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Lamotrigine is a commonly prescribed antiepileptic drug. U.S. Food and Drug Administration (FDA)-funded clinical studies have demonstrated bioequivalence (BE) for generic lamotrigine immediate-release (IR) products in epilepsy patients with generic substitution. To address the potential concerns about the risk of generic-brand substitution of lamotrigine extended-release (ER) products, considering the complexity of controlled release systems and pharmacokinetic variations associated with possible within-subject variability (WSV), this prospective study assessed (1) BE of generic and brand lamotrigine ER products in a fully replicated BE study design in healthy subjects and (2) whether such fully replicated study design and WSV data can better support the approval of generic lamotrigine ER products.This open-label, single-dose, two-treatment, four-period, two-sequence, fully replicated crossover BE study compared generic lamotrigine ER tablet to brand Lamictal XR (200 mg) in 30 healthy subjects under fed conditions. Pharmacokinetics (PK) profiles were generated based on intensive blood sampling up to 144 h.The two products showed comparable peak plasma concentration (CThe generic lamotrigine ER tablet product demonstrates BE to the brand product in a fully replicated BE study design with healthy subjects, supporting the adequacy of the two-way crossover study design to demonstrate BE and generic-brand substitution of lamotrigine ER products.

This work is the second in a series of publications outlining the fundamental principles and proposed design of a biopharmaceutics classifications system for inhaled drugs and drug products (the iBCS). Here, a mechanistic computer-based model has been used to explore the sensitivity of the primary biopharmaceutics functional output parameters: (i) pulmonary fraction dose absorbed (

For oral drugs, the formulator and discovery chemist have a tool available to them that can be used to navigate the risks associated with the selection and development of immediate release oral drugs and drug products. This tool is the biopharmaceutics classification system (giBCS). Unfortunately, no such classification system exists for inhaled drugs. The perspective outlined in this manuscript provides the foundational principles and framework for a classification system for inhaled drugs. The proposed classification system, an inhalation-based biopharmaceutics classification system (iBCS), is based on fundamental biopharmaceutics principles adapted to an inhalation route of administration framework. It is envisioned that a classification system for orally inhaled drugs will facilitate an understanding of the technical challenges associated with the development of new chemical entities and their associated new drug products (device and drug formulation combinations). Similar to the giBCS, the iBCS will be based on key attributes describing the drug substance (solubility and permeability) and the drug product (dose and dissolution). This manuscript provides the foundational aspects of an iBCS, including the proposed scientific principles and framework upon which such a system can be developed.

Background: Human pluripotent stem cell (hPSC)–derived cardiomyocytes (hPSC-CMs) have tremendous promise for application in cardiac regeneration, but their translational potential is limited by an immature phenotype. We hypothesized that large-scale manufacturing of mature hPSC-CMs could be achieved through culture on polydimethylsiloxane (PDMS)-lined roller bottles and that the transplantation of these cells would mediate better structural and functional outcomes than with conventional immature hPSC-CM populations. Methods: We comprehensively phenotyped hPSC-CMs after in vitro maturation for 20 and 40 days on either PDMS or standard tissue culture plastic substrates. All hPSC-CMs were generated from a transgenic hPSC line that stably expressed a voltage-sensitive fluorescent reporter to facilitate in vitro and in vivo electrophysiological studies, and cardiomyocyte populations were also analyzed in vitro by immunocytochemistry, ultrastructure and fluorescent calcium imaging, and bulk and single-cell transcriptomics. We next compared outcomes after the transplantation of these populations into a guinea pig model of myocardial infarction using end points including histology, optical mapping of graft- and host-derived action potentials, echocardiography, and telemetric electrocardiographic monitoring. Results: We demonstrated the economic generation of >1×10 8 mature hPSC-CMs per PDMS-lined roller bottle. Compared with their counterparts generated on tissue culture plastic substrates, PDMS-matured hPSC-CMs exhibited increased cardiac gene expression and more mature structural and functional properties in vitro. More important, intracardiac grafts formed with PDMS-matured myocytes showed greatly enhanced structure and alignment, better host-graft electromechanical integration, less proarrhythmic behavior, and greater beneficial effects on contractile function. Conclusions: We describe practical methods for the scaled generation of mature hPSC-CMs and provide the first evidence that the transplantation of more mature cardiomyocytes yields better outcomes in vivo.

Papillary thyroid carcinoma (PTC) accounts for more than 80% of all thyroid carcinoma cases. Small nuclear ribonucleoprotein polypeptides B and B1 (SNRPB) has been indicated to be carcinogenic in several cancers; however, its function and mechanism in PTC are unclarified. Real time quantitative polymerase chain reaction and western blotting revealed the upregulation of SNRPB and downregulation of tumor protein p53 in PTC tissues compared with the normal tissues. Flow cytometry and western blotting displayed that SNRPB silencing induced cell cycle arrest at G1 phase and suppressed the expression levels of Cyclin family proteins in PTC cells. In vivo experiments suggested that SNRPB silencing inhibited PTC tumor growth in mice. Bioinformatics analysis revealed that the expression of SNRPB and cell cycle-associated genes in thyroid carcinoma tissues is positively correlated. Immunofluorescence staining and co-immunoprecipitation demonstrated that SNRPB directly interacted with p53 and suppressed its expression in PTC cells. In conclusion, SNRPB facilitates cell cycle progression in PTC by inhibiting p53 expression.

Complex generics are generic versions of drug products that generally have complex active ingredients, complex formulations, complex routes of delivery, complex dosage forms, are complex drug-device combination products, or have other characteristics that can make it complex to demonstrate bioequivalence or to develop as generics. These complex products (i.e. complex generics) are an important element of the United States (U.S.) Food and Drug Administration’s (FDA’s) Generic Drug User Fee Amendments (GDUFA) II Commitment Letter. The Center for Research on Complex Generics (CRCG) was formed by a grant from the FDA to address challenges associated with the development of complex generics. To understand these challenges, the CRCG conducted a “Survey of Scientific Challenges in the Development of Complex Generics”. The three main areas of questioning were directed toward which (types of) complex products, which methods of analysis to support a demonstration of bioequivalence, and which educational topics the CRCG should prioritize. The survey was open to the public on a website maintained by the CRCG. Regarding complex products, the top three selections were complex injectables, formulations, and nanomaterials; drug-device combination products; and inhalation and nasal products. Regarding methods of analysis, the top three selections were locally-acting physiologically-based pharmacokinetic modeling; oral absorption models and bioequivalence; and data analytics and machine learning. Regarding educational topics, the top three selections were complex injectables, formulations, and nanomaterials; drug-device combination products; and data analytics, including quantitative methods and modeling & simulation. These survey results will help prioritize the CRCG’s initial research and educational initiatives. Supplementary Information The online version contains supplementary material available at 10.1007/s11095-021-03149-y.

