A pragmatic regulatory approach for complex generics through the U.S. FDA 505(j) or 505(b)(2) approval pathways.

The diverse nature of complex drug products poses challenges for the development of regulatory guidelines for generic versions. While complexity is not new in medicines, the technical capacity to measure and analyze data has increased. This requires a determination of which measurements and studies are relevant to demonstrate therapeutic equivalence. This paper describes the views of the NBCD Working Group and provides pragmatic solutions for approving complex generics by making best use of existing U.S. Food and Drug Administration's abbreviated approval pathways 505(j) and 505(b)(2). We argue that decisions on the appropriateness of submitting a 505(j) or 505(b)(2) application can build on the FDA's complex drug product classification as well as the FDA's much applauded guidance document for determining whether to submit an ANDA or a 505(b)(2) application. We hope that this paper contributes to the discussions to increase the clarity of regulatory approaches for complex generics, as well as the predictability for complex generic drug developers, to facilitate access to much-needed complex generics and to promote the sustainability of the healthcare system.

Publisher Correction: On the issue of transparency and reproducibility in nanomedicine.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Publisher Correction: On the issue of transparency and reproducibility in nanomedicine.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Report of the AAPS Guidance Forum on the FDA Draft Guidance for Industry: "Drug Products, Including Biological Products, that Contain Nanomaterials".

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of innovative and generic drug products that contain nanomaterials, the U.S. Food and Drug Administration issued the draft guidance for industry titled: "Drug Products, Including Biological Products, that Contain Nanomaterials" in December 2017.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AAPS Guidance Forum on September 11, 2018, participants from industry, academia, and regulatory bodies discussed this draft guidance in an open setting.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.

Ion quantification in liposomal drug products using high performance liquid chromatography.

A simple, straightforward analytical method based on liquid chromatography has been optimized to quantify total, internal, and external ions in drug-loaded liposomal products. The quantification of ammonium and sulfate ions in Doxil is detailed; although, the methodology has been extrapolated to quantitate a variety of ions, including calcium, acetate, and others in several different liposomal formulations. Total ion concentrations were measured after disruption of the liposome via lyophilization, to liberate all components. External ion concentrations were made following membrane centrifugation, without disruption of the liposome structure, where the permeate fraction was analyzed for external ion quantities. The internal ion fraction was derived from mass balance of the total and external ion measurements. High performance liquid chromatography (HPLC), equipped with different separation columns, and coupled to a charged aerosol detector, was employed for all ion quantifications. The analytical measurements were confirmed using simple stoichiometry based on the drug crystallization of doxorubicin within the liposome interior. The method presented herein is quick, highly accurate, and has significantly improved lower limits of detection and quantification over other traditional methods. As more follow-on versions of Doxil are being developed, this facile approach to ion quantitation can be used to help establish compositional similarity to the reference listed drug.

Evaluating Nanomedicines: Obstacles and Advancements.

Continued advancements in nanotechnology are expanding the boundaries of medical research, most notably as drug delivery agents for treatment against cancer. Drug delivery with nanotechnology can offer greater control over the biodistribution of therapeutic agents to improve the therapeutic index. In the last 20 years, a number of nanomedicines have transitioned into the clinic. As nanomedicines evolve, techniques to properly evaluate their safety and efficacy must also evolve. Characterization methods for nano-based materials must be adapted to the demands of nanomedicine developers and regulators. This second edition book provides updated characterization protocols designed to address the clinical potential of nanomedicines during their preclinical development. In this chapter, the characterization challenges of nanoparticles intended for drug delivery will be discussed, along with examples of advancements and improvements in nanomedicine characterization.

Anticoagulants Influence the Performance of In Vitro Assays Intended for Characterization of Nanotechnology-Based Formulations.

