect to skin absorption, follicular accumulation, and dermal papilla cell targeting. The particle size of the NLC type was 177 nm; that of the NE type was 194 nm. Approximately 80% of DPCP and 60% of minoxidil were entrapped into squarticles. An improved drug deposition in the skin was observed in the in vitro absorption test. Compared to the free control, the squarticles reduced minoxidil penetration through the skin. This may indicate a minimized absorption into systemic circulation. Follicular uptake by squarticles was 2- and 7-fold higher for DPCP and minoxidil respectively compared to the free control. Fluorescence and confocal images of the skin confirmed a great accumulation of squarticles in the follicles and the deeper skin strata. Vascular endothelial growth factor expression in dermal papilla cells was significantly upregulated after the loading of minoxidil into the squarticles. In vitro papilla cell viability and in vivo skin irritancy tests in nude mice suggested a good tolerability of squarticles to skin. Squarticles provide a promising nanocarrier for topical delivery of DPCP and minoxidil.
doi:10.1208/s12248-013-9550-y
PMCID: PMC3889522  PMID: 24307611
alopecia; diphencyprone; drug targeting; hair follicles; minoxidil; squarticles

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