Objectives To better understand patients' and pharmacists' preferences for and experiences with changes in pill appearance (size, shape, color, and markings). Study design Cross-sectional. Methods We conducted independent national surveys of patients 50 years and older taking generic drugs for depression, diabetes, epilepsy, HIV, hyperlipidemia, or hypertension and of licensed pharmacists practicing in chain, franchise, or independent pharmacies. Responses were collected between January and April 2016. Results Of 1000 patient respondents (30% response rate), most reported experiencing changes in pill appearance (51%) and preferred to be notified about them (82%), but less than half recalled being notified (verbally: 36%; via sticker: 45%). Among patients who reported experiencing a change, 12% reported stopping their medication or using it less frequently. Of 710 pharmacist respondents (33% response rate), many reported changes in pill appearance occurring frequently in their pharmacies (47% reported that changes occurred 6 or more times per month) and more than three-fourths reported notifying patients about them often (verbally: 88%; via sticker: 77%). Conclusions Our findings reveal opportunities to improve patients' experiences with pill appearance changes through better notification practices and patient education.

Nanomedicines are nanoparticle-based therapeutic or diagnostic agents designed for targeted delivery or enhanced stability. Nanotechnology has been successfully employed to develop various drug formulations with improved pharmacokinetic characteristics, and current research efforts are focused on the development of new innovator and generic nanomedicines. Nanomedicines, which are often denoted as complex or nonbiological complex drugs, have inherently different physicochemical and pharmacokinetic properties than conventional small molecule drugs. The tools necessary to fully evaluate nanomedicines in clinical settings are limited, which can hamper their development. One of the most successful families of nanomedicines are iron-carbohydrate nanoparticles, which are administered intravenously (IV) to treat iron-deficiency anemia. In the U.S., the FDA has approved six distinct iron-carbohydrate nanoparticles but only one generic version (sodium ferric gluconate for Ferrlecit). There is significant interest in approving additional generic iron-carbohydrate drugs; however, the lack of a direct method to monitor the fate of the iron nanoparticles in clinical samples has impeded this approval. Herein we report a novel liquid chromatography–inductively coupled plasma–mass spectrometry (LC–ICP–MS) method that allows for the direct quantification of the iron-carbohydrate drugs in clinical samples, while simultaneously measuring the speciation of the iron released from the nanoparticles in biological samples. To our knowledge, this is the first time that iron nanoparticles have been observed in clinical samples, opening the door for direct pharmacokinetic studies of this family of drugs. This method has potential applications not only for iron-nanoparticle drugs but also for any nanomedicine with an inorganic component.

BACKGROUND: Generic versions of a drug can vary in appearance, which can impact adherence. OBJECTIVE: To assess the preferences, perceptions, and responses of patients who experienced a change in the appearance of a generic medication. DESIGN: Cross-sectional survey of patients from a large commercial health plan. PARTICIPANTS: Adults receiving generic versions of lisinopril, fluoxetine, lamotrigine, or simvastatin who experienced a change in the color or shape of their pills between March 2014 and November 2015. MAIN MEASURES: Likert-scale responses to questions concerning perceptions of generic drug safety and effectiveness, reliance on and preferences for pill appearance, and responses to pill appearance changes. Multivariable logistic regression-modeled predictors of seeking advice and adjusting use following a pill appearance change. KEY RESULTS: Of 814 respondents (response rate = 41%), 72% relied on pill appearance to ensure they took the correct medication. A similar percentage wanted their pills to remain the same color (72%), shape (71%), and size (75%) upon refill, but 58% would not have paid a $1 premium on a $5 co-pay to ensure such consistency. Most respondents (86%) wanted their pharmacists to notify them about pill appearance changes, but only 37% recalled such notification; 21% thought they received the wrong medication, and 8% adjusted medication use. Younger respondents (18–33 vs. 50–57 years) were more likely to seek advice (odds ratio [OR] = 1.91; 95% confidence interval [CI],1.02–3.59), and respondents with lower household income ( $100,000) were more likely to adjust medication use (OR = 3.40; 95% CI,1.09–10.67). CONCLUSIONS: Requiring uniform pill appearance may help increase adherence but presents challenges. Standardized pharmacy notification and education policies may be a more feasible short-term solution. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s11606-018-4791-1) contains supplementary material, which is available to authorized users.

This report provides a summary of the 4th International Conference on Global Bioequivalence Harmonisation Initiative (GBHI) that was co-organised by the European Federation of Pharmaceutical Sciences (EUFEPS) and the American Association of Pharmaceutical Scientists (AAPS). The goal of the GBHI conference is to offer the most informative and up to date science and regulatory thinking of bioequivalence (BE) in global drug development to support the intended process of a scientific global harmonisation. The workshop provided an open forum for pharmaceutical scientists from academia, industry and regulatory agencies to discuss three BE topics of interest, (a) BE assessment for long-acting injectables and implants, (b) necessity of fed BE studies for immediate-release products and (c) procedures to demonstrate equivalence of orally inhaled products. Moreover, in keynote lectures, a potential road map to an international BE reference product was discussed, and visions and perspectives for future global BE harmonisation activities have been presented. The meeting delivered a cutting-edge insight into the topics in an interactive and at the same time focused way.

Background: Accumulation of systemically administered nanoparticles (NPs) in lymph nodes has been exploited clinically for diagnostic imaging (e.g., USPIOs for lymph node metastasis) and therapeutic applications (e.g., vaccine delivery). However, the combination of diagnostic and therapeutic functionalities into a single theranostic NP has obliged undesirable trade-offs between either the imaging or drug delivery of the NP and their specific accumulation in lymph nodes. To overcome these trade-offs, we conducted a screen of various lipid-based theranostic NPs focusing on differing NP design and their resulting pharmacokinetic behaviours in healthy and diseased lymph node models. Methods: Lipid-based theranostic NPs with varying physicochemical characteristics (e.g., formulation, size and morphology, surface targeting, etc.) were prepared with positron emitting Cu-64 and administered systemically at equivalent NP doses in healthy and diseased rodent models (i.e., mice and rats). NP types were assessed for time-dependent accumulation in major lymph node basins via non-invasive whole-body PET/MR imaging at two or more timepoints per animal. 72-hours post-injection the animals were sacrificed, and lymph nodes and major organs were excised for gamma counting and pathological evaluation. Pharmacokinetic behaviour of NPs in healthy versus diseased lymph nodes were calculated in individual animals and in naïvely pooled datasets using non-compartmental analysis. Results: Preliminary analysis identified a leading NP candidate with specific lymph node targeting in healthy and diseased rodents: a discoidal, 35-nm peptide-targeted HDL-mimetic. In comparison with a spherical, 100-nm PEGylated NP, the discoidal NP obtained greater absolute (%ID) and relative (%ID/g) amounts of injected dose in anatomically matched lymph nodes than the spherical NP, regardless of lymph node pathology. At greatest measured concentration in healthy lymph nodes, typically 24-hpi, the differences between the discoidal and spherical NPs were on average 3-fold greater (2.893 vs. 0.864, %ID/g). Differences in other pharmacokinetic parameters such as AUC (%ID/g*h) and MRT (h) were equally pronounced. Conclusions: Our preliminary analysis uncovered a discoidal, peptide-targeted HDL-mimetic with remarkable accumulation in lymph nodes of healthy and diseased models. Future investigations will focus on the biochemical and cellular mechanisms underlying their unique lymphatic pharmacokinetics. These preliminary results provide key insights for design of theranostic NPs for non-invasive imaging and staging lymph node pathologies, and for applications in delivery of therapeutics to lymph nodes following systemic administration. Citation Format: Michael S. Valic, Mark Zheng, Lili Ding, Michelle Lai, Chris J. Zhang, Tina Ye, Jenny Ma, Michael Halim, Pamela Schimmer, Wenlei Jiang, Juan Chen, Gang Zheng. Lymph node accumulation of theranostic lipid-based nanoparticles in healthy and diseased models: Preliminary results comparing nanoparticle morphology and targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 307.