The preclinical safety assessment of novel nanotechnology-based drug products frequently relies on in vitro assays, especially during the early stages of product development, due to the limited quantities of nanomaterials available for such studies. The majority of immunological tests require donor blood. To enable such tests one has to prevent the blood from coagulating, which is usually achieved by the addition of an anticoagulant into blood collection tubes. Heparin, ethylene diamine tetraacetic acid (EDTA), and citrate are the most commonly used anticoagulants. Novel anticoagulants such as hirudin are also available but are not broadly used. Despite the notion that certain anticoagulants may influence assay performance, a systematic comparison between traditional and novel anticoagulants in the in vitro assays intended for immunological characterization of nanotechnology-based formulations is currently not available. We compared hirudin-anticoagulated blood with its traditional counterparts in the standardized immunological assay cascade, and found that the type of anticoagulant did not influence the performance of the hemolysis assay. However, hirudin was more optimal for the complement activation and leukocyte proliferation assays, while traditional anticoagulants citrate and heparin were more appropriate for the coagulation and cytokine secretion assays. The results also suggest that traditional immunological controls such as lipopolysaccharide (LPS ) are not reliable for understanding the role of anticoagulant in the assay performance. We observed differences in the test results between hirudin and traditional anticoagulant-prepared blood for nanomaterials at the time when no such effects were seen with traditional controls. It is, therefore, important to recognize the advantages and limitations of each anticoagulant and consider individual nanoparticles on a case-by-case basis.

Zeta potential: a case study of cationic, anionic, and neutral liposomes.

Zeta potential is often used to approximate a nanoparticle's surface charge, i.e., cationic, anionic, or neutral character, and has become a standard characterization technique to evaluate nanoparticle surfaces. While useful, zeta potential values provide only very general conclusions about surface charge character. Without a thorough understanding of the measurement parameters and limitations of the technique, these values can become meaningless. This case study attempts to explore the sensitivity of zeta potential measurement using specifically formulated cationic, anionic, and neutral liposomes. This study examines zeta potential dependence on pH and ionic strength, resolving power, and highlights the sensitivity of zeta potential to charged liposomes. Liposomes were prepared with cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and varying amounts of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS). A strong linear relationship was noted between zeta potential values and the mole percentage of charged lipids within a liposome (e.g., cationic DOTAP or anionic DOPS). This finding could be used to formulate similar liposomes to a specific zeta potential, potentially of importance for systems sensitive to highly charged species. In addition, cationic and anionic liposomes were titrated with up to two mole percent of the neutral lipid 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (lipid-PEG; LP). Very small amounts of the lipid-PEG (<0.2 mol%) were found to impart stability to the DOTAP- and DOPS-containing liposomes without significantly affecting other physicochemical properties of the formulation, providing a simple approach to making stable liposomes with cationic and anionic surface charge.

Nanomedicine strategies to overcome the pathophysiological barriers of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer- related deaths. PDAC remains one of the most difficult-to-treat cancers, owing to its unique pathobiological features: a nearly impenetrable desmoplastic stroma, and hypovascular and hypoperfused tumour vessels render most treatment options largely ineffective. Progress in understanding the pathobiology and signalling pathways involved in disease progression is helping researchers to develop novel ways to fight PDAC, including improved nanotechnology-based drug-delivery platforms that have the potential to overcome the biological barriers of the disease that underlie persistent drug resistance. So-called 'nanomedicine' strategies have the potential to enable targeting of the Hedgehog-signalling pathway, the autophagy pathway, and specific RAS-mutant phenotypes, among other pathological processes of the disease. These novel therapies, alone or in combination with agents designed to disrupt the pathobiological barriers of the disease, could result in superior treatments, with increased efficacy and reduced off-target toxicities compared with the current standard-of-care regimens. By overcoming drug-delivery challenges, advances can be made in the treatment of PDAC, a disease for which limited improvement in overall survival has been achieved over the past several decades. We discuss the approaches to nanomedicine that have been pursued to date and those that are the focus of ongoing research, and outline their potential, as well as the key challenges that must be overcome.

Stable isotope method to measure drug release from nanomedicines.