We demonstrate the use of modeling and simulation to investigate bioequivalence (BE) concerns raised about generic warfarin products. To test the hypothesis that the loss of isopropyl alcohol and slow dissolution in acidic pH has significant impact on the pharmacokinetics of warfarin sodium tablets, we conducted physiologically based pharmacokinetic absorption modeling and simulation using formulation factors or in vitro dissolution profiles as input parameters. Sensitivity analyses indicated that warfarin pharmacokinetics was not sensitive to solubility, particle size, density, or dissolution rate in pH 4.5, but was affected by dissolution rate in pH 6.8 and potency. Virtual BE studies suggested that stressed warfarin sodium tablets with slow dissolution rate in pH 4.5 but having similar dissolution rate in pH 6.8 would be bioequivalent to the unstressed warfarin sodium tablets. A four-way, crossover, single-dose BE study in healthy subjects was conducted to test the same hypothesis and confirmed the simulation conclusion.

Enteral feeding tubes are used to deliver food or drugs to patients that cannot swallow. To deliver delayed-release drugs that are formulated as enteric coated granules to these patients via feeding tubes requires that they be suspended in water prior to administration. Importantly, the suspension of enteric granules in water of varying pH can cause damage to the enteric coating and affect the bioavailability of the drug. Here, analytical methods for testing acid resistance stability and particle size distribution (PSD) of esomeprazole granules were utilized to monitor the integrity of the granule enteric coating after water pretreatment and delivery through an oral syringe and nasogastric (NG) tube. Granules from esomeprazole magnesium delayed-release capsules were transferred to an oral syringe, suspended in water, and delivered on the bench through a NG tube. Subsequently, acid resistance stability, (i.e., the amount of drug released after 2-hour acid dissolution) was determined via HPLC and the particle size distributions (PSD) were measured with a laser diffraction system. All of the granules demonstrated acid resistance stability when the granules were delivered immediately (0 min incubation) through the oral syringe and NG tube. By contrast, some granules demonstrated significant drug release during acid exposure after a 15-min incubation period which mimics a possible delay in delivery of the drug from the syringe by the caregiver. A bimodal PSD was observed with these granules, which was attributed to debris from damaged enteric coating and particle agglomeration. The methods developed in this study could be used to distinguish batches with suboptimal product quality for delivery using NG tubes and to confirm the substitutability of generic drug products for this alternative route of administration.

Biotechnology and nanotechnology provide a growing number of innovator-driven complex drug products and their copy versions. Biologics exemplify one category of complex drugs, but there are also nonbiological complex drug products, including many nanomedicines, such as iron-carbohydrate complexes, drug-carrying liposomes or emulsions, and glatiramoids. In this white paper, which stems from a 1-day conference at the New York Academy of Sciences, we discuss regulatory frameworks in use worldwide (e.g., the U.S. Food and Drug Administration, the European Medicines Agency, the World Health Organization) to approve these complex drug products and their follow-on versions. One of the key questions remains how to assess equivalence of these complex products. We identify a number of points for which consensus was found among the stakeholders who were present: scientists from innovator and generic/follow-on companies, academia, and regulatory bodies from different parts of the world. A number of topics requiring follow-up were identified: (1) assessment of critical attributes to establish equivalence for follow-on versions, (2) the need to publish scientific findings in the public domain to further progress in the field, (3) the necessity to develop worldwide consensus regarding nomenclature and labeling of these complex products, and (4) regulatory actions when substandard complex drug products are identified.

The development of curative glioblastoma treatments and tumour-specific contrast agents that can overcome the blood–brain barrier (BBB) and infiltrative tumour morphology remains a challenge. Apolipoprotein E3 (apoE3) is a high density lipoprotein apolipoprotein that chaperones the transcytosis of nanoparticles across the BBB, and displays high-affinity binding with the low density lipoprotein receptor (LDLR), a cell-surface receptor overexpressed by glioblastoma cells. This LDLR overexpression and apoE3 binding capacity was exploited for the development of glioblastoma-targeted porphyrin-lipid apoE3 lipid nanoparticles (pyE-LNs) with intrinsic theranostic properties. Size-controlled discoidal and cholesteryl oleate (CO)-loaded spherical pyE-LNs were synthesized through the systematic variation of particle composition, which dictated nanoparticle size and morphology. Composition optimization yielded 30 nm pyE-LNs with stable loading of apoE3 and porphyrin-lipid that simultaneously conferred the nanoparticles with glioblastoma targeting and activatable near-infrared fluorescence imaging functionalities. A 4-fold higher uptake of pyE-LNs by LDLR-expressing U87 glioblastomas cells relative to minimally expressing ldlA7 cells was observed in vitro. This uptake was a result of receptor-mediated endocytosis, which could be inhibited through LDL competition and acetylation of particle apoE3 moieties. ApoE3-dependent delivery of pyE-LN to glioblastomas was also demonstrated in orthotopic U87-GFP tumour-bearing animals. Quantification of CO-loaded pyE-LN biodistribution demonstrated successful selective uptake of porphyrin by malignant tissue, with a 4 : 1 tumour : healthy tissue particle specificity. This allowed for the detection of strong, tumour-localized porphyrin fluorescence, which was diminished when apoE3-devoid py-LN particles were administered. Furthermore, this selective uptake yielded cell-specific potent PDT sensitization in vitro, resulting in an 83% reduction in glioblastoma cell viability. These results highlight the promising capacity of pyE-LNs to target porphyrin delivery to glioblastoma tumours for theranostic applications.

The European Federation of Pharmaceutical Sciences (EUFEPS) and American Association of Pharmaceutical Scientists (AAPS) have collaborated since 2015 to organize international conferences to support global harmonization of regulatory requirements for bioequivalence (BE) assessment. This collaboration has resulted in three Global Bioequivalence Harmonization Initiative (GBHI) workshops which provided a unique opportunity for scientists from academia, industry, and regulatory agencies to discuss current, complex BE issues. The 3rd GBHI workshop was held in April 2018 in Amsterdam/The Netherlands and covered the following topics: (a) the necessity of multiple-dose studies in BE testing; (b) BE of transdermal delivery systems, and (c) liposomal parenteral preparations. This report summarizes the extensive discussions that led to better understanding of the similarities and differences across the major regulatory agencies on these topics and paved the way for future international harmonization.

To guide developers of innovative and generic drug products that contain nanomaterials, the U.S. Food and Drug Administration issued the draft guidance for industry titled: "Drug Products, Including Biological Products, that Contain Nanomaterials" in December 2017. During the AAPS Guidance Forum on September 11, 2018, participants from industry, academia, and regulatory bodies discussed this draft guidance in an open setting. Two questions raised by the AAPS membership were discussed in more detail: what is the appropriate regulatory pathway for approval of drug products containing nanomaterials, and how to determine critical quality attributes (CQAs) for nanomaterials? During the meeting, clarification was provided on how the new FDA center-led guidance relates to older, specific nanomaterial class, or specific product-related guidances. The lively discussions concluded with some clear observations and recommendations: (I) Important lessons can be learned from how CQAs were determined for, e.g., biologics. (II) Publication of ongoing scientific discussions on strategies and studies determining CQAs of drug products containing nanomaterials will significantly strengthen the science base on this topic. Furthermore, (III) alignment on a global level on how to address new questions regarding nanomedicine development protocols will add to efficient development and approval of these much needed candidate nanomedicines (innovative and generic). Public meetings such as the AAPS Guidance Forum may serve as the place to have these discussions.

Sirolimus, an immunosuppressive agent used in renal transplantation, can prevent allograft rejection. Identification of the therapeutic index (the ratio of minimum toxic concentration to minimum therapeutic concentration) for immunosuppresants is necessary to optimize the care of patients and set standards for bioequivalence evaluation of sirolimus products. However, the therapeutic index for sirolimus has been inconsistently defined, potentially because of inconsistencies in sirolimus exposure-response relationships.The authors used retrospective therapeutic drug monitoring data from the electronic health records of patients treated in a tertiary health care system from 2008 to 2014 to (1) develop a population pharmacokinetic (PK) model, (2) use the model to simulate sirolimus concentrations, and (3) characterize the exposure-response relationship. Using Wilcoxon rank-sum and Fisher exact tests, the authors determined relationships between sirolimus exposure and adverse events (AEs) (anemia, leukopenia, thrombocytopenia, hyperlipidemia, and decline in renal function) and the composite efficacy end point of graft loss or rejection.The developed 2-compartment population PK model showed appropriate goodness of fit. In a late-phase (12 months), postrenal transplant population of 27 inpatients, the authors identified statistically significant relationships between 83 simulated peak and trough sirolimus concentrations and outcomes: graft loss or rejection (P = 0.018) and decline in renal function (P = 0.006), respectively.Use of therapeutic drug monitoring results and PK modeling permitted correlation of sirolimus concentrations with graft loss or rejection and decline in renal function. However, the method was limited in its assessment of other AEs. To better evaluate sirolimus exposure-response relationships, the method should be applied to a larger sample of newly transplanted patients with a higher propensity toward AEs or efficacy failure.

The objective was to assess the impact of larger than conventional amounts of 14 commonly used excipients on Biopharmaceutics Classification System (BCS) class 3 drug absorption in humans. Cimetidine and acyclovir were used as model class 3 drugs across three separate four-way crossover bioequivalence (BE) studies (n = 24 each) in healthy human volunteers, denoted as study 1A, 1B, and 2. In study 1A and 1B, three capsule formulations of each drug were manufactured, collectively involving 14 common excipients. Capsule formulations that incorporated hydroxypropyl methylcellulose (HPMC) or magnesium stearate exhibited lower absorption. The cimetidine commercial solution contained sorbitol and also resulted in lower absorption. Hence, in study 2, two capsule formulations with lower amounts of HPMC and magnesium stearate, the sorbitol-containing commercial solution, and a sorbitol-free solution were assessed for BE. Overall, 12 common excipients were found in large amounts to not impact BCS class 3 drug absorption in humans, such that these excipients need not be qualitatively the same nor quantitatively very similar to reference, but rather simply be not more than the quantities studied here. Meanwhile, for each HPMC and microcrystalline cellulose, BCS class 3 biowaivers require these two excipients to be qualitatively the same and quantitatively very similar to the reference.

The purpose of this study was to determine the intrapatient (within the same patient) variability of tacrolimus in adherent patients. Daily tacrolimus trough levels were obtained at home using dried blood spot technology in kidney and liver transplant recipients. Patients were randomized to receive 3 formulations of tacrolimus, each for two 1-week periods. Adherence was monitored by patient diary, pill counts, and use of the Medication Event Monitoring System (MEMS). Variability was quantified as the coefficient of variation (CV). Comparison of CV between groups was by independent t test or one-way ANOVA as appropriate. The population was found to be adherent with a rate of 99.9% with a mean interval between the evening and morning dose of tacrolimus of 11.86 hours. The median CV for the entire population was 15.2% (range 4.8%-110%). There were no differences in CV by allograft type or tacrolimus formulation. The multivariate analysis did not identify any demographic characteristics associated with a CV > 30%. In a highly adherent population, tacrolimus did not display high intrapatient variability. Given the association between IPV and poor allograft outcomes, future studies are needed to quantitate the influence of adherence and establish target IPV goals.

To understand the quality, efficacy, and safety of liposomal drugs, it is necessary to develop a robust and accurate method for the separation and the quantification of unencapsulated and liposome-associated drugs (or liposomal encapsulated drugs). Conventional methods involve separation of unencapsulated and liposome-associated drug using solid phase extraction and further drug quantification. This is a lengthy process, and sometimes solid phase extraction induces drug leakage from the liposomes causing erroneous results. In this study, a capillary electrophoresis (CE) with UV–Vis detection method was developed for the simultaneous separation and quantification of unencapsulated drug from liposome-associated drug using a doxorubicin-containing liposome formulation as the model drug. CE separates the unencapsulated drug and liposomal drugs based on their electrophoretic mobility under the electric field. Liposomal drugs were diluted to the appropriate concentrations with running buffer or 5% dextrose before hydrodynamic sample injection. Using a high-sensitivity detection cell, the doxorubicin detection sensitivity was enhanced about 10-fold compared to the conventional on-column UV–Vis detection with a 75 µm i.d. capillary column. The optimal separation of unencapsulated doxorubicin from liposome-associated doxorubicin with minimal perturbation of liposomes was accomplished using phosphate buffer (20 mM, pH 6.5) in the presence of 10% sucrose.

Head and neck cancer is the fifth most common type of cancer worldwide and remains challenging for effective treatment due to the proximity to critical anatomical structures in the head and neck region, which increases the probability of toxicity from surgery and radiotherapy, and therefore emphasizes the importance of maximizing the targeted ablation. We have assessed the effectiveness of porphysome nanoparticles to enhance fluorescence and photoacoustic imaging of head and neck tumors in rabbit and hamster models. In addition, we evaluated the effectiveness of this agent for localized photothermal ablative therapy of head and neck tumors. We have demonstrated that porphysomes not only enabled fluorescence and photoacoustic imaging of buccal and tongue carcinomas, but also allowed for complete targeted ablation of these tumors. The supremacy of porphysome-enabled photothermal therapy over surgery to completely eradicate primary tumors and metastatic regional lymph node while sparing the adjacent critical structures' function has been demonstrated for the first time. This study represents a novel breakthrough that has the potential to revolutionize our approach to tumor diagnosis and treatment in head and neck cancer and beyond.