Existing methods to measure nanomedicine drug release in biological matrices are inadequate. A novel drug release method utilizing a stable isotope tracer has been developed. Stable isotope-labeled drug is spiked into plasma containing nanomedicine. The labeled drug equilibrates with plasma components identical to the normoisotopic drug released from the nanomedicine formulation. Therefore, the ultrafilterable fraction of the isotope-labeled drug represents a reliable measure of free normoisotopic drug fraction in plasma, and can be used to calculate nanomedicine encapsulated and unencapsulated drug fractions. To demonstrate the utility of this method, we performed a plasma drug release study with both a fast releasing commercial docetaxel formulation, Taxotere®, and a delayed releasing nanomicellar formulation of a docetaxel prodrug, Procet 8. The instability of the unencapsulated prodrug in plasma allowed us to compare our calculated prodrug release and docetaxel conversion with the actual docetaxel concentration measured directly without fractionation. Drug release estimates for the fast releasing Taxotere formulation demonstrated accuracy deviation and precision (%CV) of <15%. For the controlled release Procet 8 formulation, we calculated a slow release and conversion of the prodrug in rat plasma that was highly correlated with the direct docetaxel measurement (R(2)=0.98). We believe that this method will have tremendous utility in the development and regulatory evaluation of nanomedicines, and aid in determination of generic bioequivalence.

Analyzing the influence of PEG molecular weight on the separation of PEGylated gold nanoparticles by asymmetric-flow field-flow fractionation.

Polyethylene glycol (PEG) is an important tool for increasing the biocompatibility of nanoparticle therapeutics. Understanding how these potential nanomedicines will react after they have been introduced into the bloodstream is a critical component of the preclinical evaluation process. Hence, it is paramount that better methods for separating, characterizing, and analyzing these complex and polydisperse formulations are developed. We present a method for separating nominal 30-nm gold nanoparticles coated with various molecular weight PEG moieties that uses only phosphate-buffered saline as the mobile phase, without the need for stabilizing surfactants. The optimized asymmetric-flow field-flow fractionation technique using in-line multiangle light scattering, dynamic light scattering, refractive index, and UV-vis detectors allowed successful separation and detection of a mixture of nanoparticles coated with 2-, 5-, 10-, and 20-kDa PEG. The particles coated with the larger PEG species (10 and 20 kDa) were eluted at times significantly earlier than predicted by field-flow fractionation theory. This was attributed to a lower-density PEG shell for the higher molecular weight PEGylated nanoparticles, which allows a more fluid PEG surface that can be greater influenced by external forces. Hence, the apparent particle hydrodynamic size may fluctuate significantly depending on the overall density of the stabilizing surface coating when an external force is applied. This has considerable implications for PEGylated nanoparticles intended for in vivo application, as nanoparticle size is important for determining circulation times, accumulation sites, and routes of excretion, and highlights the importance and value of the use of secondary size detectors when one is working with complex samples in asymmetric-flow field-flow fractionation.

Induction of oxidative stress by Taxol® vehicle Cremophor-EL triggers production of interleukin-8 by peripheral blood mononuclear cells through the mechanism not requiring de novo synthesis of mRNA.

Understanding the ability of cytotoxic oncology drugs, and their carriers and formulation excipients, to induce pro-inflammatory responses is important for establishing safe and efficacious formulations. Literature data about cytokine response induction by the traditional formulation of paclitaxel, Taxol®, are controversial, and no data are available about the pro-inflammatory profile of the nano-albumin formulation of this drug, Abraxane®. Herein, we demonstrate and explain the difference in the cytokine induction profile between Taxol® and Abraxane®, and describe a novel mechanism of cytokine induction by a nanosized excipient, Cremophor EL, which is not unique to Taxol® and is commonly used in the pharmaceutical industry for delivery of a wide variety of small molecular drugs. FROM THE CLINICAL EDITOR: Advances in nanotechnology have enabled the production of many nano-formulation drugs. The cellular response to drugs has been reported to be different between traditional and nano-formulations. In this article, the authors investigated and compared cytokine response induction profiles between Taxol® and Abraxane®. The findings here provided further understanding to create drugs with better safety profiles.

Safe anticoagulation when heart and lungs are "on vacation".

Circulation and oxygenation of blood outside the body is commonly required during complex surgical interventions involving coronary pulmonary bypass (CPB) and extracorporeal membrane oxygenation (ECMO). Both CPB and ECMO are life-supporting procedures utilizing a heart-lung machine, which subjects the blood to unphysiological conditions, potentially promoting undesirable blood coagulation. Traditionally, thrombotic complications from CPB and ECMO are resolved by heparin, an inexpensive broad spectrum anticoagulant that prevents blood clotting, but often results in bleeding. Despite hemostatic support therapy and constant monitoring, the lives of patients undergoing CPB and ECMO are often threatened by uncontrolled bleeding. There is an urgent need for novel strategies which provide safe anti-coagulation alternatives during CPB and ECMO procedures. Several non-traditional approaches, including nitric oxide donors as well as various protease and contact activation inhibitors, have been investigated and shown some success. More recently, Larsson et al. isolated a recombinant fully human (3F7) antibody inhibiting Factor XIIa. The antibody was shown to be both an efficacious and safe alternative to heparin. Below we will examine this study in more detail and offer considerations for translation of this novel concept to the clinic.

Immunological and hematological toxicities challenging clinical translation of nucleic acid-based therapeutics.

INTRODUCTION: Nucleic acid-based therapeutics (NATs) are proven agents in correcting disorders caused by gene mutations, as treatments against cancer, microbes and viruses, and as vaccine adjuvants. Although many traditional small molecule NATs have been approved for clinical use, commercialization of macromolecular NATs has been considerably slower, and only a few have successfully reached the market. Preclinical and clinical evaluation of macromolecular NATs has revealed many assorted challenges in immunotoxicity, hematotoxicity, pharmacokinetics (PKs), toxicology and formulation. Extensive review has been given to the PK and toxicological concerns of NATs including approaches designed to overcome these issues. Immunological and hematological issues are a commonly reported side effect of NAT treatment; however, literature exploring the mechanistic background of these effects is sparse. AREAS COVERED: This review focuses on the immunomodulatory properties of various types of therapeutic nucleic acid concepts. The most commonly observed immunological and hematological toxicities are described for various NAT classes, with citations of how to circumvent these toxicities. EXPERT OPINION: Although some success with overcoming immunological and hematological toxicities of NATs has been achieved in recent years, immunostimulation remains the main dose-limiting factor challenging clinical translation of these promising therapies. Novel delivery vehicles should be considered to overcome this challenge.

Strategy for selecting nanotechnology carriers to overcome immunological and hematological toxicities challenging clinical translation of nucleic acid-based therapeutics.

INTRODUCTION: Clinical translation of nucleic acid-based therapeutics (NATs) is hampered by assorted challenges in immunotoxicity, hematotoxicity, pharmacokinetics, toxicology and formulation. Nanotechnology-based platforms are being considered to help address some of these challenges due to the nanoparticles' ability to change drug biodistribution, stability, circulation half-life, route of administration and dosage. Addressing toxicology and pharmacology concerns by various means including NATs reformulation using nanotechnology-based carriers has been reviewed before. However, little attention was given to the immunological and hematological issues associated with nanotechnology reformulation. AREAS COVERED: This review focuses on application of nanotechnology carriers for delivery of various types of NATs, and how reformulation using nanoparticles affects immunological and hematological toxicities of this promising class of therapeutic agents. EXPERT OPINION: NATs share several immunological and hematological toxicities with common nanotechnology carriers. In order to avoid synergy or exaggeration of undesirable immunological and hematological effects of NATs by a nanocarrier, it is critical to consider the immunological compatibility of the nanotechnology platform and its components. Since receptors sensing nucleic acids are located essentially in all cellular compartments, a strategy for developing a nanoformulation with reduced immunotoxicity should first focus on precise delivery to the target site/cells and then on optimizing intracellular distribution.

The similarity question for biologicals and non-biological complex drugs.