Purpose Currently, the FDA allows biowaivers for Class I (high solubility and high permeability) and Class III (high solubility and low permeability) compounds of the Biopharmaceutics Classification System (BCS). Scientific evidence should be provided to support biowaivers for BCS Class I and Class III (high solubility and low permeability) compounds. Methods Data on the effects of excipients on drug permeability are needed to demonstrate that commonly used excipients do not affect the permeability of BCS Class III compounds, which would support the application of biowaivers to Class III compounds. This study was designed to generate such data by assessing the permeability of four BCS Class III compounds and one Class I compound in the presence and absence of five commonly used excipients. Results The permeability of each of the compounds was assessed, at three to five concentrations, with each excipient in two different models: Caco-2 cell monolayers, and in situ rat intestinal perfusion. No substantial increases in the permeability of any of the compounds were observed in the presence of any of the tested excipients in either of the models, with the exception of disruption of Caco-2 cell monolayer integrity by sodium lauryl sulfate at 0.1 mg/ml and higher. Conclusion The results suggest that the absorption of these four BCS Class III compounds would not be greatly affected by the tested excipients. This may have implications in supporting biowaivers for BCS Class III compounds in general.

• Release of levetiracetam from extended-release tablets that appear intact in patient stool.

Iron deficiency anemia is a common clinical consequence for people who suffer from chronic kidney disease, especially those requiring dialysis. Intravenous (IV) iron therapy is a widely accepted safe and efficacious treatment for iron deficiency anemia. Numerous IV iron drugs have been approved by U.S. Food and Drug Administration (FDA), including a single generic product, sodium ferric gluconate complex in sucrose. In this study, we compared the cellular iron uptake profiles of the brand (Ferrlecit®) and generic sodium ferric gluconate (SFG) products. We used a colorimetric assay to examine the amount of iron uptake by three human macrophage cell lines. This is the first published study to provide a parallel evaluation of the cellular uptake of a brand and a generic IV iron drug in a mononuclear phagocyte system. The results showed no difference in iron uptake across all cell lines, tested doses, and time points. The matching iron uptake profiles of Ferrlecit® and its generic product support the FDA’s present position detailed in the draft guidance on development of SFG complex products that bioequivalence can be based on qualitative (Q1) and quantitative (Q2) formulation sameness, similar physiochemical characterization, and pharmacokinetic bioequivalence studies.

Background: Social media offer a novel avenue to engage with and recruit research participants. Facebook in particular is a promising option given its popularity and widespread use. Objective: To explore the feasibility of using Facebook to recruit physicians and patients to participate in a survey to assess their perceptions about generic venlafaxine extended release (ER) tablet indicated for depression. Methods: Web-based surveys were developed to gauge physicians’ prescribing experiences with and patients’ perceptions of generic venlafaxine ER tablet. The surveys included questions specific to venlafaxine ER tablets, such as perceived safety and efficacy of the drug and overall comfort level with either prescribing or taking the drug. Survey links were then posted and advertised on Facebook to recruit physicians and patients. Results: Advertisement for physicians reached 1898 Facebook users and advertisement for patients reached 1144 users during a 10-day advertising period. However, only 14 and 35 users clicked on the survey for physicians and patients, respectively. No physician completed the physician survey while 3 patients completed the patient survey. Conclusions: The findings of this study suggest that Facebook may not be an effective method to recruit physicians. Facebook holds promise to recruit patients, but additional recruitment efforts, such as incentives, are needed.

Relative biodistribution of FDA-approved innovator and generic sodium ferric gluconate (SFG) drug products was investigated to identify differences in tissue distribution of iron after intravenous dosing to rats. Three equal cohorts of 42 male Sprague-Dawley rats were created with each cohort receiving one of three treatments: (1) the innovator SFG product dosed intravenously at a concentration of 40 mg/kg; (2) the generic SFG product dosed intravenously at a concentration of 40 mg/kg; (3) saline dosed intravenously at equivalent volume to SFG products. Sampling time points were 15 min, 1 h, 8 h, 1 week, two weeks, four weeks, and six weeks post-treatment. Six rats from each group were sacrificed at each time point. Serum, femoral bone marrow, lungs, brain, heart, kidneys, liver, and spleen were harvested and evaluated for total iron concentration by ICP-MS. The ICP-MS analytical method was validated with linearity, range, accuracy, and precision. Results were determined for mean iron concentrations (µg/g) and mean total iron (whole tissue) content (µg/tissue) for each tissue of all groups at each time point. A percent of total distribution to each tissue was calculated for both products. At any given time point, the overall percent iron concentration distribution did not vary between the two SFG drugs by more than 7% in any tissue. Overall, this study demonstrated similar tissue biodistribution for the two SFG products in the examined tissues.

Intravenous (IV) iron formulations are complex colloidal suspensions of iron oxide nanoparticles. Small changes in formulation can allow more labile iron to be released after injection causing toxicity. Thus, bioequivalence (BE) evaluation of generic IV iron formulations remains challenging. We evaluated labile iron release in vitro and in vivo using a high performance liquid chromatography chelatable iron assay to develop a relational model to support BE. In vitro labile iron release and in vivo labile iron pharmacokinetics were evaluated for Venofer®, Ferrlecit®, generic sodium ferric gluconate complex, InFeD®, Feraheme® and a pre-clinical formulation GE121333. Labile iron release profiles were studied in vitro in 150mM saline and a biorelevant matrix (rat serum) at 0.952 mgFe/mL. In vivo plasma labile iron concentration-time profiles (t0-240 min) were studied in rats after a 40 mgFe/kg IV dose. In vitro labile iron release in saline was significantly higher compared to rat serum, especially with InFeD®. An in vitro release constant (iKr) was calculated which correlated well with maximal plasma concentrations in the in vivo rat PK model (R(2) = 0.711). These data suggest an in vitro to in vivo correlation model of labile iron release kinetics could be applied to BE. Other generic IV iron formulations need to be studied to validate this model.

Background Although the generic drug approval process has a long-term successful track record, concerns remain for approval of narrow therapeutic index generic immunosuppressants, such as tacrolimus, in transplant recipients. Several professional transplant societies and publications have generated skepticism of the generic approval process. Three major areas of concern are that the pharmacokinetic properties of generic products and the innovator (that is, “brand”) product in healthy volunteers may not reflect those in transplant recipients, bioequivalence between generic and innovator may not ensure bioequivalence between generics, and high-risk patients may have specific bioequivalence concerns. Such concerns have been fueled by anecdotal observations and retrospective and uncontrolled published studies, while well-designed, controlled prospective studies testing the validity of the regulatory bioequivalence testing approach for narrow therapeutic index immunosuppressants in transplant recipients have been lacking. Thus, the present study prospectively assesses bioequivalence between innovator tacrolimus and 2 generics in individuals with a kidney or liver transplant. Methods and findings From December 2013 through October 2014, a prospective, replicate dosing, partially blinded, randomized, 3-treatment, 6-period crossover bioequivalence study was conducted at the University of Cincinnati in individuals with a kidney (n = 35) or liver transplant (n = 36). Abbreviated New Drug Applications (ANDA) data that included manufacturing and healthy individual pharmacokinetic data for all generics were evaluated to select the 2 most disparate generics from innovator, and these were named Generic Hi and Generic Lo. During the 8-week study period, pharmacokinetic studies assessed the bioequivalence of Generic Hi and Generic Lo with the Innovator tacrolimus and with each other. Bioequivalence of the major tacrolimus metabolite was also assessed. All products fell within the US Food and Drug Administration (FDA) average bioequivalence (ABE) acceptance criteria of a 90% confidence interval contained within the confidence limits of 80.00% and 125.00%. Within-subject variability was similar for the area under the curve (AUC) (range 12.11–15.81) and the concentration maximum (Cmax) (range 17.96–24.72) for all products. The within-subject variability was utilized to calculate the scaled average bioequivalence (SCABE) 90% confidence interval. The calculated SCABE 90% confidence interval was 84.65%–118.13% and 80.00%–125.00% for AUC and Cmax, respectively. The more stringent SCABE acceptance criteria were met for all product comparisons for AUC and Cmax in both individuals with a kidney transplant and those with a liver transplant. European Medicines Agency (EMA) acceptance criteria for narrow therapeutic index drugs were also met, with the only exception being in the case of Brand versus Generic Lo, in which the upper limits of the 90% confidence intervals were 111.30% (kidney) and 112.12% (liver). These were only slightly above the upper EMA acceptance criteria limit for an AUC of 111.11%. SCABE criteria were also met for the major tacrolimus metabolite 13-O-desmethyl tacrolimus for AUC, but it failed the EMA criterion. No acute rejections, no differences in renal function in all individuals, and no differences in liver function were observed in individuals with a liver transplant using the Tukey honest significant difference (HSD) test for multiple comparisons. Fifty-two percent and 65% of all individuals with a kidney or liver transplant, respectively, reported an adverse event. The Exact McNemar test for paired categorical data with adjustments for multiple comparisons was used to compare adverse event rates among the products. No statistically significant differences among any pairs of products were found for any adverse event code or for adverse events overall. Limitations of this study include that the observations were made under strictly controlled conditions that did not allow for the impact of nonadherence or feeding on the possible pharmacokinetic differences. Generic Hi and Lo were selected based upon bioequivalence data in healthy volunteers because no pharmacokinetic data in recipients were available for all products. The safety data should be interpreted in light of the small number of participants and the short observation periods. Lastly, only the 1 mg tacrolimus strength was utilized in this study. Conclusions Using an innovative, controlled bioequivalence study design, we observed equivalence between tacrolimus innovator and 2 generic products as well as between 2 generic products in individuals after kidney or liver transplantation following current FDA bioequivalence metrics. These results support the position that bioequivalence for the narrow therapeutic index drug tacrolimus translates from healthy volunteers to individuals receiving a kidney or liver transplant and provides evidence that generic products that are bioequivalent with the innovator product are also bioequivalent to each other. Trial registration ClinicalTrials.gov NCT01889758., In a randomized 3-treatment crossover clinical trial, Rita Alloway of the University of Cincinnati and colleagues establish bioequivalence between 3 versions of the drug tracolimus (a brand name and 2 disparate generics) in kidney and liver transplant recipients., Author summary Why was this study done? Consensus documents developed by professional transplantation societies worldwide have cautioned the use of generic immunosuppressants such as tacrolimus in individuals with a solid organ transplant. Reasons have included repeated switching between innovator (that is, “brand” products) and generics and among different generics, especially when not controlled by physicians. There was uncertainty in the transplant community as to whether tacrolimus generics that are bioequivalent to the innovator are also bioequivalent to each other. For market approval, generic drug products of the narrow therapeutic index drug tacrolimus had to be studied only in healthy individuals and not in the much more complex organ transplant population. What did the researchers do and find? We performed a randomized, prospective, 3-treatment, 6-period, crossover, replicate dose study in individuals with a kidney or liver transplant. Thirty-five individuals with a kidney transplant and 36 individuals with a liver transplant receiving tacrolimus were studied to compare the tacrolimus time concentration profiles of 3 different products in their blood: namely, Innovator (Prograf), Generic Hi (Sandoz), and Generic Lo (Dr. Reddy) 1.0 mg tacrolimus capsules. Generic products were selected based upon pharmacokinetic data from healthy volunteer studies since bioequivalence data were not available in individuals with an organ transplant. We observed bioequivalence based on average bioequivalence and scaled average bioequivalence criteria in individuals after kidney or liver transplant between tacrolimus innovator and the 2 generics on the US market as well as between the 2 generics. What do these findings mean? Similar tacrolimus exposure is expected in individuals with a kidney or liver transplant when receiving Prograf, Sandoz generic, or Dr. Reddy’s generic tacrolimus.

Importance Switching between generic antiepileptic drugs is a highly debated issue that affects both clinical care and overall health care costs. Objective To evaluate the single-dose pharmacokinetic bioequivalence of 3 (1 branded and 2 generic drugs) on-market, immediate-release lamotrigine drug products. Design, Setting, and Participants The Equivalence Among Antiepileptic Drug Generic and Brand Products in People With Epilepsy (EQUIGEN) single-dose study is a crossover, prospective, sequence-randomized, replicate pharmacokinetic study conducted at 5 US academic epilepsy centers. Fifty adults (≥18 years) with epilepsy who were taking concomitant antiepileptic drugs and not currently receiving lamotrigine were enrolled between July 18, 2013, and January 19, 2015. Every participant was randomly assigned to 1 of 3 equivalent sequences, each comprising 6 study periods, during which they had blood draws before and after medication administration. Forty-nine participants were included in intention-to-treat analyses. Interventions Participants received a single 25-mg dose of immediate-release lamotrigine at the start of each period, with the branded and the 2 most disparate generic products each studied twice. Lamotrigine was selected as the antiepileptic drug of interest because of its wide use, publications indicating problems with generic switches, and complaints to the US Food and Drug Administration regarding generic products. Both participants and study personnel were blinded to the specific generic products selected. Main Outcomes and Measures The primary outcome was bioequivalence between products. Maximum plasma concentration (Cmax) and area under the concentration–time curve (AUC) were compared, and average bioequivalence (ABE) was established if the 90% CIs of the ratios of the 2 products were within equivalence limits (80%-125%). Results Of the 50 randomized participants, 49 (98%) received all 3 lamotrigine products and completed at least 3 pharmacokinetic assessments and 46 (92%) completed all 6 pharmacokinetic assessments. Among the 49 participants, 28 (57%) were men and 21 (43%) were women, 42 (86%) self-identified as white, and 46 (16) years was the mean (SD) age. The 3 drug products were considered bioequivalent because the 90% CIs were within equivalence limits (lowest and highest CI limits for Cmax, 92.6% and 110.4%; for AUC 0-96 , 96.9% and 101.9%). Replicate testing demonstrated no significant differences in within-subject variability across the 3 products (likelihood ratios, χ 2 2 for log-transformed variables: AUC 0-96 , 2.58; Cmax, 0.64; and AUC 0-∞ , 4.05; P ≥ .13) and that the 3 products were also bioequivalent according to scaled ABE and individual bioequivalence criteria with no subject × formulation interaction (Cmax, 0.00; AUC 0-96 , 0.54; and AUC 0-∞ , 0.36; P ≥ .76). Conclusions and Relevance This study provides evidence that the disparate lamotrigine products studied are bioequivalent when tested in people with epilepsy taking concomitant antiepileptic drugs. Trial Registration clinicaltrials.gov Identifier:NCT01733394

Small changes in bioavailability of narrow therapeutic index (NTI) drugs can alter clinical outcomes, raising concern over generic NTI substitution. We surveyed pharmacists to identify their perceptions of generic NTI drugs, their frequency of performing generic NTI substitution, and predictors of this behavior. Of 710 respondents (33% response rate), 87% perceived generic NTI drugs as effective as their brand-name versions and 94% as safe. Whereas 82% almost always performed generic NTI substitution for initial prescriptions, only 60% did for refills. Pharmacists in non-chain settings (odds ratio (OR) = 2.37; 95% confidence interval (CI) = 1.40-4.02), in practice longer (per year OR = 1.04; 95% CI = 1.02-1.06), in states with affirmative patient consent laws (OR = 1.88; 95% CI = 1.06-3.32), and in states with NTI-specific substitution requirements (OR = 1.95; 95% CI = 1.16-3.26) were more likely not to substitute initial prescriptions. Education of non-chain and veteran pharmacists and elimination of affirmative patient consent and NTI-specific substitution requirements could increase generic NTI substitution.