For small - low molecular weight - molecule medicines a robust regulatory system has evolved over the years. This system guarantees high and constant quality of our (generic) medicines. Pharmaceutical equivalence and bioequivalence assessment are the pillars under that system. But there are complex medicines where the question of equivalence is more challenging to answer. For biologicals the paradigm of similarity rather than equality (the emergence of 'biosimilars') was developed in the past decade. This has been a program where an evolutionary, science based approach has been chosen by the frontrunner regulatory body, the EMA, with a 'learn and confirm' character. In addition, there is another group of complex drugs, the non-biological complex drugs, NBCDs, where the generic paradigm can be challenged as well. The NBCDs are defined as: 1. consisting of a complex multitude of closely related structures; 2. the entire multitude is the active pharmaceutical ingredient; 3. the properties cannot be fully characterized by physicochemical analysis and 4. the consistent, tightly controlled manufacturing process is fundamental to reproduce the product. NBCDs encompass product families such as the glatiramoids, liposomes, iron-carbohydrate colloids and many candidates of the group of the upcoming nanoparticulate systems. Following the main principles of regulatory pathways for biologicals (with appropriate product-by-product adjustments), instead of that for small molecules, would be the more logical strategy for these NBCDs. The status and outstanding regulatory issues for biosimilars and NBCD-similars/follow on versions were discussed at a conference in Budapest, Hungary (October 2014) and this commentary touches upon the issues brought up in the presentations, deliberations and conclusions.

Quantitative analysis of PEG-functionalized colloidal gold nanoparticles using charged aerosol detection.

Surface characteristics of a nanoparticle, such as functionalization with polyethylene glycol (PEG), are critical to understand and achieve optimal biocompatibility. Routine physicochemical characterization such as UV-vis spectroscopy (for gold nanoparticles), dynamic light scattering, and zeta potential are commonly used to assess the presence of PEG. However, these techniques are merely qualitative and are not sensitive enough to distinguish differences in PEG quantity, density, or presentation. As an alternative, two methods are described here which allow for quantitative measurement of PEG on PEGylated gold nanoparticles. The first, a displacement method, utilizes dithiothreitol to displace PEG from the gold surface. The dithiothreitol-coated gold nanoparticles are separated from the mixture via centrifugation, and the excess dithiothreitol and dissociated PEG are separated through reversed-phase high-performance liquid chromatography (RP-HPLC). The second, a dissolution method, utilizes potassium cyanide to dissolve the gold nanoparticles and liberate PEG. Excess CN(-), Au(CN)2 (-), and free PEG are separated using RP-HPLC. In both techniques, the free PEG can be quantified against a standard curve using charged aerosol detection. The displacement and dissolution methods are validated here using 2-, 5-, 10-, and 20-kDa PEGylated 30-nm colloidal gold nanoparticles. Further value in these techniques is demonstrated not only by quantitating the total PEG fraction but also by being able to be adapted to quantitate the free unbound PEG and the bound PEG fractions. This is an important distinction, as differences in the bound and unbound PEG fractions can affect biocompatibility, which would not be detected in techniques that only quantitate the total PEG fraction.

Longitudinal imaging of cancer cell metastases in two preclinical models: a correlation of noninvasive imaging to histopathology.

Metastatic spread is the leading cause of death from cancer. Early detection of cancer at primary and metastatic sites by noninvasive imaging modalities would be beneficial for both therapeutic intervention and disease management. Noninvasive imaging modalities such as bioluminescence (optical), positron emission tomography (PET)/X-ray computed tomography (CT), and magnetic resonance imaging (MRI) can provide complementary information and accurately measure tumor growth as confirmed by histopathology. Methods. We validated two metastatic tumor models, MDA-MD-231-Luc and B16-F10-Luc intravenously injected, and 4T1-Luc cells orthotopically implanted into the mammary fat pad. Longitudinal whole body bioluminescence imaging (BLI) evaluated metastasis, and tumor burden of the melanoma cell line (B16-F10-Luc) was correlated with (PET)/CT and MRI. In addition, ex vivo imaging evaluated metastasis in relevant organs and histopathological analysis was used to confirm imaging. Results. BLI revealed successful colonization of cancer cells in both metastatic tumor models over a 4-week period. Furthermore, lung metastasis of B16-F10-Luc cells imaged by PET/CT at week four showed a strong correlation (R (2) = 0.9) with histopathology. The presence and degree of metastasis as determined by imaging correlated (R (2) = 0.7) well with histopathology findings. Conclusions. We validated two metastatic tumor models by longitudinal noninvasive imaging with good histopathology correlation.