Background: The development of lipid-based nanoparticles for the delivery of drugs, imaging agents, ect. to solid tumors continues to yield promising candidates for clinical testing. However, concerns have been raised over the poor translation of preclinical datasets with nanoparticle-enabled therapies into clinical outcomes. In an effort to support the utility of preclinical tumors models for predicting nanoparticle accumulation, we have created a multi-compartmental pharmacokinetic model for lipid-based nanoparticles using experimental data in two prevalent xenograft models of ovarian cancer. Methods: Two ovarian cancer xenograft models, SK-OV-3 and OV-90, were orthotopically established in a single ovary of athymic nude mice. Once the tumors had reached a volume of 0.5-0.7 mm2, the mice were intra-venously administered equivalent doses of radio-labelled nanoparticles, either porphyrin-lipid-containing PEGylated nanovesicles (Porphysomes) and PEGylated liposomes. At six timepoints post administration (6, 12, 18, 24, 36, and 48 hours) a full necropsy was preformed, and the organs measured for radio-activity. The concentrations of nanoparticles in tumors and major organs were calculated and plotted versus time. A multi-compartmental pharmacokinetic model was constructed using the time-dependent concentration of nanoparticles for either xenograft model. Results: The pathology of the orthotopic SK-OV-3 and OV-90 models were remarkably different in their vascular-permeability and cellular density, and these differences contributed to diverging nanoparticle biokinetics. Porphysome accumulation (%I.D./g), exposure (A.U.C.), and delivery efficiency (%) were significantly greater in the SK-OV-3 tumors compared with control organs (e.g., healthy ovary, muscle, etc.). Liposomes, however, did not exhibit this trend in SK-OV-3 tumors. In the OV-90 tumors, both nanoparticles exhibited increased but statistically insignificant accumulation and exposure compared with control organs. Only Porphysomes demonstrated improved delivery efficiency in the OV-90 tumors. Modelling the pharmacokinetics of the nanoparticles in a multi-compartmental model revealed distinctive kinetic parameters specific to each nanoparticle type and tumor pathology. In particular, differences were observed in the clearance rates of Porphysomes and liposomes, providing a possible explanation for their dissimilar tumor accumulations and exposures. Conclusions: Differences in the accumulation of lipid-based nanoparticles in preclinical models of ovarian cancer appear most sensitive to vascular permeability and cellular density of the xenograft. Adjusting for xenograft-dependent kinetic parameters, the Porphysome demonstrated improved but statistically insignificant tumor exposure and delivery efficiency compared with liposomes. Citation Format: Michael S. Valic, Wenlei Jiang, Lili Ding, Juan Chen, Stéphanie Lheureux, Amit M. Oza, Gang Zheng. Modelling of lipid-based nanoparticle pharmacokinetics and tumor accumulation using preclinical models of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1947.

In the past few decades, the development of medicine at the nanoscale has been applied to oral and parenteral dosage forms in a wide range of therapeutic areas to enhance drug delivery and reduce toxicity. An obvious response to these benefits is reflected in higher market shares of complex drug products containing nanomaterials than that of conventional formulations containing the same active ingredient. The surging market interest has encouraged the pharmaceutical industry to develop cost-effective generic versions of complex drug products based on nanotechnology when the associated patent and exclusivity on the reference products have expired. Due to their complex nature, nanotechnology-based drugs present unique challenges in determining equivalence standards between generic and innovator products. This manuscript attempts to provide the scientific rationales and regulatory considerations of key equivalence standards (e.g., in vivo studies and in vitro physicochemical characterization) for oral drugs containing nanomaterials, iron-carbohydrate complexes, liposomes, protein-bound drugs, nanotube-forming drugs, and nano emulsions. It also presents active research studies in bridging regulatory and scientific gaps for establishing equivalence of complex products containing nanomaterials. We hope that open communication among industry, academia, and regulatory agencies will accelerate the development and approval processes of generic complex products based on nanotechnology.

It has been proposed that proinflammatory mechanisms are involved in the pathogenesis of brain edema in acute liver failure (ALF). The aim of this study was to assess the contribution of cerebral inflammation to the neurologic complications of ALF and to assess the antiinflammatory effect of mild hypothermia. Upregulation of CD11b/c immunoreactivity, consistent with microglial activation, was observed in the brains of ALF rats at coma stages of encephalopathy. Interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) mRNAs were increased two to threefold in the brains of ALF rats compared with that in sham-operated controls. The magnitude of increased expression of proinflammatory cytokines in the brain was correlated with the progression of encephalopathy and the onset of brain edema. Significant increases in IL-1beta, IL-6, and TNF-alpha levels were also found in the sera and cerebrospinal fluid (CSF) of these animals. Mild hypothermia delayed the onset of encephalopathy, prevented brain edema, and concomitantly attenuated plasma, brain, and CSF proinflammatory cytokines. These results show that experimental ALF leads to increases in brain production of proinflammatory cytokines, and afford the first direct evidence that central inflammatory mechanisms play a role in the pathogenesis of the cerebral complications of ALF. Antiinflammatory agents could be beneficial in the management of these complications.

Lipocalin 2 (Lcn2), a mammalian protein that is expressed and secreted in various pathologic states, binds siderophores, which are high-affinity iron chelators. Besides its role in limiting iron availability to pathogens in the setting of bacterial infection, Lcn2:siderophore complexes can also deliver iron to cells. In this study, we examined Lcn2 regulation in the liver of mice in situations of increased iron utilization, namely, during anemia. Anemia induced by phlebotomy, iron deprivation, or phenylhydrazine treatment was associated with upregulation of Lcn2 gene expression in the liver and elevation of serum Lcn2 protein levels. We further explored the participation of several factors known to co-occur during anemia, including hypoxia, changes in iron levels, and erythropoietic drive, in the regulation of Lcn2 by anemia. We found that hypoxia, but not iron or erythropoietin, caused an induction of Lcn2 expression. The upregulation of Lcn2 levels by anemia and hypoxia, which is not directly mediated by iron or erythropoietin, suggests a possible physiological role for Lcn2 during increased iron utilization and mobilization from stores.

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United States. Tacrolimus is a narrow therapeutic index drug and as such requires therapeutic drug monitoring and dose adjustment based on its whole blood trough concentrations. To facilitate home therapeutic drug and adherence monitoring, the collection of dried blood spots is an attractive concept. After a finger stick, the patient collects a blood drop on filter paper at home. After the blood is dried, it is mailed to the analytical laboratory where tacrolimus is quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in combination with a simple manual protein precipitation step and online column extraction. For tacrolimus analysis, a 6-mm disc is punched from the saturated center of the blood spot. The blood spot is homogenized using a bullet blender and then proteins are precipitated with methanol/0.2 M ZnSO4 containing the internal standard D2,(13)C-tacrolimus. After vortexing and centrifugation, 100 µl of supernatant is injected into an online extraction column and washed with 5 ml/min of 0.1 formic acid/acetonitrile (7:3, v:v) for 1 min. Hereafter, the switching valve is activated and the analytes are back-flushed onto the analytical column (and separated using a 0.1% formic acid/acetonitrile gradient). Tacrolimus is quantified in the positive multi reaction mode (MRM) using a tandem mass spectrometer. The assay is linear from 1 to 50 ng/ml. Inter-assay variability (3.6%-6.1%) and accuracy (91.7%-101.6%) as assessed over 20 days meet acceptance criteria. Average extraction recovery is 95.5%. There are no relevant carry-over, matrix interferences and matrix effects. Tacrolimus is stable in dried blood spots at RT and at +4 °C for 1 week. Extracted samples in the autosampler are stable at +4 °C for at least 72 hr.

Doxil® is a complex parenteral doxorubicin (DOX) liposome formulation approved by the FDA. For generic doxorubicin liposomes, analyzing the release profile of DOX is important for quality control and comparability studies. However, there is no robust standard drug release assay available for doxorubicin liposomes. In this study, we describe a USP-4 apparatus assay capable of discriminating DOX liposomal formulations based on release profile. Establishment of the assay was hindered by limited DOX release from liposomes in physiological conditions at 37°C. The addition of NH4HCO3 to the release media facilitated DOX release proportionally to the salt concentration added but caused precipitation of released drug in USP-4 apparatus. Precipitation of DOX was avoided by adding hydroxypropyl-cyclodextrin (HP-CD) to the release medium. We optimized conditions for DOX release by varying a number of parameters such as: concentration of HP-CD, testing temperature, and concentration of tested samples. The optimized release medium contained: 100 mM NH4HCO3, 75 mM 2-(N-morpholino) ethanesulfonic acid (MES) and 5% w/v HP-CD, 5% w/v sucrose, 0.02% w/v NaN3 (pH 6). The drug release assay was performed at 45°C. The optimized release assay can discriminate between DOX liposomal formulations of different compositions, physicochemical properties, and prepared by different manufacturing methods. This indicates that the assay could be used to compare DOX release from generic DOX formulations to the innovator product Doxil®.

OBJECTIVE To test the current U.S. Food and Drug Administration (FDA) bioequivalence standard in a comparison of generic and brand-name drug pharmacokinetic (PK) performance in "generic-brittle" patients with epilepsy under clinical use conditions. METHODS This randomized, double-blind, multiple-dose, steady-state, fully replicated bioequivalence study compared generic lamotrigine to brand-name Lamictal in "generic-brittle" patients with epilepsy (n = 34) who were already taking lamotrigine. Patients were repeatedly switched between masked Lamictal and generic lamotrigine. Intensive PK blood sampling at the end of each 2-week treatment period yielded two 12-h PK profiles for brand-name and generic forms for each patient. Steady-state area under the curve (AUC), peak plasma concentration (Cmax ), and minimum plasma concentration (Cmin ) data were subjected to conventional average bioequivalence (ABE) analysis, reference-scaled ABE analysis, and within-subject variability (WSV) comparisons. In addition, generic-versus-brand comparisons in individual patients were performed. Secondary clinical outcomes included seizure frequency and adverse events. RESULTS Generic demonstrated bioequivalence to brand. The 90% confidence intervals of the mean for steady-state AUC, Cmax , and Cmin for generic-versus-brand were 97.2-101.6%, 98.8-104.5%, and 93.4-101.0%, respectively. The WSV of generic and brand were also similar. Individual patient PK ratios for generic-versus-brand were similar but not identical, in part because brand-versus-brand profiles were not identical, even though subjects were rechallenged with the same product. Few subjects had seizure exacerbations or tolerability issues with product switching. One subject, however, reported 267 focal motor seizures, primarily on generic, although his brand and generic PK profiles were practically identical. SIGNIFICANCE Some neurologists question whether bioequivalence in healthy volunteers ensures therapeutic equivalence of brand and generic antiepileptic drugs in patients with epilepsy, who may be at increased risk for problems with brand-to-generic switching. Bioequivalence results in "generic-brittle" patients with epilepsy under clinical conditions support the soundness of the FDA bioequivalence standards. Adverse events on generic were not related to the small, allowable PK differences between generic and brand.

Hereditary hemochromatosis (HH), an iron overload disease associated with mutations in the HFE gene, is characterized by increased intestinal iron absorption and consequent deposition of excess iron, primarily in the liver. Patients with HH and Hfe-deficient (Hfe-/-) mice manifest inappropriate expression of the iron absorption regulator hepcidin, a peptide hormone produced by the liver in response to iron loading. In this study, we investigated the contribution of Hfe expression in macrophages to the regulation of liver hepcidin levels and iron loading. We used bone marrow transplantation to generate wild-type (wt) and Hfe-/- mice chimeric for macrophage Hfe gene expression. Reconstitution of Hfe-deficient mice with wt bone marrow resulted in augmented capacity of the spleen to store iron and in significantly decreased liver iron loading, accompanied by a significant increase of hepatic hepcidin mRNA levels. Conversely, wt mice reconstituted with Hfe-deficient bone marrow had a diminished capacity to store iron in the spleen but no significant alterations of liver iron stores or hepcidin mRNA levels. Our results suggest that macrophage Hfe participates in the regulation of splenic and liver iron concentrations and liver hepcidin expression. (Blood. 2005;106:2189-2195)

The development of curative glioblastoma treatments and tumour-specific contrast agents that can overcome the blood-brain barrier (BBB) and infiltrative tumour morphology remains a challenge. Apolipoprotein E3 (apoE3) is a high density lipoprotein apolipoprotein that chaperones the transcytosis of nanoparticles across the BBB, and displays high-affinity binding with the low density lipoprotein receptor (LDLR), a cell-surface receptor overexpressed by glioblastoma cells. This LDLR overexpression and apoE3 binding capacity was exploited for the development of glioblastoma-targeted porphyrin-lipid apoE3 lipid nanoparticles (pyE-LNs) with intrinsic theranostic properties. Size-controlled discoidal and cholesteryl oleate (CO)-loaded spherical pyE-LNs were synthesized through the systematic variation of particle composition, which dictated nanoparticle size and morphology. Composition optimization yielded 30 nm pyE-LNs with stable loading of apoE3 and porphyrin-lipid that simultaneously conferred the nanoparticles with glioblastoma targeting and activatable near-infrared fluorescence imaging functionalities. A 4-fold higher uptake of pyE-LNs by LDLR-expressing U87 glioblastomas cells relative to minimally expressing ldlA7 cells was observed